presentation of herpes simplex virus

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Sores associated with genital herpes can be small bumps, blisters or open sores. Scabs eventually form and the sores heal, but they tend to recur.

Most people infected with HSV don't know they have it. They may have no symptoms or have very mild symptoms.

Symptoms start about 2 to 12 days after exposure to the virus. They may include:

Pain or itching around the genitals
Small bumps or blisters around the genitals, anus or mouth
Painful ulcers that form when blisters rupture and ooze or bleed
Scabs that form as the ulcers heal
Painful urination
Discharge from the urethra, the tube that releases urine from the body
Discharge from the vagina

During the first outbreak, you may commonly have flu-like symptoms such as:

Swollen lymph nodes in the groin

Differences in symptom location

Sores appear where the infection enters the body. You can spread the infection by touching a sore and then rubbing or scratching another area of your body. That includes your fingers or eyes.

Sore can develop on or in the:

Repeat outbreaks

After the first outbreak of genital herpes, symptoms often appear again. These are called recurrent outbreaks or recurrent episodes.

How often recurrent outbreaks happen varies widely. You'll usually have the most outbreaks the first year after infection. They may appear less often over time. Your symptoms during recurrent outbreaks usually don't last as long and aren't as severe as the first.

You may have warning signs a few hours or days before a new outbreak starts. These are called prodromal symptoms. They include:

Genital pain
Tingling or shooting pain in the legs, hips or buttocks

When to see a doctor

If you suspect you have genital herpes, or any other STI , see your health care provider.

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Genital herpes is caused by two types of herpes simplex virus. These types include herpes simplex virus type 2 (HSV-2) and herpes simplex virus type 1 (HSV-1). People with HSV infections can pass along the virus even when they have no visible symptoms.

HSV-2 is the most common cause of genital herpes. The virus can be present:

On blisters and ulcers or the fluid from ulcers
The moist lining or fluids of the mouth
The moist lining or fluids of the vagina or rectum

The virus moves from one person to another during sexual activity.

HSV-1 is a version of the virus that causes cold sores or fever blisters. People may be exposed to HSV-1 as children due to close skin-to-skin contact with someone infected.

A person with HSV-1 in tissues of the mouth can pass the virus to the genitals of a sexual partner during oral sex. The newly caught infection is a genital herpes infection.

Recurrent outbreaks of genital herpes caused by HSV-1 are often less frequent than outbreaks caused by HSV-2 .

Neither HSV-1 nor HSV-2 survives well at room temperature. So the virus is not likely to spread through surfaces, such as a faucet handle or a towel. But kissing or sharing a drinking glass or silverware might spread the virus.

More Information

Genital herpes: Can you get it from a toilet seat?

Risk factors

A higher risk of getting genital herpes is linked to:

Contact with genitals through oral, vaginal or anal sex. Having sexual contact without using a barrier increases your risk of genital herpes. Barriers include condoms and condom-like protectors called dental dams used during oral sex. Women are at higher risk of getting genital herpes. The virus can spread more easily from men to women than from women to men.
Having sex with multiple partners. The number of people you have sex with is a strong risk factor. Contact with genitals through sex or sexual activity puts you at higher risk. Most people with genital herpes do not know they have it.
Having a partner who has the disease but is not taking medicine to treat it. There is no cure for genital herpes, but medicine can help limit outbreaks.
Certain groups within the population. Women, people with a history of sexually transmitted diseases, older people, Black people in in the United States and men who have sex with men diagnosed with genital herpes at a higher than average rate. People in groups at higher risk may choose to talk to a health care provider about their personal risk.

Complications

Complications associated with genital herpes may include:

Other sexually transmitted infections. Having genital sores raises your risk of giving or getting other STI s, including HIV / AIDS .
Newborn infection. A baby can be infected with HSV during delivery. Less often, the virus is passed during pregnancy or by close contact after delivery. Newborns with HSV often have infections of internal organs or the nervous system. Even with treatment, these newborns have a high risk of developmental or physical problems and a risk of death.
Internal inflammatory disease. HSV infection can cause swelling and inflammation within the organs associated with sexual activity and urination. These include the ureter, rectum, vagina, cervix and uterus.
Finger infection. An HSV infection can spread to a finger through a break in the skin causing discoloration, swelling and sores. The infections are called herpetic whitlow.
Eye infection. HSV infection of the eye can cause pain, sores, blurred vision and blindness.
Swelling of the brain. Rarely, HSV infection leads to inflammation and swelling of the brain, also called encephalitis.
Infection of internal organs. Rarely, HSV in the bloodstream can cause infections of internal organs.

Prevention of genital herpes is the same as preventing other sexually transmitted infections.

Have one long-term sexual partner who has been tested for STI s and isn't infected.
Use a condom or dental dam during sexual activity. These reduce the risk of disease, but they don't prevent all skin-to-skin contact during sex.
Don't have sex when a partner with genital herpes has symptoms.

Pregnancy precautions

If you are pregnant and know you have genital herpes, tell your health care provider. If you think you might have genital herpes, ask your provider if you can be tested for it.

Your provider may recommend that you take herpes antiviral medicines late in pregnancy. This is to try to prevent an outbreak around the time of delivery. If you have an outbreak when you go into labor, your provider may suggest a cesarean section. That is a surgery to remove the baby from your uterus. It lowers the risk of passing the virus to your baby.

Genital herpes: CDC detailed fact sheet. U.S. Centers for Disease Control and Prevention. https://www.cdc.gov/std/herpes/stdfact-herpes-detailed.htm. Accessed Sept. 28, 2022.
Genital herpes. Sexually Transmitted Infections Treatment Guidelines, 2021. U.S. Centers for Disease Control and Prevention. https://www.cdc.gov/std/treatment-guidelines/herpes.htm. Accessed Sept. 28, 2022.
AskMayoExpert. Simplex herpes virus (SHV) (adult). Mayo Clinic; 2022.
Loscalzo J, et al., eds. Herpes simplex virus infections. In: Harrison's Principles of Internal Medicine. 21st ed. McGraw Hill; 2022. https://accessmedicine.mhmedical.com. Accessed Sept. 28, 2022.
Schiffer JT et al. Herpes simplex virus. In: Mandell, Douglas, and Bennett's Principles and Practice of Infectious Diseases. 9th ed. Elsevier; 2020. https://www.clinicalkey.com. Accessed Sept. 28, 2022.
FAQs: Genital herpes. American College of Obstetricians and Gynecologists. https://www.acog.org/Patients/FAQs/Genital-Herpes. Accessed Sept. 28, 2022.
Dinulos JGH. Sexually transmitted viral infections. In: Habif's Clinical Dermatology. 7th ed. Elsevier; 2021. https://www.clinicalkey.com. Accessed Oct. 31, 2022.
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Genital Herpes – CDC Detailed Fact Sheet

What is genital herpes.

Genital herpes is a sexually transmitted disease (STD) caused by the herpes simplex virus type 1 (HSV-1) or type 2 (HSV-2).

How common is genital herpes?

Genital herpes infection is common in the United States. CDC estimated that there were 572,000 new genital herpes infections in the United States in a single year. 1 Nationwide, 11.9 % of persons aged 14 to 49 years have HSV-2 infection (12.1% when adjusted for age). 2 However, the prevalence of genital herpes infection is higher than that because an increasing number of genital herpes infections are caused by HSV-1. 3 Oral HSV-1 infection is typically acquired in childhood; because the prevalence of oral HSV-1 infection has declined in recent decades, people may have become more susceptible to contracting a genital herpes infection from HSV-1. 4

HSV-2 infection is more common among women than among men; the percentages of those infected during 2015-2016 were 15.9% versus 8.2% respectively, among 14 to 49 year olds. 2 This is possibly because genital infection is more easily transmitted from men to women than from women to men during penile-vaginal sex. 5 HSV-2 infection is more common among non-Hispanic blacks (34.6%) than among non-Hispanic whites (8.1%). 2 A previous analysis found that these disparities, exist even among persons with similar numbers of lifetime sexual partners. Most infected persons may be unaware of their infection; in the United States, an estimated 87.4% of 14 to 49 year olds infected with HSV-2 have never received a clinical diagnosis. 6

The age-adjusted percentage of persons in the United States infected with HSV-2 decreased from 18.0% in 1999–2000 to 12.1% in 2015-2016. 2

How do people get genital herpes?

Infections are transmitted through contact with HSV in herpes lesions, mucosal surfaces, genital secretions, or oral secretions. 5 HSV-1 and HSV-2 can be shed from normal-appearing oral or genital mucosa or skin. 7 ,8 Generally, a person can only get HSV-2 infection during genital contact with someone who has a genital HSV-2 infection. However, receiving oral sex from a person with an oral HSV-1 infection can result in getting a genital HSV-1 infection. 4 Transmission commonly occurs from contact with an infected partner who does not have visible lesions and who may not know that he or she is infected. 7 In persons with asymptomatic HSV-2 infections, genital HSV shedding occurs on 10.2% of days, compared to 20.1% of days among those with symptomatic infections. 8

What are the symptoms of genital herpes?

Most individuals infected with HSV are asymptomatic or have very mild symptoms that go unnoticed or are mistaken for another skin condition. 9 When symptoms do occur, herpes lesions typically appear as one or more vesicles, or small blisters, on or around the genitals, rectum or mouth. The average incubation period for an initial herpes infection is 4 days (range, 2 to 12) after exposure. 10 The vesicles break and leave painful ulcers that may take two to four weeks to heal after the initial herpes infection. 5,10 Experiencing these symptoms is referred to as having a first herpes “outbreak” or episode.

Clinical manifestations of genital herpes differ between the first and recurrent (i.e., subsequent) outbreaks. The first outbreak of herpes is often associated with a longer duration of herpetic lesions, increased viral shedding (making HSV transmission more likely) and systemic symptoms including fever, body aches, swollen lymph nodes, or headache. 5 ,10 Recurrent outbreaks of genital herpes are common, and many patients who recognize recurrences have prodromal symptoms, either localized genital pain, or tingling or shooting pains in the legs, hips or buttocks, which occur hours to days before the eruption of herpetic lesions. 5 Symptoms of recurrent outbreaks are typically shorter in duration and less severe than the first outbreak of genital herpes. 5 Long-term studies have indicated that the number of symptomatic recurrent outbreaks may decrease over time. 5 Recurrences and subclinical shedding are much less frequent for genital HSV-1 infection than for genital HSV-2 infection. 5

What are the complications of genital herpes?

Genital herpes may cause painful genital ulcers that can be severe and persistent in persons with suppressed immune systems, such as HIV-infected persons. 5 Both HSV-1 and HSV-2 can also cause rare but serious complications such as aseptic meningitis (inflammation of the linings of the brain). 5 Development of extragenital lesions (e.g. buttocks, groin, thigh, finger, or eye) may occur during the course of infection. 5

Some persons who contract genital herpes have concerns about how it will impact their overall health, sex life, and relationships. 5, 11 There can also be considerable embarrassment, shame, and stigma associated with a herpes diagnosis that can substantially interfere with a patient’s relationships. 10 Clinicians can address these concerns by encouraging patients to recognize that while herpes is not curable, it is a manageable condition. 5 Three important steps that providers can take for their newly-diagnosed patients are: giving information, providing support resources, and helping define treatment and prevention options. 12 Patients can be counseled that risk of genital herpes transmission can be reduced, but not eliminated, by disclosure of infection to sexual partners, 5 avoiding sex during a recurrent outbreak, 5 use of suppressive antiviral therapy, 5, 10 and consistent condom use. 7 Since a diagnosis of genital herpes may affect perceptions about existing or future sexual relationships, it is important for patients to understand how to talk to sexual partners about STDs. One resource can be found here: www.gytnow.org/talking-to-your-partner

There are also potential complications for a pregnant woman and her newborn child. See “ How does herpes infection affect a pregnant woman and her baby? ” below for information about this.

What is the link between genital herpes and HIV ?

Genital ulcerative disease caused by herpes makes it easier to transmit and acquire HIV infection sexually. There is an estimated 2- to 4-fold increased risk of acquiring HIV, if individuals with genital herpes infection are genitally exposed to HIV. 13-15 Ulcers or breaks in the skin or mucous membranes (lining of the mouth, vagina, and rectum) from a herpes infection may compromise the protection normally provided by the skin and mucous membranes against infections, including HIV. 14 In addition, having genital herpes increases the number of CD4 cells (the target cell for HIV entry) in the genital mucosa. In persons with both HIV and genital herpes, local activation of HIV replication at the site of genital herpes infection can increase the risk that HIV will be transmitted during contact with the mouth, vagina, or rectum of an HIV-uninfected sex partner. 14

How does genital herpes affect a pregnant woman and her baby?

Neonatal herpes is one of the most serious complications of genital herpes. 5, 16 Healthcare providers should ask all pregnant women if they have a history of genital herpes. 11 Herpes infection can be passed from mother to child during pregnancy or childbirth, or babies may be infected shortly after birth, resulting in a potentially fatal neonatal herpes infection. 17 Infants born to women who acquire genital herpes close to the time of delivery and are shedding virus at delivery are at a much higher risk for developing neonatal herpes, compared with women who have recurrent genital herpes . 16, 18-20 Thus, it is important that women avoid contracting herpes during pregnancy. Women should be counseled to abstain from intercourse during the third trimester with partners known to have or suspected of having genital herpes. 5, 11

While women with genital herpes may be offered antiviral medication late in pregnancy through delivery to reduce the risk of a recurrent herpes outbreak, third trimester antiviral prophylaxis has not been shown to decrease the risk of herpes transmission to the neonate. 11, 21, 22 Routine serologic HSV screening of pregnant women is not recommended. 11 However, at onset of labor, all women should undergo careful examination and questioning to evaluate for presence of prodromal symptoms or herpetic lesions. 11 If herpes symptoms are present a cesarean delivery is recommended to prevent HSV transmission to the infant. 5, 11, 23 There are detailed guidelines for how to manage asymptomatic infants born to women with active genital herpes lesions. 24

How is genital herpes diagnosed?

HSV nucleic acid amplification tests (NAAT) are the most sensitive and highly specific tests available for diagnosing herpes. However, in some settings viral culture is the only test available. The sensitivity of viral culture can be low, especially among people who have recurrent or healing lesions. Because viral shedding is intermittent, it is possible for someone to have a genital herpes infection even though it was not detected by NAAT or culture. 11

Type-specific virologic tests can be used for diagnosing genital herpes when a person has recurrent symptoms or lesion without a confirmatory NAAT, culture result, or has a partner with genital herpes. Both virologic tests and type-specific serologic tests should be available in clinical settings serving patients with, or at risk for, sexually transmitted infections. 11

Given performance limitations with commercially available type-specific serologic tests (especially with low index value results [<3]), a confirmatory test (Biokit or Western Blot) with a second method should be performed before test interpretation. If confirmatory tests are unavailable, patients should be counseled about the limitations of available testing before serologic testing. Healthcare providers should also be aware that false-positive results occur. In instances of suspected recent acquisition, serologic testing within 12 weeks after acquisition may be associated with false negative test results. 11

HSV-1 serologic testing does not distinguish between oral and genital infection, and typically should not be performed for diagnosing genital HSV-1 infection. Diagnosis of genital HSV-1 infection is confirmed by virologic tests from lesions. 11

CDC does not recommend screening for HSV-1 or HSV-2 in the general population due to limitations of the type specific serologic testing. 11 Several scenarios where type-specific serologic HSV tests may be useful include:

Patients with recurrent genital symptoms or atypical symptoms and negative HSV NAAT or culture;
Patients with a clinical diagnosis of genital herpes but no laboratory confirmation; and
Patients who report having a partner with genital herpes. 11

Patients who are at higher risk of infection (e.g., presenting for an STI evaluation, especially those with multiple sex partners), and people with HIV might need to be assessed for a history of genital herpes symptoms, followed by serology testing in those with genital symptoms. 11

Providers are strongly encouraged to look at CDC’s STI Treatment Guidelines for further diagnostic considerations.

Is there a cure or treatment for herpes?

There is no cure for herpes. Antiviral medications can, however, prevent or shorten outbreaks during the period of time the person takes the medication. 11 In addition, daily suppressive therapy (i.e., daily use of antiviral medication) for herpes can reduce the likelihood of transmission to partners. 11

There is currently no commercially available vaccine that is protective against genital herpes infection. Candidate vaccines are in clinical trials.

How can herpes be prevented?

Correct and consistent use of latex condoms can reduce, but not eliminate, the risk of transmitting or acquiring genital herpes because herpes virus shedding can occur in areas that are not covered by a condom. 25, 26

The surest way to avoid transmission of STDs, including genital herpes, is to abstain from sexual contact, or to be in a long-term mutually monogamous relationship with a partner who has been tested for STDs and is known to be uninfected.

Persons with herpes should abstain from sexual activity with partners when herpes lesions or other symptoms of herpes are present. It is important to know that even if a person does not have any symptoms, he or she can still infect sex partners. Sex partners of infected persons should be advised that they may become infected and they should use condoms to reduce the risk. Sex partners can seek testing to determine if they are infected with HSV.

Daily treatment with valacyclovir decreases the rate of HSV-2 transmission in discordant, heterosexual couples in which the source partner has a history of genital HSV-2 infection. 27 Such couples should be encouraged to consider suppressive antiviral therapy as part of a strategy to prevent transmission, in addition to consistent condom use and avoidance of sexual activity during recurrences.

Counseling those with genital herpes, as well as their sex partners, is critical. It can help patients cope with the infection and prevent further spread into the community. The STI Treatment Guidelines includes messaging broken down by herpes type. 11

1. Kreisel KM, Spicknall IH, Gargano JW, Lewis FM, Lewis RM, Markowitz LE, Roberts H, Satcher Johnson A, Song R, St. Cyr SB, Weston EJ, Torrone EA, Weinstock HS. Sexually transmitted infections among US women and men: Prevalence and incidence estimates, 2018. Sex Transm Dis 2021; in press.

2. McQuillan G, Kruszon-Moran D, Flagg EW, Paulose-Ram R. Prevalence of herpes simplex virus type 1 and type 2 in persons aged 14–49: United States, 2015–2016. NCHS Data Brief, no 304 . Hyattsville, MD: National Center for Health Statistics. 2018

3. Xu F, Sternberg MR, Kottiri BJ, et al. Trends in herpes simplex virus type 1 and type 2 seroprevalence in the United States. JAMA , 2006. 296 (8): 964–73.

4. Bradley H, Markowitz L, Gibson T, et al. Seroprevalence of herpes simplex virus types 1 and 2—United States, 1999–2010. J Infect Dis , 2014. 209 (3):325-33.

5. Corey L, Wald A. Genital Herpes. In: Holmes KK, Sparling PF, Stamm WE, et al. (editors). Sexually Transmitted Diseases . 4th ed. New York: McGraw-Hill; 2008: 399–437.

6. Fanfair RN, Zaidi A, Taylor LD, Xu F, Gottlieb S, Markowitz L. Trends in seroprevalence of herpes simplex virus type 2 among non-Hispanic blacks and non-Hispanic whites aged 14 to 49 years–United States, 1988 to 2010. Sex Transm Dis , 2013. 40 (11):860-4.

7. Mertz GJ. Asymptomatic shedding of herpes simplex virus 1 and 2: implications for prevention of transmission. J Infect Dis , 2008. 198 (8): 1098–1100.

8. Tronstein E, Johnston C, Huang M, et al. Genital shedding of herpes simplex virus among symptomatic and asymptomatic persons with HSV-2 infection. JAMA , 2011. 305 (14): 1441–9.

9. Wald A, Zeh J, Selke S, et al. Reactivation of genital herpes simplex virus type 2 infection in asymptomatic seropositive persons. New Engl J Med , 2000. 342 (12): 844–50.

10. Kimberlin DW, Rouse DJ. Genital Herpes. N Engl J Med , 2004. 350 (19): 1970–7.

11. Workowski, KA, Bachmann, LH, Chang, PA, et. al. Sexually Transmitted Infections Treatment Guidelines, 2021 . MMWR Recomm Rep 2021; 70(No. 4): 1-187.

12. Alexander L, Naisbett B. Patient and physician partnerships in managing genital herpes. J Infect Dis , 2002. 186 (Suppl 1): S57–S65.

13. Freeman EE, Weiss HA, Glynn JR, Cross PL, Whitworth JA, Hayes RJ. Herpes simplex virus 2 infection increases HIV acquisition in men and women: systematic review and meta-analysis of longitudinal studies. AIDS , 2006. 20 (1): 73–83.

14. Barnabas RV, Celum C. Infectious co-factors in HIV-1 transmission. Herpes simplex virus type-2 and HIV-1: new insights and interventions. Curr HIV Res , 2012. 10 (3): 228–37

15. Corey L, Wald A, Celum CL, Quinn TC. The effects of herpes simplex virus-2 on HIV-1 acquisition and transmission: a review of two overlapping epidemics. JAIDS , 2004. 35 (5): 435–45.

16. Brown ZA, Selke S, Zeh J, et al. The acquisition of herpes simplex virus during pregnancy. N Engl J Med , 1997. 337 (8): 509–15.

17. Kimberlin DW. Herpes simplex virus infections in the newborn. Semin Perinatol , 2007. 31 (2): 19–25.

18. Brown ZA, Wald A, Morrow RA, Selke S, Zeh J, Corey L. Effect of serologic status and cesarean delivery on transmission rates of herpes simplex virus from mother to infant. JAMA , 2003. 289 (2):203–9

19. Brown ZA, Benedetti J, Ashley R, et al. Neonatal herpes simplex virus infection in relation to asymptomatic maternal infection at the time of labor. N Engl J Med , 1991. 324 (18):1247–52

20. Brown ZA, Vontver LA, Benedetti J, et al. Effects on infants of a first episode of genital herpes during pregnancy. N Engl J Med , 1987. 317 (20):1246–51

21. Hollier LM, Wendel GD. Third trimester antiviral prophylaxis for preventing maternal genital herpes simplex virus (HSV) recurrences and neonatal infection. Cochrane Database Syst Rev , 2008. Issue 1: Art. No. CD004946.

22. Pinninti SG, Angara R, Feja KN, et al. Neonatal herpes disease following maternal antenatal antiviral suppressive therapy: a multicenter case series. J Pediatr , 2012. 161 (1):134-8.

23. American College of Obstetricians and Gynecologists (ACOG). ACOG Practice Bulletin. Clinical management guidelines for obstetrician-gynecologists. No. 82 June 2007. Management of herpes in pregnancy. Obstet Gynecol, 2007. 109 (6): 1489–98.

24. Kimberlin DW, Balely J, Committee on Infectious Diseases, Committee on Fetus and Newborn. Guidance on management of asymptomatic neonates born to women with active genital herpes lesions. Pediatrics, 2013. 131 (2):e635-46.

25. Martin ET, Krantz A, Gottlieb SL, et al. A pooled analysis of the effect of condoms in preventing HSV-2 acquisition. Arch Intern Med , 2009. 169 (13): 1233–40.

26. Wald A, Langenberg AGM, Link K, et al. Effect of condoms on reducing the transmission of herpes simplex virus type 2 from men to women. JAMA , 2001. 285 (24): 3100–6.

27. Corey L, Wald A, Patel R, et al. Once-daily valacyclovir to reduce the risk of transmission of genital herpes. N Engl J Med , 2004. 350 :11–20.

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Herpes Simplex Virus

Microbiology.

Herpes simplex virus 1 and 2 (HSV-1, HSV-2): members of the Herpes DNA virus family, Herpesviridae , aka Human Herpes Virus 1 and 2 (HHV-1 and HHV-2).
After primary infection, the virus establishes latency in neurons, with potential for reactivation--usually near the site of initial acquisition.
Tissue culture using Vero cell culture line or similar with tube or shell vial technique. Growth is often quick within 1-2d, with a cytopathic effect—confirmation of virus by immunologic staining.
Blood viral culture: use whole blood in a heparinized tube to obtain a buffy coat, an insensitive method.
HSV-1: 50-80% of adults seropositive (U.S. data)
HSV-2: 20-40% of adults seropositive
HSV-1: herpes labialis is the most common form of recurrent HSV-1, but 30% of genital HSV is nowadays HSV-1.
Asymptomatic in two-thirds of both HSV-1 and 2. Most HSV is acquired from an infected but asymptomatic person.
Primary gingivostomatitis: fever, sore throat, cervical lymphadenopathy , oral cavity vesicular enanthem that may involve the lips, tongue and mucosal surfaces [ Fig 1] .
Mononucleosis syndrome: pharyngitis , fever, cervical lymphadenopathy ; not uncommon primary infection of adolescents.
Genital infection: see the section below.
Neonatal infection: risk ~40% if primary genital HSV infection occurs in the mother during the third trimester.
Diagnostics: note that serotype-specific serology helps confirm seroconversion in primary infection; the role in non-primary infection diagnosis is poorly defined.
Women: infection is often asymptomatic, but when clinically active, may include sores or vesicles around the vaginal region, including the vulva and cervix, perianal, buttocks or thighs [ Fig 2 ]. Dysuria or difficulty voiding may accompany.
Men: often asymptomatic, less commonly with ulcers or vesicles on the penis, including glans and shaft [ Fig 3 ], perianal or on buttocks/thighs.
Primary infection may be associated with constitutional symptoms, often with urinary retention (in women), with or without aseptic meningitis (30% women; 10% men) and takes longer to resolve than recurrent disease.
HSV-2 accounts for 70-80% of cases; HSV-1 for 20-30% of cases.
HSV-2 is more likely to have clinical recurrences.
Genital ulcer disease, including that caused by genital HSV, increases the risk of acquiring and transmitting HIV infection.
Obtain scraping of cells or fluid by unroofing intact blister with a sterile needle.
HSV DNA PCR from lesions 98-100% sensitive.
Viral shedding is intermittent, so negative PCR does not exclude the diagnosis. Specificity is 97%.
1) Recurrent genital or atypical symptoms with negative HSV PCR or culture.
2) Clinical diagnosis of genital herpes without laboratory confirmation.
3) A patient whose partner has genital herpes.
See the Genital Ulcer Disease module for additional details.
The psychological impact of genital HSV cannot be overstated; 60% report being "devastated" when first told of their dx.
Recurrent genital herpes (>9 episodes/yr) in non-immunosuppressed may be due to persistently lower levels of IgG1, IgG3, and complement compared with infected persons without recurrent disease [15] .
Condom use and valacyclovir reduce transmission of genital herpes in serodiscordant couples [22] (this observation was not replicated in HIV/HSV-2 discordant couples [10] ).
Shedding may occur without clinically evident lesions.
CSF viral culture is insensitive.
Rarely CSF PCR negative, yet virus identified on brain biopsy/autopsy.
The prognosis is poor if the brain MRI shows edema with a shift.
Mortality is 70% without therapy.
Definition: >2 recurrences of fever and meningismus lasting 2-5 d with spontaneous recovery.
If presentation follows genital HSV recurrence, onset typically 5-7 days later.
Other viruses ( EBV , VZV , echoviruses) have been implicated.
M:F is 1:2 with mean age = 35 years.
Recurrences are usually less frequent over time.
Syndrome: fever, headache (can be severe), photophobia, meningismus; symptoms reach maximum intensity in a few hours.
50% have transient neurologic signs/symptoms, including cranial nerve palsies, diplopia, hallucinations, seizures, and altered consciousness. Thus, BRLM must be a dx of exclusion.
Hallmark is large granular plasma cells (Mollaret cells) seen by Papanicolaou stain.
Patients are completely well between episodes with CSF normalizing and spontaneous clinical resolution.
A study showed no benefit in risk reduction of meningitis recurrence at 2 years with suppressive valacyclovir (500mg twice daily ) [12] .
Approximately 50,000 new and recurrent ocular HSV/yr in the U.S., a leading cause of corneal opacification and infection-related visual loss.
Atopy increases the risk of ocular herpes [9] .
The recurrence rate is 20% by two years, 40% by five years, and 67% by seven years.
Herpetic Eye Disease Study Group has shown that oral acyclovir suppression following initial ocular herpes decreases recurrence by 45% in the first year; the most significant suppressive effect may be seen in those with concomitant history of atopy. This approach, however, may increase the risk of refractory disease due to the emergence of acyclovir resistance [11] .
Acute follicular conjunctivitis and kerato- conjunctivitis : foreign body sensation, lacrimation, photophobia, conjunctival hyperemia followed by vesicular blepharitis , ulceration, blurring of vision secondary to keratitis, and ultimate healing without scarring.
It begins with foreign body sensation, lacrimation, photophobia and decreased vision that is slow to heal; repeated recurrences can lead to scarring. It can also be sight-threatening.
Caused by viral reactivation, previously dormant in trigeminal ganglia.
Patients with atopy may have unusually severe keratitis due to impaired cell-mediated immunity.
Response to topical antivirals poor; use oral agents.
Herpes retinitis: rarer than VZV-related retinal necrosis, can lead to acute retinal necrosis secondary to occlusive vasculitis, sight-threatening.
HIV-infected persons: 60-70% in the U.S. are infected with HSV-2; disseminated infection with visceral involvement can be seen when CD4 count < 200 cells/mL and is potentially life-threatening.
Acyclovir is the most studied and is the preferred drug to use.
HSV outbreak in pregnancy, recommend treating for 7-10 days.
Pregnant women w/ HSV or a history of genital HSV are recommended to take oral antiviral suppression at 36 weeks.
Acute immunosuppression: may reactivate HSV within 2 wks of immunosuppression onset.
HSV esophagitis: seen in immunocompromised patients and must be differentiated from other causes of esophagitis, including CMV and Candida .
Usually seen in those with significant anti-HSV medication exposure and immunosuppression.
Acyclovir resistance is primarily due to TK-deficient strains.
Severe, recurrent ano- genital herpes : commonly seen in patients with AIDS with low CD4 counts (< 200 cells/mL) and high viral loads. HIV-infected, especially w/ AIDS, need more prolonged treatment and/or higher doses for episodic cutaneous HSV.
HSV tracheobronchitis: rare but most commonly seen in immunosuppressed or elderly intubated patients.
Herpes dermatitis: seen in athletes (herpes gladiatorum), health care workers (herpetic whitlow) and patients with eczema who become superinfected with HSV (Kaposi’s varicelliform eruption).
Usually due to HSV-1, but HSV-2 has increasingly been identified.
It may reactivate after exposure to sunlight, wind, cold, emotional stressors or the late stage of the menstrual cycle.
Erythema multiforme: a condition in ~5% following recurrent or symptomatic HSV.
Mean duration of recurrence (vesicles to the healing of lesions): 7-8 d.
Mean duration of viral shedding: approximately 60h (measured by PCR) with a peak viral load during the vesicle/ulcer stage.
Data suggest that lesion size, progression to ulcers and duration are improved with topical acyclovir 5%/hydrocortisone 1% combination compared to topical acyclovir 5% alone.
Occurs in 1:3,000-20,000 live births, 50% of cases are due to HSV type 1 and 50% to type 2.
The vertical transmission risk to the neonate is 40-80% if primary transmission occurs at the delivery time.
Congenital herpes occurs by the transfer of infection in utero and is extremely rare.
Cesarean section if active disease at the time of labor.
Cutaneous/ocular/oral: lesions may be seen.
CNS: encephalitis may occur without mucocutaneous findings.
Disseminated infection: multiple organs involved, including the above and hepatitis, pneumonitis, and DIC.
Recurrent genital herpes is associated with a very low risk of neonatal herpes (0-3% for vaginal delivery).
Most neonates with CNS infection will not have skin/eye or mouth manifestations.
Neonates with CNS herpes may have better neurological development if continued on suppressive acyclovir after initial treatment [13] .
Elective Cesarean section and suppressive therapy with acyclovir (400mg TID) at or beyond 36 weeks of gestation are recommended for women with first-episode genital lesions during the third trimester.
Vaginal delivery and suppressive acyclovir therapy are recommended for recurrent genital lesions during the third trimester.
Note that ~15% of women with "primary" genital herpes present with recurrent infections.
Re-testing is not needed for prior confirmed history.
Active lesions: obtain a specimen for culture, antigen or molecular testing.
History of ulcers, no active lesions: obtain type-specific serology.
Insufficient data to recommend routine screening of all pregnant women for HSV.
Oral antiviral suppression is recommended at 36 weeks for those with genital HSV history.

SITES OF INFECTION

Oro-facial: primary gingivostomatitis; recurrent stomatitis; herpes labialis
Genital: genital ulcer disease
Eye: follicular conjunctivitis , keratitis , acute retinal necrosis syndrome, endophthalmitis
Other skin areas: eczema herpeticum, herpetic whitlow, herpes gladiatorum
Central nervous system: sporadic encephalitis , meningoencephalitis, aseptic meningitis ; sacral radiculopathy, benign recurrent lymphocytic meningitis (Mollaret’s meningitis)
Esophagus: esophagitis
Respiratory system: pneumonia , tracheobronchitis
Liver: hepatitis
Rectum: proctitis
Multiple organs: disseminated infection

Mucocutaneous Infections

Acyclovir 400 mg PO three times daily
Acyclovir 200 mg PO five times daily
Famciclovir 250 mg PO three times daily
Valacyclovir 1g PO twice daily
Acyclovir 400 mg PO three times daily x 2d
Acyclovir 800 mg PO twice daily x 5d
Acyclovir 800 mg PO three times daily x 5d
Valacyclovir 500mg PO twice daily x 3d
Famciclovir 125 mg PO three times daily x 5d
Famciclovir 1000mg PO twice daily x 1 day alternative short-course regimen
Acyclovir 400 mg PO three times daily x 5-10d
Famciclovir 500 mg PO twice daily x 5-10d
Valacyclovir 1g PO twice daily x 5-10d
Acyclovir 400 mg PO twice daily
Famciclovir 250 mg PO twice daily
Valacyclovir 500 mg PO daily
Valacyclovir 1g PO daily (recommended for persons with >10 recurrences/year)
400 mg three times daily used in pregnancy due to enhanced renal clearance rather than twice daily dosing
Not suggested in pregnancy
Above dose in pregnancy.
Use oral acyclovir per the above regimen with initial HSV infection or if highly symptomatic recurrent HSV.
Parenteral acyclovir is needed for life-threatening infection.
Acyclovir 400 mg PO three times a day x 7-10d
Acyclovir 200 mg PO five times a day x 7-10d
Famciclovir 250 mg PO q8h X 7-10d
Valacyclovir 1g PO twice daily x 7-10d
Valacyclovir 250 mg PO twice daily
Valacyclovir 500 mg PO once daily
Valacyclovir 1000 mg PO once daily
Acyclovir 400-800 mg PO five times daily
Famciclovir 500mg PO twice or thrice daily
Acyclovir 5 mg/kg IV q8h

Central Nervous System Infection

Duration of treatment x 21d advocated by some to minimize relapse.
Neonatal CNS infection is usually maintained on acyclovir suppression.
HSV meningitis often with hemorrhagic features in CSF fluid.
Recent data suggests that 11% have sequelae six months after infection [6] .
Antiviral treatment is not necessary to use as the condition is usually self-limiting.
A study showed no benefit of suppressive valacyclovir in preventing the recurrence of meningitis [12] .

Severe, non-CNS infections

May convert to an oral regimen upon sufficient clinical response.

Ocular Infection

Requires ophthalmological consultation. Suggestions adapted from [3] .
Use a therapeutic oral or topical agent. • Acyclovir 400 mg 3–5 times daily for 7–10 days • Valacyclovir 500 mg twice daily for 7–10 days • Famciclovir 250 mg twice daily for 7–10 days • Trifluridine 1% 1 drop 9 times daily for 7 days (not to exceed 21 days) • Acyclovir 3% ointment 5x daily for 7 days • Ganciclovir ointment 0.15% 1 drop 5 times daily for 7 days
Epithelial debridement
Taper slowly to the lowest dose required to control inflammation
PLUS prophylactic oral antiviral • Acyclovir 400 mg 2 times daily • Valacyclovir 500 mg daily • Famciclovir 250 mg daily
Use Prednisolone acetate 1% 1 to 4 times daily
PLUS • Acyclovir 800 mg 5 times daily for 7–10 days • Valacyclovir 1 gm 3 times daily for 7–10 days • Famciclovir 500 mg 2–3 times daily for 7–10 days
Acyclovir 800 mg 5 times daily
Valacyclovir 1 gm 3 times daily
Famciclovir 500 mg 2 times daily
PLUS prednisolone acetate 1% 4 times daily

Prophylaxis for Immunosuppressed Patients

Initiation: acyclovir 5 mg/kg IV q8h X 7d.
Maintenance: acyclovir 200-400 mg PO 3-5x daily x 1-3 mos.
Acyclovir 400-800 mg PO twice or thrice daily
Valacyclovir 500 mg daily
Famciclovir 500 mg twice daily
Burn pts: acyclovir 5 mg/kg IV q8h x 7d, then 200 mg PO 5x/d x 7-14d.

Acyclovir-Resistant Strains

Acyclovir resistance should be suspected in unresponsive cases.

Foscarnet 40-80mg/kg IV q8h until clinical resolution.
Topical cidofovir gel 1% for genital or perirectal lesions daily X 5 d may be tried (local pharmacy must compound).
Imiquimod 5% q8H 3 times/week applied to the lesion until clinical resolution is an alternative therapy for HSV-resistant genital infections.
Parenteral cidofovir : rarely used option due to toxicities, but may be considered for resistant systemic HSV infection at 5mg/kg once weekly.

Selected Drug Comments

Resistance testing is not routinely recommended unless there is an apparent clinical failure.
Suppression therapy does not obviate recurrences but reduces frequency and severity. Relapse if on suppressive therapy is not solved by escalating the dose.
Moderate 40%
Mild or normal 30%
< 30 years of age
< 4 days of symptoms
Glasgow coma scale (GCS) score > 6
If GCS < 6 and > 30 years, only 35% with normal or mild impairments if they survive.
Neonatal HSV-2 infection is worse than HSV-1.

OTHER INFORMATION

HSV suppressive therapy does NOT decrease the risk of HIV acquisition among HSV-infected, HIV-uninfected women.
Breastfeeding is not contraindicated in women with active herpes simplex virus unless there is a lesion in the breast. Given that postnatally-acquired herpes can be as lethal as that acquired during delivery special consideration of handwashing should be taken by mothers and family members with active lesions in any part of the body.
HSV serologic testing should be considered for persons presenting for an STD evaluation (especially for those persons with multiple sex partners), persons with HIV infection, and MSM at increased risk for HIV acquisition. Screening for HSV-1 and HSV-2 in the general population is not indicated.
Abstinence during the presence of symptoms or signs is not an effective strategy to reduce the risk of transmission since asymptomatic shedding plays a significant role in HSV transmission.
Transmission can be reduced with either suppressive therapy or condom use.

Basis for recommendation

Comment: Updated CDC STI Treatment Guidelines used in this module.

Comment: Recommendations for diagnosis and management in pregnancy are used in this module.

Comment: The spectrum of disease and dosing recommendations, also photos.

Comment: 2014 update of the 2007 guidelines on the management of anogenital herpes.

Comment: Clinically useful overview focusing on neurological disease ranging from pregnancy/neonatal to adults.d

Comment: Database analysis from Denmark looked at 205 patients and found less favorable outcomes in 31%, evaluated at discharge. More concerning was 11% after 6 months, unrelated to any clinical or lab-based factors. Almost all received antiviral therapy (96%, majority oral ), so unclear whether it helped.

Comment: Negative trial to see if patients with prolonged ventilation and (+) HSV secretions benefited from antiviral suppression.

Comment: Review of 1028 infants with HSV PCR performed in blood and CSF specimens. Of the 21 who had positive CSF PCR, 76% also had positive HSV PCR in their blood. The important conclusion is that a blood PCR in this population cannot be used to exclude CNS HSV infection.

Comment: This was a retrospective, population-based case-control study of 114 patients with HSV ocular disease and 137 with herpes-zoster ocular disease (HZO) in Hawaii. Authors found that patients with atopy had a 2.6-fold (95% CI, 1.6-4.2) higher odds of having HSVocular disease and 1.8-fold (95% CI, 1.2-2.8) increased odds of having HZO compared to patients without atopy. Patients with 2 or more atopic conditions had an 8.9-fold (95% CI, 3.5-22.6) higher odds of having HSVocular disease and a 2.9-fold (95% CI, 1.1-7.7) higher odds of having HZO. Rating: Important

Comment: This randomized controlled trial evaluated the impact of acyclovir 400mg twice daily on the prevention of transmission of HSV-2 genital herpes in HIV-1/HSV-2 discordant couples in Africa. Key findings : Treatment of HSV-2/HIV-1-infected persons with daily suppressive acyclovir did not decrease the risk of HSV-2 transmission to susceptible partners. Rating: Important

Comment: This retrospective study analyzed 169 corneal swabs from 78 immunocompetent patients with recurrent herpetic keratitis for acyclovir resistance. Key findings : 1) 26% of the isolates were acyclovir-resistant, 2) acyclovir prophylaxis x ≥12 m and recurrence duration of ≥45 days were associated with acyclovir resistance and acyclovir refractory disease, 3) acyclovir-resistant isolates were a risk factor for acyclovir refractory disease (OR 2.28; 95% CI, 1.06–4.89). Rating: Important

Comment: This Sweedish randomized, double-blind, placebo-controlled multicenter trial investigated the effect of valacyclovir on the prevention of recurrence of HSV meningitis. Patients received valacyclovir 500 mg twice daily (n=50) or placebo (n=51) for 1 year after primary or recurrent, confirmed or probable, HSV meningitis. Patients were followed for 2 years. Key finding : no difference between the 2 groups during the first year however, during the second year, the risk of recurrence was higher among patients exposed to valacyclovir (HR, 3.29 [95% confidence interval, 10.06–10.21]). Rating: Important

Comment: A study of 74 infants with neonatal HSV: 45 with CNS involvement were enrolled in a study; 29 with skin, eye and mouth involvement (enrolled in a different study). All 45 neonates with CNS involvement received 14-21 d of parenteral acyclovir and were randomly assigned to receive acyclovir suppression TID x 6 mo vs. placebo. The Mental Development Index of the Bayley Scales of Infant Development (in which scores range from 50 to 150, with a mean of 100 and with higher scores indicating better neurodevelopmental outcomes) was assessed in 28 of the 45 infants with CNS involvement (62%) at 12 months of age. Infants surviving neonatal HSV disease with CNS involvement had significantly improved neurodevelopmental outcomes after receiving suppressive therapy with oral acyclovir for 6 months. Rating: Important

Comment: This randomized, double-blind, placebo-controlled multicenter, multinational phase III clinical trial among HIV-uninfected, HSV-2 seropositive heterosexual women (n=1358) and men who have sex with men (MSM; n=1814) examined the primary outcome of new HIV-1 acquisition and the secondary outcome of the incidence of genital ulcers amongst those receiving twice daily acyclovir (400 mg) and placebo. Amongst participants from all countries, no reduction in HIV-1 incidence was noted between the treatment and control groups. HSV-2 positive ulcers were reduced by 63% in the treatment group compared with the control group (Relative risk = 0.37, Confidence Interval 0.31-0.45). No serious drug effects were noted in the study. Rating: Important

Comment: This prospective case-control study examined immunogenetic risk factors for recurrent genital herpes. the study population included 52 consecutive eligible patients, without immunodeficiency, with culture-confirmed HSV-2 from an active lesion >12 months before enrollment and >9 recurrences per year and 80 HSV seropositive and 70 HSV seronegative controls. Anti-HSV-2 antibodies did not correlate with protection from recurrence. Risk factors for recurrence included lower IgG1 antibody -Confidence Interval (CI), 2.0-12.5; p< 0.001 and IgG3 antibody - CI 1.7-7,8, p< .001. Complement levels were lower in patients with recurrent symptomatic infections. Rating: Important

Comment: This Australian community-based cohort study of 1,427 HIV-negative gay men examined risk factors for herpes simplex virus type 1 (HSV-1) and HSV type 2 (HSV-2) over a median follow-up period of 2 years. At enrolment, the prevalence of HSV-1 was 75%, and HSV-2 was 23%, and both infections had a lower prevalence in those < 25 years. The incidence of HSV-1 infection was 5.58/100 person-years (PY) and 1.45/100 PY for HSV-2. Using multivariate analysis, significant independent risk factors for HSV-1 infection were insertive oral intercourse with casual sex partners (hazards ratio = 3.91; 95% confidence interval [CI] =1.23-12.44) and younger age (p< 0.03). A significant risk factor for HSV-2 acquisition was anal sex with casual partners. Rating: Important

Comment: This randomized, controlled Phase IIb trial of a 10-session behavioral intervention vs. brief counseling session (control group) to reduce HIV acquisition among 4295 high-risk HIV-uninfected men who have sex with men (MSM). Sera and behavioral data collected during this trial were subsequently examined to determine risk factors for herpes simplex virus type 2 (HSV-2, ) evaluate the role of prevalent and incident HSV-2 infection in HIV infection acquisition, and determine the impact of the behavioral intervention on HSV acquisition (already shown not to have a role in HIV acquisition). 91% of subjects had evaluable data; 20.3% were HSV-2 positive (by serology) at enrolment; 4.3% acquired infection over the 24-month study period, and 75.4% remained uninfected with HSV-2. Risk factors for seroconversion included unprotected anal receptive intercourse in the prior 6 months, having at least 1 HIV-infected partner in the past 6 months, and having >5 male sex partners in the last 6 months. HIV risk was increased among MSM with recent HSV-2 infection identified compared with HSV-2 uninfected MSM. The intense behavioral intervention did not increase the risk of HSV-2 infection. Rating: Important

Comment: This is the report of a 27-site multicenter randomized, double-blinded parallel placebo control trial examining the efficacy of 1-gram valacyclovir (VAC) in reducing HSV-2 viral shedding in both clinical and asymptomatic infections among immunocompetent persons. 152 persons were randomized--43 placebo (40 completed) and 109 VAC (94 completed. Over 60 days, each participant reported daily on the presence or absence of genital lesions and collected daily genital and anorectal samples. VAC significantly decreased total days of viral shedding amongst clinical and subclinical cases and a viral load when shedding compared with the controls. In the intent-to-treat group, a 71% reduction in total shedding was noted (p< 0.001), a 58% reduction in subclinical shedding (p< 0.001), and a 64% reduction in clinical shedding (p< 0.01) was seen in the VAC group. There were no major adverse effects noted with VAC over the 60-day study period. Rating: Important

Comment: Excellent review on RBLM.

Comment: The authors report the largest case series of 11 patients/12 eyes with HSV-2 acute retinal necrosis (ARN) and review the world’s literature. Although other infections are associated with ARN, the authors identify some HSV-2-specific features, including young age at DX, hx of HSV at birth, and a preexisting chorioretinal scar in the ARN eye, triggering events such as trauma or steroids. Also, the clinical syndrome described with HSV-2 is more aggressive and rapid. This is a sight-threatening condition and requires prompt consultative referral to an ophthalmologist. Rating: Important

Comment: The authors report the findings of a study among 89 pts with HSV-like lesions--81 with genital and 8 with cutaneous lesions. Specimens were collected for quantitative duplex PCR and culture; 64% were PCR positive, 51% were cx positive. PCR detected 30 of 34 primary and 24 of 29 recurrent infections. 2 HSV-1 samples were positive on cx only despite repeated PCR attempts. Symptomatic pts had significantly higher copy numbers on PCR. In this study, duplex qPCR for HSV-1 and HSV-2 was more sensitive than the gold standard cx for mucocutaneous HSV. Rating: Important

Comment: The authors conducted a study among 528 mutually-monogamous heterosexual couples discordant for HSV-2 infection. Although the antiviral was the intervention under observation, data were also collected re: condom use. When condoms were used more than 70% of the time by the discordant pairs with a positive man and a negative woman, transmission risk was reduced by 60%, even in the absence of antiviral suppression. Acquisition of infection by the seronegative partner and recurrence and shedding by the positive partner were significantly reduced when valacyclovir was used. Rating: Important

Comment: Report of a chart review of 170 patients seen on referral to a dermatology clinic found to have culture-confirmed HSV. This specialty practice was likely to see "outliers" in presentation as only 49% had "typical" cluster genital lesions. Single ulcers, erosion, crusts, fissures, edema, and erythema were seen. Women were more likely to have extragenital lesions than men. Rating: Important

Comment: The author reviewed 29 published clinical trials. Notable that ACV ointment did cause superficial punctate keratitis in 9.8% of 998 pts and 4% noted burning of the eye with the application of the agent. Found to compare favorably with other topical antiherpetics available. Rating: Important

Primary Herpes Simplex virus infection

Descriptive text is not available for this image

Primary Herpes Simplex virus infection involving lips and tongue.

Source: CDC

Genital HSV female

Ulcers and vesicle from genital HSV are seen around vaginal introitus due to HSV-2.

Genital HSV male

Vesicle seen on penile shaft due to HSV-2.

Source: CDC/Suan Lindsley

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Herpes Simplex Virus (HSV) Infections

Symptoms and Complications |
Diagnosis |
Treatment |
Prevention |

Herpes simplex virus infection causes recurring episodes of small, painful, fluid-filled blisters on the skin, mouth, lips (cold sores), eyes, or genitals.

This very contagious viral infection is spread by direct contact with sores or sometimes contact with an affected area when no sores are present.

Herpes causes blisters or sores in the mouth or on the genitals and, often with the first infection, a fever and general feeling of illness.

The virus sometimes infects other parts of the body, including the eyes and brain.

Usually, doctors easily recognize the sores caused by herpes, but sometimes analysis of material from a sore or blood tests are necessary.

No medication can get rid of the virus, but antiviral medications can help relieve symptoms and help symptoms resolve a little sooner.

Herpes simplex is one of several types of herpesviruses . There are two types of herpes simplex virus (HSV):

HSV-1, which is the usual cause of cold sores on the lips (herpes labialis) and sores on the cornea of the eye ( herpes simplex keratitis )

HSV-2, which is the usual cause of genital herpes (although HSV-1 can also cause genital herpes)

Infection can also occur in other parts of the body such as the brain (a serious illness) or gastrointestinal tract. Widespread infection may occur in newborns or in people with a weakened immune system , particularly those who have HIV infection .

HSV is very contagious and can be spread by direct contact with sores and sometimes by contact with the mouth (oral area) or genitals of people who have HSV infection even when no sores are can be seen.

Primary and recurrent (reactivated) infection

After the first (primary) infection, HSV, like other herpesviruses, remains inactive (dormant or latent) in the body for life. A latent infection may not cause symptoms again, or it may periodically reactivate and cause symptoms.

The primary HSV infection produces an eruption of tiny blisters. After the eruption of blisters subsides, the virus remains in a dormant state inside the collection of nerve cells (ganglia) near the spinal cord that supply the nerve fibers to the infected area. Periodically, the virus reactivates, begins multiplying again, and travels through the nerve fibers back to the skin—causing eruptions of blisters in the same area of skin as the earlier infection. Sometimes the virus is present on the skin or mucous membranes even when no blisters can be seen.

The virus may reactivate many times. Reactivation of a latent oral or genital HSV infection may be triggered by the following:

Emotional stress

Suppression of the immune system (for example, by a medication taken to prevent rejection of an organ transplant )

Physical trauma, such as a dental procedure

Overexposure of the lips to sunlight

Often, the trigger is unknown.

Symptoms and Complications of HSV Infections

Tiny blisters appear on the following:

Skin: Particularly around the mouth or on the genitals

Mucous membranes: Including those lining the eyes, vagina, cervix, or inside of the mouth.

The skin around the blisters is often red.

Oral infection

The first oral infection with HSV usually causes many painful sores inside the mouth (herpetic gingivostomatitis). Herpetic gingivostomatitis most commonly develops in children.

People usually feel sick and have a fever, a headache, and body aches.

The mouth sores last 10 to 19 days and are often very severe, making eating and drinking extremely uncomfortable. As a result, people may become dehydrated. Occasionally, no symptoms develop.

Recurrences usually produce a cluster of sores on the rim of the lip.

The lip sore is called a cold sore or fever blister (so named because they are often triggered by colds or fevers). Other triggers include over-exposure to sun on the lips, anxiety, certain dental procedures, and any condition that reduces the body's resistance to infection.

Image courtesy of Dr. Herrmann via the Public Health Image Library of the Centers for Disease Control and Prevention.

Before a cold sore appears, people usually feel a tingling at the site, lasting from minutes to a few hours, followed by redness and swelling. Usually, fluid-filled blisters form and break open, leaving sores. The sores quickly form a scab. After about 5 to 10 days, the scab falls off and the episode ends. Less often, tingling and redness occur without blister formation.

Other infections and complications

Genital herpes causes painful blisters in the genital and/or anal area. In women, internal blisters may develop in the vagina or on the cervix. Internal blisters are less painful and are not visible. The blisters develop 4 to 7 days after people are infected. The blisters go away but may come back (recur) because the virus never truly leaves the body. Blisters caused by a first genital infection are usually more painful, last longer, and are more widespread than those caused by a recurrent infection.

In people with a weakened immune system, recurrences of oral or genital herpes can result in progressive, gradually enlarging sores that take weeks to heal. The infection may progress inside the body, moving into the esophagus, lungs, or colon. Ulcers in the esophagus cause pain during swallowing, and lung infection causes pneumonia with cough and shortness of breath.

Sometimes HSV-1 or HSV-2 enters through a break in the skin of a finger, causing a swollen, painful, red fingertip ( herpetic whitlow ). Health care workers who are exposed to saliva or other body secretions (such as dentists) when not wearing gloves are most commonly affected.

DR P. MARAZZI/SCIENCE PHOTO LIBRARY

HSV-1 can infect the cornea of the eye. This infection (called herpes simplex keratitis ) causes a painful sore, tearing, sensitivity to light, and blurred vision. Over time, particularly without treatment, the cornea can become cloudy, causing a significant loss of vision.

Infants or adults with a skin disorder called atopic eczema can develop a potentially severe HSV infection in the area of skin that has the eczema (eczema herpeticum). Therefore, people with atopic eczema should avoid being near anyone with an active herpes infection.

HSV can infect the brain. This infection (called herpes encephalitis ) begins with confusion, fever, and seizures and can be fatal.

Infrequently, a pregnant woman can transmit HSV infection to her baby (called neonatal herpes ). Transmission usually occurs at birth, when the baby comes into contact with infected secretions in the birth canal. Rarely, HSV is transmitted to the fetus during pregnancy. Transmission during birth is more likely when

The mother has recently acquired the herpes infection

The mother has visible herpes sores in the vaginal area, although babies may become infected from mothers who have no apparent sores

When acquired at birth, the infection appears between the first and fourth week of life. Newborns with HSV infection become very ill. They may have widespread disease, brain infection, or skin infection. Without treatment, about 85% of those with widespread disease and about half of those with a brain infection die. Even with treatment, many have brain damage.

Diagnosis of HSV Infections

Testing of a sample taken from the sore

If doctors suspect a brain infection, magnetic resonance imaging and a spinal tap

Herpes simplex virus infection is usually easy for doctors to recognize. If unsure, doctors may use a swab to take a sample of material from the sore and send the swab to a laboratory to grow (culture) and identify the virus.

Material from the sore can also be tested using polymerase chain reaction (PCR) to identify the herpes simplex DNA. PCR is a more sensitive test than viral culture, which means it will miss fewer cases of the virus.

Sometimes doctors examine material scraped from the blisters under a microscope. Although the virus itself cannot be seen, scrapings sometimes contain enlarged infected cells (giant cells) that are characteristic of infection by a herpes-type virus.

Blood tests to identify antibodies to HSV can also be helpful. ( Antibodies are produced by the immune system to help defend the body against a particular attacker, such as HSV.)

Certain blood tests can distinguish between HSV-1 infection and HSV-2 infection.

If a brain infection is suspected, magnetic resonance imaging (MRI) of the brain and a spinal tap (lumbar puncture) to obtain a sample of cerebrospinal fluid for analysis may be done.

Treatment of HSV Infections

Antiviral medications.

Some Antiviral Medications for Herpesvirus Infections ). Treatment is most effective if started early, usually within a few hours after symptoms start—preferably at the first sign of tingling or discomfort, before blisters appear. For people who have frequent, painful attacks, the number of outbreaks can be reduced by taking antivirals every day indefinitely (called suppressive therapy). However, taking antivirals does not necessarily prevent infected people from transmitting the infection. Most antivirals are available by prescription only.

Severe HSV infections, including herpes encephalitis and infections in newborns

People with herpes simplex keratitis

Other treatments

For people who have minimal discomfort, the only treatment needed for recurring herpes of the lips or genitals is to keep the infected area clean such as by gentle washing with soap and water. Applying ice may be soothing and reduce swelling.

Pain relievers may be taken for pain.

Prevention of HSV Infections

People with HSV infection should avoid activities and other things known to trigger recurrences. For example, people with oral HSV infection triggered by sunlight should avoid exposure to sunlight as much as possible or use sunscreen when sunlight cannot be avoided.

Because HSV infection is contagious, people with infection of the lips should avoid kissing as soon as they feel the first tingling (or, if no tingling is felt, when a blister appears) until the sore has completely healed. They should not share a drinking glass and, if possible, should not touch their lips. They should also avoid oral sex.

People with genital herpes should use condoms at all times. Even when there are no visible blisters and no symptoms, the virus may be present on the genitals and can be spread to sex partners.

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Genital herpes: diagnosis and treatment

Coloured transmission electron micrograph (TEM) of herpes virus particles in infected tissue

DR KLAUS BOLLER/SCIENCE PHOTO LIBRARY

After reading this article, you should be able to:

Recognise the signs and symptoms of genital herpes, including the risk factors;
Understand how the condition is transmitted and how it is diagnosed ;
Know how the condition is managed and be able to provide patients with self-care advice .

Introduction

Genital herpes is a common sexually transmitted infection caused by the herpes simplex virus (HSV). There are two types of herpes: herpes simplex type 1 (HSV-1) and herpes simplex type 2 (HSV-2) [1] . HSV-1 is the usual cause of herpes around the mouth and lips (orolabial) and now the most common cause of genital herpes in the UK. HSV-2 was historically the most common cause of genital herpes in the UK and the virus type that is more likely to cause recurrent anogenital symptoms [2] .

In the UK, seven out of ten people have contracted at least one type of HSV by the age of 25 years [1] . Only one third of individuals will develop symptoms and so most people do not know that they have it [2] .

Herpes simplex is part of the herpes virus family that also includes chickenpox and glandular fever. These viruses can remain dormant in the body and may lead to recurring symptoms in the future, which is why they are often referred to as ‘incurable’. However, when symptoms resurface, the immune system works to heal or ‘cure’ them. Typically, herpes simplex infections are localised to a specific area (or dermatome) and do not commonly spread to other parts of the body [1] . The focus of this article is primarily on infections in the genital region.

Pharmacists have a role in providing information, support and treatment options for individuals with genital herpes. This article will outline the symptoms and risk factors, diagnosis and management of the condition.

Signs and symptoms

Genital herpes can present with symptoms such as blisters, tingling, itching, and pain in the genital area. This is notably different from the appearance of genital warts, which present as flesh-coloured lumps [3] . These symptoms may vary between men and women, with women often experiencing more severe symptoms [3] . It is important to note that some individuals may be asymptomatic carriers of the virus [3] . The interval between infection and the appearance of symptoms can be between two days and two weeks. Many people may have mild symptoms or no symptoms at all when they are first infected, which can make it difficult to realise they have contracted the virus. Symptoms may not appear for many years, which may potentially cause suspicion o f infidelity in a long-term relationship [1] .

Some individuals may never experience symptoms at all (known as asymptomatic carriers), while others may initially feel tingling or itching in the genital area. Swollen and sensitive glands in the groin, along with flu-like symptoms, may also occur. Small blisters or ulcers may appear, which can be numerous or there may only be one. These blisters eventually burst, leaving behind small, red, often painful sores that crust over on dry skin and heal within three to ten days [1] .

Symptoms of genital herpes can include painful ulceration, dysuria and vaginal or urethral discharge. Systemic symptoms, such as fever and myalgia, are more common in primary infections than in non-primary or recurrent disease. Rarely, systemic symptoms may be the only indication of infection [2] .

On examination, signs of genital herpes may include tender swollen lymph nodes around the groin (inguinal lymphadenitis). In first episodes of the disease, lesions and lymphadenitis are typically bilateral. Recurrent episodes may have lesions that affect favoured sites on one side. Lymphadenitis occurs in approximately 30% of patients with genital herpes [2] .

Risk factors

The following risk factors can increase the likelihood of contracting the virus and developing symptoms:

Oral, anal or vaginal sex without the use of barrier protection (e.g. condom, dental dam);
Sexual contact with a person with HSV-1 or HSV-2;
Multiple or anonymous sexual partners;
Presence or history of another sexually transmitted or blood-borne infection;
Females are at higher risk of acquiring genital herpes from a male partner than vice versa [4] .

Transmission

Herpes is passed directly from the affected area of skin, which could be the genitals, face or hands, by direct skin-to-skin contact with friction when the virus is present. Subsequent outbreaks may return at or near the place the virus was contracted, meaning that if it is contracted, it will not travel through the body and appear on the face or be present in saliva [1] .

Transmission is very likely if there are lumps, blisters or sores present, but unlikely when there are no symptoms. It is contracted on the genitals by having sex with someone when the virus is active on their genitals or by receiving oral sex from someone with a facial cold sore. The virus is not contracted from objects such as towels, sheets, cups, cutlery, baths, swimming pools or blood, and it is easier to infect the thin skin of the lips, genital and anal region rather than normal skin [1] .

Herpes simplex can easily enter through mucous membranes or through a cut or break in the skin on other parts of the body if there is direct contact with the affected area. In some cases, the virus can enter the body through damaged skin owing to conditions like eczema or sunburn. It is important to avoid skin contact with the affected area when symptoms or warning signs such as itching, stabbing pains, tingling, burning, or aching are present [1] .

Using condoms can help prevent transmission if they are put on before any skin contact with the affected area and cover the right area. It is crucial not to allow a sexual partner to come into contact with the affected area during times of symptoms or warning signs to minimise the risk of transmission [1] .

Patients with symptoms of genital herpes typically seek evaluation at sexual health clinics, primary care settings or pharmacies. Diagnosing the condition involves a physical examination, medical history and laboratory tests, such as viral culture or polymerase chain reaction (PCR) testing. Differential diagnoses for genital herpes include conditions such as genital warts, which can be ruled out through visual inspection and testing, or monkeypox, impetigo, scabies, which would require referral to a sexual health clinic or GP [1] .

Viral identification techniques such as nucleic acid amplification tests (NAATs) or viral culture are used to diagnose herpes [4] .

Confirming the infection and identifying the type through direct detection of HSV in genital lesions is essential for diagnosis and management. PCR is recommended as the preferred diagnostic method for genital herpes owing to its increased detection rates compared to viral culture. NAAT methods are now considered the test of choice for diagnosing herpes. HSV culture is still used in some centres but may miss around 30% of PCR-positive samples [2] .

The use of antiviral drugs differs depending on whether the patient is presenting with a first episode of anogenital herpes or a recurrence [2,5] . Oral antivirals are indicated if started within five days of the episode but can still be used whilst new lesions are forming or if systemic symptoms persist [2,5] . It should be noted that while antiviral drugs can reduce severity and duration of episodes, they do not alter the frequency or severity of subsequent episodes [2,5] .

First episode

Aciclovir 400mg three times a day orally for five days [6] ;
Valaciclovir 500mg twice a day orally for five days [7] .

Alternative regimens

Aciclovir 200mg five times a day for five days [6] ;
Famciclovir 250mg three times a day for five days [8] .

Topical agents are less effective and there is no evidence to show combining oral and topical treatments is any more effective than oral alone [2] . Both aciclovir and valaciclovir demonstrate similar efficacy in suppressing the frequency and quantity of genital HSV shedding; however, valaciclovir demonstrates better absorption and more prolonged serum concentrations [9] . Treatment choice should be guided by local formulary restrictions and patient preference regarding frequency of dosing [5] .

Recurrent episodes

Whilst recurrent episodes are self-limiting and generally milder than initial presentation, episodic antiviral treatment can be considered:

Aciclovir 400mg three times a day for three to five days [6] ;
Valaciclovir 500mg twice daily for three days [7] ;

There is little evidence to suggest an advantage of one treatment over another.

If patients have at least six recurrences per year, suppressive antiviral therapy can be considered. A common regimen is aciclovir 400mg twice daily, increasing to three times a day if breakthrough episodes occur [5] . This should be discontinued after a maximum of one year to assess recurrence [5] . As recurrence can be triggered by discontinuation, an assessment period should include at least two recurrences before resuming suppressive therapy. Patient-initiated treatment at the first signs of recurrence is most beneficial [2] .

Patient prognosis

HSV-2 is more likely to cause recurrence of symptoms, with a median recurrence rate of four per year, compared with one per year for HSV-1 and this generally decreases over time. Complications can include superinfection of lesions with streptococcal and candida, neuropathy leading to urinary retention, and aseptic meningitis, but these are rare [2] .

Non-pharmacological management, such as saline bathing and use of topical anaesthetics, including 5% lidocaine ointment, can be used in addition to antiviral medication. Patients should be advised to keep sores clean and dry and wear loose-fitting cotton underwear during episodes. Applying petroleum jelly to the sores can be used if urination is painful. For recurrent episodes, it is beneficial for patients to review and address any potential triggers. These may include exposure to UV light, tight clothing, smoking and other factors, such as stress [10] .

Sexual activity with the same partner can be resumed when symptoms have improved. For new partners, sex should be delayed until ulcers have fully healed. Patients should be reminded it is still possible to transmit the virus even if no symptoms are showing and using condoms can reduce this risk [11] .

Patient counselling points

Consistent use of male condoms may reduce risk of genital herpes [12] ;
Care should be taken when discussing herpes to avoid alarmist language (e.g. incurable) as this can worsen distress caused by diagnosis. Patients can be signposted to groups such as the Herpes Viruses Association for additional support;
Ensuring privacy (e.g. a consultation room) and adopting a non-judgemental approach when discussing herpes symptoms and treatment with patients is essential to reduce stigma;
Disclosure to all sexual partners is advised and can be supported by sexual health clinics using the Partner Notification service, as symptomatic partners may need treatment. Anyone who thinks that they have genital herpes symptoms should visit a sexual health clinic for a diagnosis. Asymptomatic patients cannot be tested. Patients can find their nearest clinic by visiting here . Visits are confidential: clinics do not pass on information without patients’ permission.
1 Genital herpes – your questions answered. Herpes Viruses Association . https://herpes.org.uk/frequently-asked-questions/ (accessed May 2024)
2 Patel R, Green J, Clarke E, et al. 2014 UK national guideline for the management of anogenital herpes. Int J STD AIDS. 2015;26:763–76. https://doi.org/10.1177/0956462415580512
3 Albrecht MA. Patient education: Genital herpes (Beyond the Basics). Up To Date. 2024. https://www.uptodate.com/contents/genital-herpes-beyond-the-basics (accessed May 2024)
4 Genital herpes guide: Risk factors and clinical manifestations. Public Health Agency of Canada. https://www.canada.ca/en/public-health/services/infectious-diseases/sexual-health-sexually-transmitted-infections/canadian-guidelines/herpes-simplex-virus/risk-factors-clinical-manifestation.html (accessed May 2024)
5 Herpes simplex – genital. National Institute for Health and Care Excellence. 2023. https://cks.nice.org.uk/topics/herpes-simplex-genital/ (accessed May 2024)
6 Aciclovir 400mg tablets: Summary of Product Characteristics. Electronic Medicines Compendium. https://www.medicines.org.uk/emc/product/11451/smpc (accessed May 2024)
7 Valaciclovir 500mg tablets, Summary of Product Characteristics. Electronic Medicines Compendium. 2023. https://www.medicines.org.uk/emc/product/15071/smpc (accessed May 2024)
8 Famciclovir 250mg tablets: Summary of Product Characteristics. Electronic Medicines Compendium. 2018. https://www.medicines.org.uk/emc/product/9448/smpc (accessed May 2024)
9 Gupta R, Wald A, Krantz E, et al. Valacyclovir and Acyclovir for Suppression of Shedding of Herpes Simplex Virus in the Genital Tract. J INFECT DIS. 2004;190:1374–81. https://doi.org/10.1086/424519
10 Genital herpes. NHS. 2023. https://www.nhs.uk/conditions/genital-herpes/ (accessed May 2024)
11 Genital herpes – the basics. British Association of Sexual Health and HIV. 2018. https://www.bashh.org/_userfiles/pages/files/resources/hsv_pil_2015_screen_friendly.pdf (accessed May 2024)
12 Magaret AS, Mujugira A, Hughes JP, et al. Effect of Condom Use on Per-act HSV-2 Transmission Risk in HIV-1, HSV-2-discordant Couples. Clin Infect Dis. 2015;civ908. https://doi.org/10.1093/cid/civ908

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Open access
Published: 10 May 2024

Herpes simplex encephalitis due to a mutation in an E3 ubiquitin ligase

Stéphanie Bibert ORCID: orcid.org/0000-0001-5170-7506 1 ,
Mathieu Quinodoz 2 , 3 , 4 na1 ,
Sylvain Perriot 5 na1 ,
Fanny S. Krebs 6 , 7 ,
Maxime Jan ORCID: orcid.org/0000-0001-6483-7430 8 ,
Rita C. Malta 9 ,
Emilie Collinet 1 ,
Mathieu Canales 5 ,
Amandine Mathias ORCID: orcid.org/0009-0005-3604-5096 5 ,
Nicole Faignart ORCID: orcid.org/0000-0003-0039-3396 10 ,
Eliane Roulet-Perez 10 ,
Pascal Meylan 11 ,
René Brouillet 11 ,
Onya Opota 11 ,
Leyder Lozano-Calderon 1 ,
Florence Fellmann ORCID: orcid.org/0000-0003-3156-4768 12 ,
Nicolas Guex ORCID: orcid.org/0000-0001-6023-0519 8 ,
Vincent Zoete 6 , 7 , 13 ,
Sandra Asner 1 , 9 na2 ,
Carlo Rivolta ORCID: orcid.org/0000-0002-0733-9950 2 , 3 , 4 na2 ,
Renaud Du Pasquier 5 , 14 na2 &
Pierre-Yves Bochud ORCID: orcid.org/0000-0002-2208-4757 1

Nature Communications volume 15 , Article number: 3969 ( 2024 ) Cite this article

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Herpes virus
Immunological deficiency syndromes
Viral infection

Encephalitis is a rare and potentially fatal manifestation of herpes simplex type 1 infection. Following genome-wide genetic analyses, we identified a previously uncharacterized and very rare heterozygous variant in the E3 ubiquitin ligase WWP2 , in a 14-month-old girl with herpes simplex encephalitis. The p.R841H variant (NM_007014.4:c.2522G > A) impaired TLR3 mediated signaling in inducible pluripotent stem cells-derived neural precursor cells and neurons; cells bearing this mutation were also more susceptible to HSV-1 infection compared to control cells. The p.R841H variant increased TRIF ubiquitination in vitro. Antiviral immunity was rescued following the correction of p.R841H by CRISPR-Cas9 technology. Moreover, the introduction of p.R841H in wild type cells reduced such immunity, suggesting that this mutation is linked to the observed phenotypes.

Antisense oligonucleotide therapeutic approach for Timothy syndrome

Brain endothelial GSDMD activation mediates inflammatory BBB breakdown

CRISPR/Cas9 therapeutics: progress and prospects

Introduction.

Herpes simplex virus type 1 (HSV-1) is a ubiquitous neurotropic human alpha herpesvirus usually causing mild mucocutaneous lesions 1 . In rare circumstances, HSV-1 can reach the central nervous system (CNS) via the olfactory bulb or the trigeminal nerve, causing life-threatening herpes simplex encephalitis (HSE) 1 , 2 . HSE occurs at a rate of 2–4 per million individuals per year 3 with a bimodal age distribution that includes children (at ages of 6 months to 3 years), mainly as a result of a first infection in life 4 , and adults over the age of 50 years, as a result of viral reactivation. Antiviral treatments such as acyclovir have reduced HSE mortality from about 75% to about 20%, but important sequelae with significant and life-long neurological impairment are common 5 , 6 , 7 .

The reasons why HSV-1 causes such a devastating disease in a very limited number of individuals have been unclear for a long time. Genetic studies have shown that childhood HSE results from monogenic inborn errors of immunity, leading to uncontrolled primary infection or reactivation 8 , most of which with a weak clinical penetrance 9 , 10 . While a number of pattern recognition receptors are involved in the innate immune detection of HSV-1, these studies have identified germline loss-of-function mutations of genes specifically involved in the Toll-like receptor 3 (TLR3) signaling pathway 11 , 12 , 13 , 14 , 15 , 16 , 17 that lead to impaired interferon (IFN) production. Indeed, human induced pluripotent stem cells (iPSCs)-derived neurons and oligodendrocytes from TLR3-deficient patients were more permissive to HSV-1 infection compared to those from controls 18 . Since individuals who develop HSE do not present infections due to pathogens other than HSVs and usually have normal antibody titers and T-cell functions 19 , 20 , 21 , 22 , the TLR3/IFN pathway was proposed to be crucial for CNS immunity against HSV-1 23 . However, immune deficiencies involving pathways unrelated to the TLR3/IFN pathway have also been associated with impaired CNS neuron immunity against HSV-1 24 , 25 , 26 , 27 , 28 , 29 .

In the present work, we identified a heterozygous mutation in WWP2 , the gene encoding the WW domain containing E3 ubiquitin protein ligase 2 in a child with HSE. This genetic defect impaired IFN and interferon-stimulated genes (ISGs) production in response to both HSV-1 and TLR3 agonists by targeting the TIR-domain-containing adapter-inducing IFNB (TRIF) protein, the TLR3-adaptor, for ubiquitination.

Clinical presentation

The patient was a 14-month-old, previously healthy girl, admitted to the emergency ward for prolonged focal seizures with bilateral propagation, occurring in the setting of microbiologically documented varicella that started 4 days earlier. Cerebrospinal fluid PCR was positive for HSV-1 and negative for VZV. Serological immunoglobulin tests were positive against both HSV (IgG > IgM) and VZV (IgM > IgG). Cerebral MRIs revealed left temporal edema, which progressively evolved towards extensive necrosis of the left temporal lobe and focal epilepsy.

Identification of a heterozygous WWP2 missense mutation

We analyzed the genome of the patient and her parents by exome sequencing and a customized computer pipeline aimed at detecting rare DNA mutations. A total of 231,859 variants were identified in the proband, among which 155 were retained after filtering for quality, allele frequency (AF, <0.1%, gnomAD version v2.1.1) 30 , and impact at the protein level (missense, stop-gain or canonical splicing, Fig. 1A , Supplementary Table 1 ). The 155 variants were all at the heterozygous state, including 5 heterozygous compound variants. Those were further selected based on deleteriousness (CADD 31 and MutScore 32 ) and/or corresponding inheritance (DOMINO 33 ) prediction, leaving 4 potentially deleterious variants in 3 genes (2 compound heterozygous variants in 1 gene and 2 otherwise heterozygous variants predicted to be associated with a dominant phenotype in 2 genes). The gene carrying heterozygous variants and one of the 2 genes carrying otherwise heterozygous variants were previously associated with genetic disorders, but both were unrelated to immunity. The last variant was found in WW Domain containing E3 Ubiquitin protein ligase 2 gene ( WWP2 ), a gene that was not previously associated with a genetic disorder. Since WWP2 was shown to downregulate the TLR3/IFN pathway 34 , which is essential in the immune response against HSV-1, it was considered our primary target.

A Filtering process of the variants identified by whole exome sequencing. B Sanger sequencing profiles for the WWP2 p.R841H mutation in genomic DNA from the patient (P1) and her father (as a control). C Schematic representation of WWP2, featuring the different domains as well as the location of the missense variant p.R841H. WWP2 is composed of well-defined domains comprising the C2, WW, and Homologous to the E6-AP Carboxyl Terminus (HECT) domains. D Family pedigree with allele segregation. The patient is indicated by the arrow sign, whereas “mut” refers to the p.R841H allele and “WT” to a wild-type allele. GQ genotype quality, FS Fisher strand, VQSLOD variant quality score log odds ratio, ExcessHet Phred-scaled p -value for exact test of excess heterozygosity, AF allele frequency, gnomAD genome aggregation database, CADD combined annotation dependent depletion.

This WWP2 variant consisted in a guanine-to-adenine heterozygous nucleotide replacement (NM_007014.4:c.2522G > A), which resulted in the substitution of an arginine residue for a histidine residue at amino acid position 841 in the Homologous to the E6-AP Carboxyl Terminus (HECT) catalytic domain (p.R841H, Fig. 1B, C ). The p.R841H variant has been detected with an allelic frequency of 1.2 × 10 − 5 among ~125,000 controls (gnomAD version v2.1.1) and was predicted to be deleterious by 14 out of 18 pathogenicity scores including CADD. Furthermore, the variant was classified as likely pathogenic by MutScore with a score of 0.933 (maximum is 1.0), and arginine at position 841 appeared to be conserved in all of the 46 vertebrate WWP2 orthologues we could analyze as well as in 38 paralogs (Supplementary Figs. 1 , 2 ). The patient was the only known member of this family to have developed HSE. Direct Sanger sequencing of this DNA region in members of her family revealed that her mother, sister, and brother also carried the heterozygous p.R841H variant. The mother and the sister had HSV-1-specific serum antibodies, whereas her brother was seronegative (Fig. 1D ).

Molecular modeling analysis of the WWP2 missense mutation

The WWP2 HECT domain consists of a large N-lobe that interacts with E2 proteins, linked to a smaller C-lobe, which catalyzes ubiquitin transfer (Fig. 2A ). Arg841 is in a strand of the C-terminal part of the HECT domain, close to Cys838, namely the catalytic site within a network of hydrogen bonds and ionic interactions, mainly involving charged residues (Arg767, Asp824, His836, Asp843, Fig. 2B ). Due to the aliphatic part of its side chain, Arg831 can also make hydrophobic interactions with Cys822, Phe839 and His836. In addition, Phe839 can interact with Arg841 via a cation-π interaction upon conformational changes accessible via thermal fluctuations. Without directly interacting with Arg841, Glu645, and Arg834 also contribute to the tightness of the region through ionic interactions. The substitution of an arginine residue for a histidine residue (p.R841H), much smaller and with environment-dependent protonation states, could compromise the structural integrity of the region by impacting the wild-type interaction network.

A Overview of the HECT domain structure and its two main lobes. B Zoom on Arg841 and its neighbors. Arg841 and Cys838 are respectively shown in orange and black ball and stick. The residues involved in the ionic and hydrogen bond network are shown in sticks. Residues with carbons colored in yellow are within 5 A from Arg841 (pdb id:4y07). C The expression of p.R841 and p.R841H WWP2 protein was determined by SDS-PAGE/immunoblotting under reducing conditions in cell lysates of TLR3-expressing 293 T cells after different times of transfection. Results represent 1 experiment among 3. TLR3 FL TLR3 full length.

Energetic impact of the WWP2 missense mutation

The changes of the folding free energy, ∆∆G were calculated using FoldX 35 for each crystal structure of human HECT domains sharing at least 50% identity with the HECT domain of WWP2 (see methods for details). For each of them, without exception, the replacement of arginine by histidine at position 841 led to energetic perturbations and higher ∆∆G values (Supplementary Fig. 3 ), meaning that it destabilizes the structure of the region. This is in line with the structural analysis. Altogether, the disruption of the polar interaction network might lead to a more flexible region and could facilitate the accessibility of the ubiquitination site at position 838 (Cys838).

Transient gene expression

To characterize the p.R841H variant in vitro, both wild-type (WT, WWP2 p.R841) and mutant ( WWP2 p.R841H) cDNAs were cloned and expressed in TLR3-expressing human embryonic kidney (HEK) 293 T cells. The expression of WWP2 in cell lysates was analyzed by western blotting (Fig. 2C ). The p.R841H form of WWP2 was expressed at the same level as the WT form.

Impaired PBMCs responses to Poly(I:C) and HSV-1

The mRNA production of IFNβ and ISGs (ISG56, MX1, and IFIT2) was then measured in PBMCs from individuals carrying the WWP2 p.R841H variant (patient with HSE and 3 family members) and from controls (patient’s father and 7 unrelated individuals) after stimulation with Poly(I:C). PBMCs from individuals carrying the WWP2 p.R841H variant expressed significantly lower mRNA levels of IFNβ and IFIT2 2 h after poly(I:C) stimulation (Supplementary Fig. 4A , B ). ISG56 tended to be less expressed in PBMCs from p.R841H carriers compared to those from WT individuals (Supplementary Fig. 4C ). In contrast, PBMCs from individuals carrying the WWP2 p.R841H expressed similar levels of MX1 and TNFα than those from WT controls (Supplementary Fig. 4D , E ). Of note, the baseline mRNA expression of TLR3, TRIF, and WWP2 was similar among p.R841H carriers compared to WT (Supplementary Fig. 4G ), showing that this impaired response was not due to decreased expression of genes implicated in TLR3 signaling.

In order to further characterize the impact of the p.R841H variant, we measured viral levels (copies/ml) in the supernatant of PBMCs from WWP2 p.R841H carriers and controls 18 h after HSV-1 infection (Supplementary Fig. 4F ). Viral levels were significantly increased in p.R841H carriers compared to those from controls ( P < 0.0001). Although poly(I:C) stimulation in PBMCs is not specific to TLR3, these data suggest that p.R841H carriers have a lower capacity to establish IFN responses after poly(I:C) stimulation at early time points which leads to a reduced control of HSV-1 infection.

Impaired iPSCs-derived neuronal cells responses to TLR3 agonist and HSV-1

To test whether the WWP2 p.R841H was associated with impaired TLR3 signaling in the CNS, we used brain-specific cell differentiation technologies from human induced pluripotent stem cells (iPSCs). Briefly, iPSCs were derived from the patient, her family members, and three unrelated donors by reprogramming erythroblasts (Fig. 3A ). Generated iPSCs exhibited normal karyotype, expressed pluripotency markers and were capable to differentiate into cell types from all three germ layers (Fig. 3B, C ). Three additional previously described iPSCs from WWP2 WT individuals were also used as controls 36 . Human iPSCs were then differentiated into non-hematopoietic CNS-resident cells, comprising neural stem cells/neural precursor cells (NPCs), neurons, and astrocytes 37 . The level of maturation of each cell type, specifically neurons and astrocytes, was assessed by the expression of selected marker genes and compared to the ones of NPCs (Fig. 3D, E ).

A Schematic diagram of the reprogrammation and differentiation protocols used. B Immunocytochemistry analysis of iPSCs colonies revealed the expession of pluripotent markers OCT4, TRA-1-60, and SOX2. C Immunocytochemistry analysis of the ability of iPSCs-derived embryonic bodies to differentiate into the 3 germ layers. PAX6, SMA, and AFP are markers for ectoderm, mesoderm, and endoderm respectively. Results represent the analysis of 1 iPSCs colony and is representative of results obtained for all iPSCs colonies ( n = 2 per donor). D Characterization of neurons. Compared to neuronal precursors, neurons exhibit increased expression of typical neuronal markers assessed by qPCR. E Characterization of astrocytes. Compared to neuronal precursors, astrocytes exhibit increased expression of typical astrocyte markers, assessed by qPCR.

We then focused on the time-dependent expression of IFNβ, ISG56, and MX1 mRNA in different cell types. The expression of IFNβ, ISG56, and MX1 mRNA after Poly(I:C) stimulation was reduced in iPSCs-derived NPCs (Fig. 4A ) and neurons (Fig. 4B ) from WWP2 p.R841H carriers compared to those from WT controls. In contrast, iPSCs-derived astrocytes produced normal amount of IFNβ, ISG56, and MX1 mRNA after Poly(I:C) stimulation as shown by comparison with controls (Fig. 4C, F ). As a control, the expression of IFNβ was not reduced in p.R841H carriers compared to WT controls when iPSCs-derived NPCs (Fig. 4D ) and neurons (Fig. 4E ) were stimulated with LPS (TLR4 agonist), CpG (TRL9 agonist) and R848 (TLR7/8 agonists), suggesting that reduced IFNβ in p.R841H carriers was specific to the TLR3/IFN axis. Altogether, these data indicated that reduced expression of IFNβ, ISG56, and MX1 mRNA after stimulation with Poly(I:C) relied on the WWP2 p.R841H mutation, both on NPCs and neurons, but not in astrocytes.

Induction of IFNβ, ISG56 and Mx1 mRNA after different time of Poly(I:C) stimulation in neural precursor cells ( A ), neurons ( B ) and astrocytes ( C ) from WWP2 p.R841 individuals [black, N = 5 ( A ), N = 4 ( B ), N = 5 ( C )] and from WWP2 p.R841H individuals [red, N = 8 ( A , B ), N = 4 ( C )]. As a control, the expression of IFNβ was measured in neural precursor cells ( D ), neurons ( E ) and astrocytes ( F ) from WWP2 p.R841 individuals (black) and from WWP2 p.R841H individuals (red), stimulated with LPS [WWP2 p.R841 individuals N = 5 ( D , E ), N = 4 ( F ); WWP2 p.R841H individuals N = 8 ( D , E ), N = 2 ( F )], CPG [WWP2 p.R841 individuals N = 5 ( D , E ), N = 4 ( F ); WWP2 p.R841H individuals N = 8 ( D , E ), N = 2 ( F )] and R848 [WWP2 p.R841 individuals N = 5 ( D , E ), N = 4 ( F ); WWP2 p.R841H individuals N = 8 ( D , E ), N = 2 ( F )] for 2, 3 and 4 h. A.U. stands for arbitrary unit. Results represent the mean ± standard error of 1 representative experiment among 2 ( A , B ), 3 ( C ), and 1 ( D – F ). Statistical analyses were performed using an unpaired two-tailed Student t -test. A Left *, **, *** mean for P = 0.0129, P = 0.0011 and P = 0.0001, ( A ) middle *, ** mean for P = 0.0501 and P = 0.0413, ( A ) right *, **, *** mean for P = 0.0010, P = 0.0025, P = 0.0021. B Left *, **, *** mean for P = 0.0026, P = 0.0006 and P = 0.0187, ( B ) middle *, ** mean for P = 0.0299 and P = 0.0022. D * means for P = 0.0475. E *, ** mean for P = 0.0121 and P = 0.0005.

In order to provide a comprehensive analysis of the impact of WWP2 p.R841H on the TLR3 pathway, we performed RNA sequencing in neurons from both WWP2 p.R841H carriers and controls under basal conditions and after 3 h poly(I:C) stimulation. Alignment metrics are detailed in the Supplementary Table 3 . Genes downstream NF-kB in the canonical pathway (Kyoto Encyclopedia of Genes and Genomes, KEGG has06604) as well as genes downstream AP-1 (hsa04668) had a lower expression in poly(I:C) stimulated neurons from WWP2 p.R841H carriers compared to WWP2 p.R841 neurons (false discovery rate [FDR] adjusted P = 3.02E-02 and P = 3.55E-05, respectively, Supplementary Figs. 5 and 6 , Supplementary Table 4 ). In contrast, no significant difference was found in genes of the NF-kB non-canonical and atypical pathways (both P = 4.02E-01).

In order to further characterize the impact of the WWP2 p.R841H variant, we measured viral levels by qPCR (copies/ml) in both NPCs (Fig. 5A ), neurons (Fig. 5B ), and astrocytes (Fig. 5C ) from WWP2 p.R841H carriers and controls after HSV-1 infection. Viral levels were significantly increased in NPCs and neurons from p.R841H carriers compared to those from controls. A treatment of NPCs and neurons with recombinant IFN-α2b prior to HSV-1 infection did not impact on viral level in NPCs and neurons from controls but restricted viral level in NPCs and neurons from WWP2 p.R841H carriers to a level similar to that obtained in NPCs from controls. In astrocytes from p.R841H carriers, viral levels were similar to those obtained in astrocytes from controls with or without an IFN-α2b pre-treatment. Since viral levels by qPCR do not necessarily reflect the amount of reproductive viral particles, those were measured (Pfu/ml) in neurons at different time-points (12 h, 24 h, and 48 h) after HSV-1 infection (Fig. 6A ). Neurons from p.R841H carriers exhibited higher viral replication rates at all time-points, compared to those controls. A treatment of neurons with recombinant IFN-α2b prior to HSV-1 infection restricted viral replication in neurons from WWP2 p.R841H carriers to a level similar to that obtained in neurons from controls (Fig. 6B ).

Quantification by polymerase chain reaction of the number of viral genome in neural precursor cells ( A ), neurons ( B ) and astrocytes ( C ) from WWP2 p.R841 individuals [black, N = 5 ( A ), N = 6 ( B ), N = 3 ( C )] and from WWP2 p.R841H individuals [red, N = 8 ( A ), N = 9 ( B ), N = 8 ( C )] without or with an IFNα2b pretreatment (+IFN). Results represent the mean ± standard error of 1 experiment among 2 ( A ) and 1 ( B , C ). Statistical analyses were performed using an unpaired two-tailed Student t -test. A *, ** mean for P = 0.0028 and P < 0.0001. B *, ** mean for P = 0.0046 and P = 0.0001. The purple triangle and the grey square correspond to patient with HSE and to patient’s father respectively.

A Quantification of the number of plaque-forming units (pfu) by plaque-titration at different time after infection of neurons from WWP2 p.R841 individuals (black, N = 3) and from WWP2 p.R841H individuals (red, N = 6). (B) Quantification of the number of plaque-forming units (pfu) by plaque-titration at 48 h after infection of neurons from WWP2 WT individuals (black, N = 3) and from WWP2 p.R841H individuals (red, N = 5) without or with an IFNα2b pretreatment (+IFN). Figure shows median of one experiment. Statistical analyses were performed using an unpaired two-tailed Student t -test. The purple triangle and the grey square correspond to patient with HSE and to patient’s father respectively. A *, ** mean for P = 0.0005 and P = 0.0002. B *, **, *** mean for P = 0.0239, P = 0.0256 and P = 0.0039.

TRIF ubiquitination is increased by WWP2 p.R841H compared to WWP2 p.R841

Since WWP2 p.R841H was predicted to facilitate the accessibility of the ubiquitination site at position 838 (Cys838), we performed an in vitro assay to compare the level of TRIF ubiquitination by WWP2 variants. These experiments showed that WWP2 p.R841H induces increased TRIF ubiquitination compared to WWP2 p.R841 (Fig. 7 ).

TRIF, p.R841 WWP2, and p.R841H WWP2 were in vitro translated. An in vitro ubiquitination assay of TRIF was conducted by using an E1 ubiquitin-activating enzyme and UbcH5c together with either p.R841 WWP2 or p.R841H WWP2. Ubiquitin-conjugated TRIF was detected by immunoblot with an anti-ubiquitin antibody. The expression levels of WWP2 were also verified by immunoblots with an anti-WWP2 antibody. Results represent 1 experiment among 2.

CRISPR-mediated genome editing restores WWP2 function in the mutant and aborts it in the WT

We used the CRISPR-Cas9 technology to generate wild type iPSCs from the patient, naturally carrying p.R841H, and to establish mutant iPSCs from the patient’s father, who carried no mutations at this site. We then differentiated them into neurons to estimate the TLR3 response. Impaired Poly(I:C) responsiveness in the patient’s neurons (p.R841H) was rescued by expression of only wild type form of WWP2 through CRISPR-Cas9 genome editing (p.H841R), with a level of induction of IFNβ mRNA similar to that of WT controls (p.R841) (Fig. 8A ). Upon poly(I:C) stimulation, neurons from the patient’s father harboring the heterozygous WWP2 p.R841H presented a lower level of induction of IFNβ mRNA compared to that obtained in previously WT neurons from the patient’s father (p.R841), but similar to that of WWP2 p.H841 carriers (Fig. 8B ). Viral levels were decreased in neurons from the patient expressing the wild type WWP2 p.R841 compared to those from the patient harboring the heterozygous WWP2 p.R841H (Fig. 8C ) as well as the viral replication (Fig. 8G ). Similarly, viral levels (Fig. 8D ) and viral replication (Fig. 8H ) were both increased in neurons from the patient’s father harboring the heterozygous WWP2 p.R841H compared to those from the patient’s father expressing the wild type WWP2 p.R841. A treatment of neurons with recombinant IFN-α2b prior to HSV-1 infection did not impact on viral replication in neurons from individuals expressing the wild type WWP2 p.R841, but restricted viral replication in neurons from WWP2 p.R841H carriers to a level similar to that obtained in neurons from WWP2 p.R841 individuals (Fig. 8C– F ). Altogether, these data showed that WWP2 p.R841H carriers had a lower capability to control HSV-1 infection in neuronal cells compared to controls due to an impaired IFN production.

A Induction of IFNβ mRNA after 1 h of Poly(I:C) stimulation in neurons from WWP2 p.R841H (red, N = 2), WWP2 p.H841R (grey, N = 2) individuals and WWP2 p.R841 controls (black, N = 3). B Induction of IFNβ mRNA after 1 h of Poly(I:C) stimulation in neurons from WWP2 p.R841 (black, N = 2), WWP2 p.R841H (grey, N = 2) individuals and WWP2 p.H841 controls (red, N = 4). Results represent the mean ± standard error of 1 experiment among 2. C Quantification of the number of viral genome in neurons from WWP2 p.R841H (red, N = 2), WWP2 p.H841R (grey, N = 2) individuals, and WWP2 p.R841 controls (black, N = 4), without or with an IFNα pretreatment (+IFN). D Quantification of the number of viral genome in neurons from WWP2 p.R841 (black, N = 2), WWP2 p.R841H (grey, N = 1) individuals and WWP2 p.H841 controls (black, N = 7) without or with an IFNα pretreatment (+IFN). E Quantification of the number of plaque-forming units (pfu) by plaque-titration after infection of neurons from WWP2 p.R841H (red, N = 2), WWP2 p.H841R (grey, N = 2) individuals and WWP2 p.R841 controls (black, N = 3) without or with an IFNα2b pretreatment (+IFN). F Quantification of the number of plaque-forming units (pfu) by plaque-titration after infection of neurons from WWP2 p.R841 (black, N = 2), WWP2 p.R841H (grey, N = 2) individuals and WWP2 p.H841 controls (black, N = 4) without or with an IFNα2b pretreatment (+IFN). G Quantification of the number of plaque-forming units (pfu) by plaque-titration at different time after infection of neurons from WWP2 p.R841H (red, N = 2), WWP2 p.H841R (grey, N = 2) individuals and WWP2 p.R841 controls (black, N = 3). A significant effect (*) of the rescue (grey) compared to allele carrier (red) ( p = 2.41e-5) was found using a linear regression on log10 + 1 transformed pfu with a time-dependent effect. H Quantification of the number of plaque-forming units (pfu) by plaque-titration at different time after infection of neurons from WWP2 p.R841 (black, N = 2), WWP2 p.R841H (grey, N = 2) individuals and WWP2 p.H841 controls (black, N = 4). A significant effect (*) of the mutant induced (pink) compared to control (black) ( p = 2.06e-3) was found using a linear regression on log10 + 1 transformed pfu with a time-dependent effect.

Here, we report a heterozygous missense WWP2 variant associated with HSE in a 14-month-old child. WWP2, also called Atrophin-1 Interacting Protein 2 (AIP2), is an E3 ubiquitin ligase, which plays a central role in protein ubiquitination, a post-translational mechanism of protein modification catalyzed by the sequential action of ubiquitin-activating (E1), ubiquitin-conjugating (E2) and ubiquitin-ligating (E3) enzymes. The human genome encodes more than 600 E3 ligases and this relatively high diversity enables their specificity in targeting protein substrates. Recently, in vitro ubiquitination assays, performed in both 293-TLR3 and A549 cells, showed that WWP2 mediates the K48-linked ubiquitination and degradation of the TLR3 adaptor TRIF following poly(I:C) stimulation. Moreover, in reporter assays, overexpression of WWP2 inhibited poly(I:C) induced transcription of IFNB1, CCL5, and ISG15 while its knockdown increased the expression of these genes 34 .

Previous studies have convincingly shown that susceptibility to HSE can be due to mutations in members of the TLR3 signaling pathway. Similar to our results, such mutations displayed a very low degree of penetrance, i.e., a substantial percentage of carriers of them did not develop disease, despite their unfavorable genotype 11 , 12 , 13 , 14 , 15 , 16 , 17 . This phenomenon highlights the importance of specific interactions between the environment (exposure to pathogens) and the host (immune status, co-infections) during a given phase of life, for these mutations to produce clinical consequences, while in fact they result in clear deficiencies at the molecular level, as shown for instance by cells derived from the mother and the siblings of our patient. In the present case, co-infection by both VZV and HSV-1, together with WWP2 p.R841H carriage, may have been required for the development of HSE. Intriguingly, WWP2 is not directly involved in the signaling cascade of the TLR3/IFN axis but is known to regulate this pathway through K48-linked ubiquitination of TRIF following Poly(I:C) stimulation 34 . WWP2 p.R841H carriers had a reduced expression of genes downstream NF-kB, AP-1, and IRF3, including IFNβ and ISGs, after Poly(I:C) stimulation. This was observed in both iPSC-derived NPCs and neurons, but not astrocytes, and was associated with an enhanced susceptibility to HSV-1 infection. These observations overlap with those from two children with TRIF deficiency (autosomal dominant p.S186L and autosomal recessive p.R141X), in which dermal fibroblasts stimulated with Poly(I:C) exhibited an impaired production of IFNβ as well as higher levels of HSV-1 replication 14 . The observed phenotype was linked to the WWP2 p. R841H, as allelic reversion (H841 > R) rescued the control of HSV-1 replication in iPSC-derived neurons from the patient, while the introduction of a single mutant allele (R841 > H) abolished IFN-mediated immunity against HSV-1 in iPSC-derived neurons from her wild type father. Interestingly, IFN production following LPS stimulation was conserved in both the WWP2 p.R841H and the TRIF p.S186L patients, but not in the TRIF p.R141X patient, suggesting that heterozygous mutations are sufficient to impact TRIF signaling in the TLR3, but not in the TLR4, pathway, and that homozygous mutations are required to abolish TRIF signaling in both TLR3 and TLR4 pathways 14 .

Interestingly, WWP2 can regulate its own catalytic activity through an intramolecular interaction mechanism involving trapping of the HECT domain 38 . Molecular modeling analyses and calculation of the change in the folding free energy indicate that the WWP2 p.R841H mutation leads to a more flexible region and may facilitate the accessibility of the ubiquitination site, thereby promoting a constitutive activation of the enzyme. This is in agreement with our in vitro assay showing an increased TRIF ubiquitination by the WWP2 p.R841H variant compared to the control.

Ubiquitination is a complex post-translational mechanism which is critical for protein homeostasis and cell signaling and regulates various cellular processes including immune responses 39 . It can modulate immune pathways by targeting for degradation key proteins involved in pathogen’s recognition, such as PRRs and their adaptor molecules 34 , 40 , their downstream signaling proteins 41 , 42 , or transcription factors 43 , 44 . Ubiquitination can also act on immune functions without altering protein stability by impairing the assembly of protein complexes or by promoting the activation of specific proteins, separating them from their binding partner(s) 39 , 40 , 42 , 45 , 46 .

Due to its crucial role in the TLR3-mediated signaling, the expression of TRIF must be tightly controlled as well as the expression of its HECT E3 ubiquitin ligases in order to ensure normal cell behavior and to prevent illnesses 47 , 48 , 49 . At a post-transcriptional level, TRIF is heavily modified and regulated by polyubiquitination, leading to the inhibition of the TLR3- and TLR4-mediated innate immune and inflammatory responses, through different mechanisms, depending on the type of E3 ubiquitin ligase. While polyubiquitination of TRIF by WWP2 results in its proteasomal degradation (36), polyubiquitination by TRIM38 34 , 50 , TRIM8 46 , or USP19 (44) are thought to impair its complex assembly without degradation.

Our study has limitations. First, the molecular modeling analysis of the mutation focuses on the free energy change and the interaction network within the active site of the HECT domain. It does not explain the activating effect of p.R841H mutation. Since WWP2 HECT domain adopts auto-inhibitory conformation, it would be interesting to test whether the p.R841H mutation activates WWP2 by releasing autoinhibition. Molecular dynamics simulations or normal mode analysis would certainly decipher the exact effect of the p.R841H mutation. Second, although we show that the WWP2 p.R841H variant is associated with an increased TRIF ubiquitination in vitro, we do not investigate whether it enhances TRIF ubiquitination in iPSC-derived neurons, whether this leads to TRIF degradation and how it leads to change in the control of HSV-1 infection. Lastly, although genome editing experiments show significant differences on viral replication in a time-dependent manner, the limited number of genetically modified iPSCs colonies and/or time points did not allow for statistical analyses regarding IFN induction. However, introduction and rescue of the mutant allele provided consistent trends towards decreased and increased IFN production, respectively, in repeated experiments.

Altogether, our study further confirms the role of the TLR3/IFN pathway in controlling HSV-1 infection in the CNS during childhood. We show that a mutation in WWP2 leading to increased TRIF ubiquitination in vitro is associated with a phenotype similar to what is observed in TRIF deficient patients.

DNA extraction

DNA from either PBMCs or whole blood was isolated using the QIAamp DNA mini kit (Qiagen) visualized by gel electrophoresis to verify quality and then quantified by Picogreen or on a NanoDrop 1000 spectrophotometer (Thermo Fischer Scientific).

Exome sequencing

Exome was performed for the proband and her parents using the Twist Exome 2.0 capture kit (Twist Bioscience, San Francisco, USA) and Illumina NovaSeq 6000 (Illumina, San Diego, USA), resulting in sequences of 100 bases. Raw reads were mapped to the human genome reference sequence (build hg19) with BWA mem (v0.7.17). Duplicate reads were removed using Picard (v. 2.14.0-SNAPSHOT). Base quality score recalibration was performed with GATK dedicated functions and variant calling done with HaplotypeCaller (GATK, v.4.1.4.1). The variants called were annotated with a custom pipeline, adding information about effect of the variants at the protein level, frequency in various databases, conservation across species, effect on splicing and deleteriousness predictors, using mainly ANNOVAR . More details on the annotation and filtering can be found in Peter et al. 51 .

Molecular modeling analysis

All experimental 3-dimensional (3D) structures were extracted from the Protein Data Bank 52 . Structures and sequence alignments were analyzed using USCF Chimera version 1.13.1 visualization software 53 . FoldX version 4 was used to generate structural models of the mutant and to estimate the energetic impact of the mutation (∆∆G values in kcal/mol) 35 . SCWRL4 was used to create models based on experimental structures, with optimized amino acid sidechain positions 54 . Protein sequences were extracted from the UniProt database 55 and multiple sequence alignments (MSAs) were performed using MUSCLE version 3.8.31 56 . All structural images were generated in-house, using USCF Chimera 53 .

Sequence conservation analysis

To study the sequence conservation among human paralogs, UniProt database 55 was used. A total of 133 human protein sequences with a HECT domain was retrieved. A multiple sequence alignment (MSA) was performed using MUSCLE 56 . Sequences covering the human WWP2 HECT region, based on UniProt delineation of human WWP2 HECT domain, were extracted and sequence identities with the human WWP2 HECT domain were calculated using UCSF Chimera 53 . 38 sequences with larger than 50% sequence identity to WWP2 were selected for further analysis (Supplementary Fig. 2 ).

Estimation of the energetic impact of the variant compared to the wild type structure

Only 5 experimental 3D structures of the HECT domain of WWP2 are published and publicly available from the Protein Data Bank (4y07 57 , 5tj7 38 , 5tj8 38 , 5tjq 38 , 6j1z 58 ). To increase this number, we retrieved human structures of the HECT domain of proteins that share a sequence identity higher than 50% with human WWP2. 23 experimental structures were selected (Supplementary Table 2 ), for ITCH, NEDD4, NEDD4L, SMURF2, WWP1 and WWP2. The chains containing the HECT domain have been retained for the following steps. To further increase our sampling, we created a collection of 3D structural models based on the experimental structures, using SCWRL4 54 . SCWRL4 checks and, if needed, corrects the side chain positions of structures. Indeed, a side chain position quality depends on the electron density detected and can be doubtful in some experimental structures. FoldX was used to generate structural models of the mutant and to estimate the energetic impact of the mutation on the Arg841 environment 35 . FoldX is an efficient software for predicting mutation-dependent folding-free energy changes, whose predictive efficiency has been trained on a large mutation set 59 . The predicted energy perturbation (∆∆G, kcal/mol) induced by the mutant is calculated. Several experimental structures were used for FoldX calculations, to strengthen the interpretation of the predicted energy perturbation results, as the approach is sensitive to small conformational changes observed between different experimental 3D structures or accessible via thermal fluctuations.

PBMCs isolation

Peripheral blood mononuclear cells (PBMCs) were prepared from fresh EDTA (1.6 mg/ml) blood from patient, patient’s relatives, and healthy donors with written consent and approval of the Ethics committee. Briefly, whole blood diluted in PBS was overlaid above Ficoll-Paque Plus (GE Healthcare, Uppsala, Sweden), and mononuclear cells extracted by gradient density centrifugation. Viability, determined by trypan blue exclusion, was >90%.

HSV-1 amplification and HSV-1 plaque assay

Human herpesvirus 1 strain MacIntyre used in viral neutralization studies was purchased from ATCC (ATCC® VR-539) and was grown on a 80% confluency African green monkey kidney (Vero, ATCC® CCL-81) cell line in DMEM/GlutaMax supplemented with 10% FBS. Briefly, Vero cells were infected with HSV-1 at a multiplicity of infection of 1, during at least 2 h at 37 °C. Medium was then replaced and supernatant was collected after 3–4 days incubation at 37 °C, until the appearance of cytopathic effect, and clarified at 4500× g for 30 min. HSV-1 titers were determined by a standard plaque assay on Vero cells. Briefly, serial dilutions of HSV-1 suspension were incubated on confluent monolayers of cells during at least 2 h at 37 °C. Supernatant was then aspirated and cells washed with PBS. Cells were then incubated in DMEM containing 1% MethylCellulose and 0.33% Sodium Bicarbonate at 37 °C for several days, until the plaque formation. Medium was then aspirated and the cells were fixed and stained with a mixture of Paraformaldehyde 2% and crystal violet 0.5% for 15 min at room temperature. HSV-1 titer was evaluated from plaque counting and expressed as plaque-forming units (PFU)/ml.

The virus preparation was aliquoted and stored at −80 °C.

TLRs agonists, HSV-1 infection, and quantification

Primary or neural stem cells and cell lines were stimulated with 50 µg/ml poly inosine polycytidylic acid (poly(I:C)), 0.1 µg/ml LPS, 5 µg/ml R848, 8 µg/ml CpG, and 1 PFU/cell HSV-1, for various periods of time. Primary or neural stem cells were treated or not with 10’000 units/ml IFNα2b (pbl assay science) during 24 h before being infected with HSV-1 at a multiplicity of infection of 1. Between 12 and 48 h post infection, cell supernatant was collected. Total viral DNA were extracted with the MagNA Pure 96®, according to the manufacturer’s instructions, and were quantified by RT-PCR 60 . The quantification of infectious virus were done by using plaque assay as detailed above.

RNA extraction

Total RNAs were extracted, using an RNeasy Mini kit and the automated QIAcube (Qiagen, Hombrechtikon, Switzerland) from patient/patient family/healthy volunteer PBMCs and/or iPSCs-derived cells, treated or not with different stimuli including pattern recognition receptor agonists or HSV-1.

Messenger RNA expression

Total RNAs were reverse transcribed in the presence of random primers using the QuantiTect Reverse Transcription Kit (Qiagen). The relative levels of TLR3, WWP2, TRIF, IFNB, ISG56, Mx1, IFIT2, and TNF transcripts were determined by RT-PCR, with a QuantStudio 12 K Flex Real-Time PCR system, using the Power SYBR green PCR master mix (Thermo Fisher) with primers described in Supplementary Table 5 . Primers were designed using the Primer 3 software and validated by BLAST analysis. The relative levels of mRNA expression to HPRT were determined by the 2 (−ΔΔCt) method as described by the manufacturer and expressed in arbitrary units (A.U.). HPRT expression was not influenced by cell stimulation.

RNA quality control and library preparation

RNA quality was assessed on a Fragment Analyzer (Agilent Technologies, Santa Clara, CA, USA), and all RNAs had an RNA quality number (RQN) comprised between 7.8 and 10. Library preparation and RNA-seq were performed at the Lausanne Genomic Technologies Facility, University of Lausanne, Switzerland ( https://www.unil.ch/gtf ). Briefly, the Truseq Stranded mRNA reagents (Illumina, San Diego, CA, USA) were used for the library preparation with 108 ng of total RNA as input. RNA-seq libraries were quantified by a fluorimetric method (QubIT, Life Technologies, Carlsbad, CA, USA). Their quality was assessed on a Fragment Analyzer. Sequencing was performed on an Illumina Novaseq 6000 for 100 cycles (single end). Sequencing data was demultiplexed using the bcl2fastq2 Conversion Software (version 2.20, Illumina). Sequencing yield was 877 M pass-filter reads with 41–61 M for each library.

RNA sequencing data processing

Purity-filtered reads were adapters and quality trimmed with Cutadapt (v. 2.5 61 ). Reads matching to ribosomal RNA sequences were removed with fastq_screen (v. 0.11.1). Reads were aligned against Homo_sapiens GRCh38 genome using STAR (v. 2.5.3a 62 ). The number of read counts per gene locus was summarized with htseq-count (v. 0.9.1 63 ) using gene annotation from the Homo_sapiens_GRCh38.102.gtf file. Quality of the RNA-seq data alignment was assessed using RSeQC (v. 2.3.7 64 ). Statistical analysis was performed in R (R version 4.2.2). Genes with low counts were filtered out according to the rule of 1 count per million (cpm) in at least 1 sample. Library sizes were scaled using TMM normalization. Subsequently, the normalized counts were transformed to cpm values, and a log2 transformation was applied by means of the function cpm with the parameter setting prior counts = 1 (EdgeR v 3.30.3 65 ). After filtering, the number of genes used for further analysis was 15,306. A quality control analysis was performed through samples correlation/clustering and PCA. Possible confounding factors were considered and removed using the RUVr method from the RUVseq_1.30.0 R package with k = 2.

Differential expression was computed with the R Bioconductor package limma 66 by fitting data to a linear model. P-values were adjusted using the Benjamini-Hochberg (BH) method (false discovery rate (FDR) control). For the DE analysis with the RUV-corrected scores, the two RUVseq factors were directly included in the design as control variables. After testing the DE genes for an FDR < 0.05, the genes were also, in a second step, filtered according to both the FDR (> 0.05) and the fold-change (> 1.5) between “medium” and “stimulation”. The log2 fold-change cutoff was set to 0.5849625 so that only genes with an absolute fold change superior to 1.5 were kept.

AP-1 and NF-kB downstream enrichment for the difference in stimulation between wild-type [WT] and mutant [MUT] were computed as follows. A stimulation effect was calculated as log2 fold-change of CPMs between Poly(I:C) stimulation and Medium (ΔStimulation) within each pair of samples. The effect of mutation was then calculated as the difference between stimulations in mutant samples and wild-type samples (ΔΔStimulation = ΔStimulation[Mut] − ΔStimulation[WT]). AP-1 and NF-kB downstream genes were taken from the TNF signaling pathways and NF-kB signaling pathway in KEGG (hsa:04064 and hsa:04668 respectively). Enrichment for ΔΔStimulation in NF-kB-atypical, NF-kB-non-canonical, NF-kB-canonical and AP-1 downstream genes in TNF pathways were computed with Bioconductor R package fgsea.

Production of recombinant proteins

The WT and mutant WWP2 cDNAs were amplified by PCR from total RNA isolated from PBMCs. Purified products were subcloned into a pGEM®-T Easy vector by T4 DNA ligase (Promega) and sequenced. We generated pcDNA3.1 expression vectors (Thermofisher) encoding the WT and mutant WWP2. Plasmids containing cDNAs were used for the transient transfection of TLR3-expressing human embryonic kidney (HEK) 293 T cells (ATCC® CRL-3216). Briefly, plasmids were mixed with JetPEI (Polyplus transfection, USA), according to the manufacturer’s instructions. Cells were cultured for 24 h, 48 h, or 72 h and washed 2 times with phosphate-buffered saline (PBS) before being lysed with a lysis buffer containing Tris 10 mM, pH 8, NaCl 150 mM, NP-40 0.5%, NaF 10 mM, Na-orthovanadate 1 mM and protease inhibitors.

Expression of recombinant proteins

Cells were harvested with the lysis buffer previously described and centrifuged at 10,000× g at 4 °C, for 5 min. Protein content was determined by the Biorad Protein assay (Biorad). Proteins (20 μg–40 μg) were then subjected to SDS-PAGE under reducing and then transferred onto a nitrocellulose membrane. The rabbit anti-human WWP2 antibody (1/1000, A302-935, Bethyl Laboratories) and mouse anti-B actin antibody (1/1000, sc-8432, Santa Cruz Biotechnology) were used as primary antibodies and were detected using anti-rabbit or mouse IgG antibodies conjugated to horseradish peroxidase (1/10,000, Amersham Biosciences). Detection was done with the ECL chemiluminescence kit (Pierce) according to the manufacturer’s protocol.

Human inducible pluripotent stem cells (iPSC)

Human iPSCs were generated from the HSE patient, family members as well as healthy controls by reprogramming erythroblasts via ectopic expression of a minimal set of transcription factors as previously described 67 . Briefly, erythroblasts were amplified from PBMCs and nucleofected with episomal plasmids pCXLE-hOCT3/4-shp53-F, pCXLE-hSK, pCXLE-hUL (gifts from Shinya Yamanaka, Addgene #27077, #27080 and #27078) 68 , plated on Matrigel-coated plates and cultured in a reprogramming medium (ReproTeSR, STEMCELL Technologies) until iPSC colonies started to appear. Human iPSC clones were then picked and lines expanded using StemMACS iPSC-Brew XF medium (Miltenyi Biotec, Bergisch Gladbach). Human iPSCs were characterized by immunofluorescence for pluripotency (expression of OCT4, TRA-1-60, and SOX2) and differentiation capacity (The three-germ layer differentiation capacity and expression of AFP, Pax6, SMA) as previously described 67 and following standard quality controls for iPSCs. The genomic integrity of iPSCs was assessed by G-banding karyotype. The p.R841H mutation was confirmed by the Sanger sequencing of iPSCs’ genomic DNA. Two lines per donor were used in this study. Healthy control iPSC lines ( n = 3, each from a different individual) were previously described 36 and assessed to be WT for WWP2.

Inducible pluripotent stem cell culture and differentiation

Human iPSCs were maintained in StemMACS human iPSC Brew medium (Miltenyi) or TeSR-E8 medium (Stemcell Technologies). The differentiation of iPSCs into neuronal precursor cells (NPCs) was promoted as previously described 69 . Subsequent differentiation into astrocytes was performed as detailed elsewhere 69 . Differentiation of NPCs into neurons was conducted by neurogenin 2 overexpression (NGN2). Briefly, NGN2 coding sequence was cloned into the pCW57.1 backbone (gift from David Root (Addgene plasmid # 41393). Human iPSC-derived NPCs were transduced with a VSV-G pseudotyped lentivirus carrying the LV-NGN2 transgene and amplified as previously described 69 in the presence of puromycin (2 μg/ml). Cells were then plated on plates coated with poly-L-ornithine and Laminin in DMEM/F-12 medium supplemented with N-2 and B-27 supplements (1x) (Thermofisher), doxycycline (2 μg/ml) and Laminin (2 μg/ml). Medium was changed every two to three days. At day 8, medium was supplemented with BDNF (10 μg/ml) and GDNF (10 μg/ml). Neurons were used for the experiments after 14 days of differentiation.

CRISPR-Edited iPSCs

CRISPR Cas9-mediated knockout cells were generated by Synthego Corporation (Redwood City, CA, USA). Briefly, ribonucleoproteins containing the Cas9 protein and synthetic chemically modified sgRNA produced at Synthego were electroporated into the cells along with a single-stranded oligodeoxynucleotide (ssODN) donor using Synthego’s optimized protocol. Editing efficiency was assessed upon recovery, 48 h post electroporation. Genomic DNA was extracted from a portion of the cells, PCR amplified, and sequenced using Sanger sequencing. The resulting chromatograms were processed using Synthego Inference of CRISPR edits software (ice.synthego.com). To create monoclonal cell populations, edited cell pools were seeded at 1 cell/well using a single cell printer into 96 or 384 well plates. All wells were imaged every 3 days to ensure expansion from a single-cell clone. Clonal populations were screened and identified using the PCR-Sanger-ICE genotyping strategy described above. Genomic stability was assessed by KaryoStat+ TM , G-banding and Short tandem repeat analysis. Pluripotency was verified by PluriTest TM and immunohistochemical analysis. Cells were tested for bacterial and fungal contamination.

In vitro ubiquitination assay

Both p.R841 WWP2, p.R841H WWP2, and TRIF were expressed by using the TNT Quick Coupled Transcription Translation Systems kit (Promega), following manufacturer’s information. In vitro ubiquitination assay was determined with a ubiquitination kit (Enzo Life Science) according to manufacturer’s instructions. Briefly, E1 (100 nM) and UbcH5c (50 µg/ml) as an E2 were added for ubiquitination assays. The reactions were incubated for 90 min at 37 °C and stopped by adding 2x non-reducing gel loading buffer. Ubiquitin-conjugated TRIF was detected by immunoblot with an anti-ubiquitin antibody (1/1000, UBCJ2, ENZ-ABS840, ENZO). The expression levels of WWP2 were also verified by immunoblots with an anti-WWP2 antibody (1/1000, A302-935, Bethyl Laboratories).

Statistics and reproducibility

No statistical method was used to predetermine sample size. No data were excluded from the analyses. The experiments were not randomized. The Investigators were not blinded to allocation during experiments and outcome assessment.

Reporting summary

Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.

Data availability

The raw data generated in this study are provided in the Supplementary Information/Source Data file. The raw patient’s sequences are protected and are not publicly available due to data privacy laws. This data is available from the corresponding author upon request and subject to a data transfer agreement. Source data are provided with this paper.

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Acknowledgements

We thank the patient and her family who agreed to participate and made this study possible. We thank Aurélie Guillet, Corine Guyat-Jacques, and Ghislaine Aubel who contributed to the management of the study patient and volunteers. P.Y.B. is supported by the Swiss National Science Foundation (31CA30_196036, 33IC30_179636, and 314730_192616), the Leenaards Foundation, the Santos-Suarez Foundation as well as grants allocated by Carigest. CR is supported by the Swiss National Science Foundation (31CA30_196036 and 31003A_176097). RDP is supported by the Swiss National Science Foundation 320030_179531.

Author information

These authors contributed equally: Mathieu Quinodoz, Sylvain Perriot.

These authors jointly supervised this work: Sandra Asner, Carlo Rivolta, Renaud Du Pasquier.

Authors and Affiliations

Infectious Diseases Service, Department of Medicine, University Hospital and University of Lausanne, Lausanne, Switzerland

Stéphanie Bibert, Emilie Collinet, Leyder Lozano-Calderon, Sandra Asner & Pierre-Yves Bochud

Institute of Molecular and Clinical Ophthalmology Basel (IOB), Basel, Switzerland

Mathieu Quinodoz & Carlo Rivolta

Department of Ophthalmology, University of Basel, Basel, Switzerland

Department of Genetics and Genome Biology, University of Leicester, Leicester, UK

Department of Clinical Neurosciences, Laboratory of Neuroimmunology, Neuroscience Research Centre, University Hospital and University of Lausanne, Lausanne, Switzerland

Sylvain Perriot, Mathieu Canales, Amandine Mathias & Renaud Du Pasquier

Department of Oncology UNIL-CHUV, Computer-Aided Molecular Engineering, University of Lausanne, Lausanne, Switzerland

Fanny S. Krebs & Vincent Zoete

Ludwig Institute for Cancer Research, Lausanne, Switzerland

Bioinformatics Competence Center, University of Lausanne, Lausanne, Switzerland

Maxime Jan & Nicolas Guex

Pediatric Infectious Diseases and Vaccinology Unit, Woman-Mother-Child Department, University Hospital and University of Lausanne, Lausanne, Switzerland

Rita C. Malta & Sandra Asner

Department of Pediatrics, Child Neurology Unit, University Hospital and University of Lausanne, Lausanne, Switzerland

Nicole Faignart & Eliane Roulet-Perez

Institute of Microbiology, University Hospital and University of Lausanne, Lausanne, Switzerland

Pascal Meylan, René Brouillet & Onya Opota

The ColLaboratory, University of Lausanne, Lausanne, Switzerland

Florence Fellmann

Molecular Modelling Group, SIB Swiss Institute of Bioinformatics, Lausanne, Switzerland

Vincent Zoete

Department of Clinical Neurosciences, Service of Neurology, University Hospital and University of Lausanne, Lausanne, Switzerland

Renaud Du Pasquier

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Contributions

S.B., M.Q., S.P., C.R., R.D.P., P.Y.B. conceived the study. S.B., M.Q., S.P., A.M., F.S.K., V.Z., N.G., M.J., R.D.P., C.R., P.Y.B. designed the experiments. M.Q., C.R., N.G., and M.J. performed statistical analysis. F.S.K., V.Z. performed molecular modeling. R.C.M., N.F., E.R.P., P.M., F.F., S.A., P.Y.B. performed clinical investigations. S.B., S.P., E.C., M.C. and L.L.C. performed laboratory investigations. E.C., M.C., A.M., P.M., R.B., O.O., L.L.C., F.F., R.D.P., P.Y.B. performed administrative, technical, or material support. S.B., M.Q., C.R. and P.Y.B. wrote the manuscript with input from all authors. C.R., R.D.P., P.Y.B. supervised. P.Y.B. obtained funding for the work.

Corresponding author

Correspondence to Pierre-Yves Bochud .

Ethics declarations

Competing interests.

The authors declare no competing interests.

Informed consent

The patient and healthy volunteers included in this study signed an informed consent form for genetic and functional testing, according to protocols approved by the Cantonal Ethics Committee of the state of Vaud (CER-VD 479/13, CER-VD 2019-02283, CER-VD 2020-01108, CER-VD 2018-01622). Samples were stored within a dedicated biobank fulfilling quality standards according to the Swiss Biobanking Platform criteria (“Vita label”, certificate CHUV_2004_3 and CHUV_2103_14).

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Bibert, S., Quinodoz, M., Perriot, S. et al. Herpes simplex encephalitis due to a mutation in an E3 ubiquitin ligase. Nat Commun 15 , 3969 (2024). https://doi.org/10.1038/s41467-024-48287-0

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DOI : https://doi.org/10.1038/s41467-024-48287-0

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The Progression of Genital Herpes: its Stages and Impact

This essay about genital herpes explores the virus from initial infection to long-term management, outlining its stages and the profound impacts on individuals. It highlights the physical symptoms, recurrent nature, and psychological effects, emphasizing the importance of prevention, awareness, and community support. The narrative also discusses the challenges and opportunities for empowerment, aiming to reduce stigma and improve understanding and support for those living with the condition.

How it works

Genital herpes, an often-misunderstood sexually transmitted infection (STI) stemming from the herpes simplex virus (HSV), embarks on a unique journey from its initial encounter to its management. Despite its prevalence, misconceptions and stigma shroud its trajectory and impact. Delving into the intricate stages of genital herpes and its multifaceted consequences unveils a narrative rich in insight and imperative for individuals grappling with the virus and society at large.

The genesis of genital herpes commences with the stealthy invasion of the herpes simplex virus into the body.

This covert infiltration occurs through intimate contact with an infected individual, often without either party realizing. Once inside, the virus establishes a stronghold in nerve cells near the point of entry, typically in the genital or anal region. Initial symptoms may be subtle or overlooked, leaving the host unaware of the impending storm. However, within days to weeks post-exposure, the virus reveals its presence with the onset of the primary outbreak.

The primary outbreak of genital herpes announces the arrival of the unexpected guest, catching individuals off guard with its intensity. Painful blisters or sores emerge in the genital area, accompanied by flu-like symptoms such as fever, headache, and swollen lymph nodes. The severity and duration of this initial assault vary, with some experiencing mild discomfort while others endure severe pain and distress.

Following the primary outbreak, genital herpes retreats into a phase of dormancy, lying dormant within the nerve cells. Periodic reactivations, known as recurrent outbreaks, serve as reminders of the past, bringing back echoes of the initial infection. Triggered by stress, illness, hormonal fluctuations, or certain medications, these outbreaks, while typically less severe, disrupt daily life and perpetuate the cycle of the condition.

Genital herpes poses a chronic challenge, persisting in the body indefinitely with no definitive cure. While antiviral medications offer symptomatic relief and can reduce outbreak frequency, they do not eradicate the virus. This perpetual presence presents a significant dilemma for individuals, necessitating ongoing management and acceptance of the condition as a lifelong companion.

Beyond its physical manifestations, genital herpes leaves an invisible wound on the psyche of those affected. The stigma surrounding STIs casts a shadow of shame and isolation, hindering individuals from disclosing their diagnosis and seeking support. Anxiety, depression, and diminished self-esteem often accompany this journey, compounding the burden of the condition.

Prevention and awareness emerge as beacons of hope in the fight against genital herpes. Comprehensive sex education, regular testing, and open communication foster informed decision-making and reduce transmission risk. By dispelling myths, challenging stigma, and fostering empathy, we can create a more supportive environment for those navigating the complexities of genital herpes.

Finding solace in community and resources is crucial for individuals grappling with genital herpes. Online forums, support groups, and healthcare providers offer invaluable guidance, understanding, and solidarity. Together, they form the pillars of strength that uphold individuals in their journey towards acceptance and resilience.

Despite its challenges, the journey of genital herpes is not devoid of hope. Through education, advocacy, and compassion, we can empower individuals to reclaim their narrative and thrive despite the presence of the virus. By fostering a culture of acceptance and support, we pave the way for a future where genital herpes is recognized not as a mark of shame, but as a part of the diverse tapestry of human experience.

In essence, the progression of genital herpes is a testament to the resilience of the human spirit in the face of adversity. By unraveling its complexities and embracing empathy, we can forge a path towards a brighter, more inclusive future for all individuals affected by this condition.

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Dendritic-cell-targeting virus-like particles as potent mRNA vaccine carriers

Affiliations.

1 Key Laboratory of Systems Biomedicine (Ministry of Education), Shanghai Center for Systems Biomedicine, Shanghai Jiao Tong University, Shanghai, China.
2 BDGENE Therapeutics, Shanghai, China.
3 MOE/NHC/CAMS Key Laboratory of Medical Molecular Virology, School of Basic Medical Sciences, Fudan University, Shanghai, China.
4 MOA Key Laboratory of Animal Virology, Zhejiang University Center for Veterinary Sciences, Hangzhou, China.
5 Department of Ophthalmology and Vision Science, Shanghai Eye, Ear, Nose and Throat Hospital, Fudan University, Shanghai, China.
6 Bio-X Institutes, Key Laboratory for the Genetics of Developmental and Neuropsychiatric Disorders, Shanghai Jiao Tong University, Shanghai, China.
7 National Beijing Center for Drug Safety Evaluation and Research, State Key Laboratory of Medical Countermeasures and Toxicology, Institute of Pharmacology and Toxicology, Academy of Military Sciences, Beijing, China.
8 CAS Key Laboratory of Special Pathogens and Biosafety, Center for Biosafety Mega-Science, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan, China.
9 University of Chinese Academy of Sciences, Beijing, China.
10 Hubei Jiangxia Laboratory, Wuhan, China.
11 Department of Ophthalmology and Vision Science, Shanghai Eye, Ear, Nose and Throat Hospital, Fudan University, Shanghai, China. [email protected].
12 Key Laboratory of Systems Biomedicine (Ministry of Education), Shanghai Center for Systems Biomedicine, Shanghai Jiao Tong University, Shanghai, China. [email protected].
PMID: 38714892
DOI: 10.1038/s41551-024-01208-4

Messenger RNA vaccines lack specificity for dendritic cells (DCs)-the most effective cells at antigen presentation. Here we report the design and performance of a DC-targeting virus-like particle pseudotyped with an engineered Sindbis-virus glycoprotein that recognizes a surface protein on DCs, and packaging mRNA encoding for the Spike protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) or for the glycoproteins B and D of herpes simplex virus 1. Injection of the DC-targeting SARS-CoV-2 mRNA vaccine in the footpad of mice led to substantially higher and durable antigen-specific immunoglobulin-G titres and cellular immune responses than untargeted virus-like particles and lipid-nanoparticle formulations. The vaccines also protected the mice from infection with SARS-CoV-2 or with herpes simplex virus 1. Virus-like particles with preferential uptake by DCs may facilitate the development of potent prophylactic and therapeutic vaccines.

Grants and funding

AF4150049/Shanghai Jiao Tong University (SJTU)
31971364/National Natural Science Foundation of China (National Science Foundation of China)

Review 1: "MX2 Restricts HIV-1 and Herpes Simplex Virus Type 1 by Forming Cytoplasmic Biomolecular Condensates that Mimic Nuclear Pore Complexes"

Reviewers found the study compelling, clearly demonstrating the mechanism by which MX2 forms cytoplasmic condensates with host factors to trap viral capsids and prevent proper nuclear targeting by HIV-1 and HSV-1.

Show Description
Published on Mar 30, 2024
www.biorxiv.org

Summary Human myxovirus resistance 2 (MX2) can potently restrict HIV-1 and herpesviruses at a post-entry step by a process that requires MX2 interaction with the capsids of these viruses. The involvement of other host cell factors in this process, however, remains poorly understood. Here, we mapped the proximity interactome of MX2 revealing strong enrichment of phenylalanine-glycine (FG)-rich proteins related to the nuclear pore complex as well as proteins that are part of cytoplasmic ribonucleoprotein granules. MX2 interacted with these proteins to form multiprotein cytoplasmic biomolecular condensates that were essential for its anti-HIV-1 and -herpes simplex virus-1 (HSV-1) activity. MX2 condensate formation required the disordered N-terminal region of MX2 and its dimerization. Incoming HIV-1 and HSV-1 capsids associated with MX2 at these dynamic cytoplasmic biomolecular condensates. Our results demonstrate that MX2 forms cytoplasmic condensates that act as nuclear pore decoys, which trap capsids and induce premature viral genome release, and thereby interfere with nuclear targeting of HIV-1 and HSV-1.

RR:C19 Evidence Scale rating by reviewer:

Strong. The main study claims are very well-justified by the data and analytic methods used. There is little room for doubt that the study produced has very similar results and conclusions as compared with the hypothetical ideal study. The study’s main claims should be considered conclusive and actionable without reservation.

***************************************

Review: The authors investigate the function of the IRG MX2 in resisting infection by the human lentivirus HIV-1 and human herpesvirus HSV-1. They identify regions of the MX2 molecule that are important for interacting with host factors to form cytoplasmic biomolecular condensates that act as a decoy to prevent viral capsids from binding to the nuclear pores of infected cells.

In the manuscript, the authors have investigated, in depth, how the human myxovirus resistance 2 (MX2) molecules, activated in cells in response to interferon (hence, an interferon response gene – IRG), is able to restrict infection of human lentivirus (HIV-1) and human herpesvirus (HSV-1). The authors have provided a strong testable hypothesis, and a detailed experimental plan that addresses protein interactions using proteomics, functional interactions through the demonstration of antiviral activity of the authentic and mutant forms of MX2, and cell imaging level, to demonstrate the interaction between MX2 molecules with identified host factors, resulting in the targeting of incoming virions to cytoplasmic biomolecular condensates containing molecules usually associated with the nuclear pores. The authors have done an outstanding job of identifying the regions of the MX2 molecules important for the interactions with the host factors, and have distinguished between the functions involved in inhibiting HIV-1 infections, and the distinct functions involved in the inhibition of HSV-1 infections. The experimental designs and presentation of the findings is outstanding, while very dense in presentation. The conclusions reached are strongly supported by the presented data, and the discussion is thoughtfully written. The study provides an important step forward in the knowledge regarding the function and versatility of the MX2 protein in antiviral responses.

Connections

Review 2: "MX2 Restricts HIV-1 and Herpes Simplex Virus Type 1 by Forming Cytoplasmic Biomolecular Condensates that Mimic Nuclear Pore Complexes"

Published on May 08, 2024
rrid.mitpress.mit.edu

Review 3: "MX2 Restricts HIV-1 and Herpes Simplex Virus Type 1 by Forming Cytoplasmic Biomolecular Condensates that Mimic Nuclear Pore Complexes"

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CRISPR gene therapy EBT-101 does not prevent HIV viral rebound

A CRISPR-based gene-editing therapy called EBT-101 was safe and well tolerated but did not prevent viral rebound in three participants who stopped antiretroviral treatment in an early study, according to a presentation last week at the American Society of Gene & Cell Therapy annual meeting.

As aidsmap previously reported , researchers presented findings at a conference last October showing that EBT-101 was well distributed in the body and did not cause serious side effects in the first three treated study participants. Although the presentation did not include data about whether the treatment actually worked to control HIV, that didn’t stop the Daily Mail from proclaiming that a cure for HIV “could be months away” – one of the many exaggerated claims over the years about the state of HIV cure research.

A unit of heredity, that determines a specific feature of the shape of a living organism. This genetic element is a sequence of DNA (or RNA, for viruses), located in a very specific place (locus) of a chromosome.

To eliminate a disease or a condition in an individual, or to fully restore health. A cure for HIV infection is one of the ultimate long-term goals of research today. It refers to a strategy or strategies that would eliminate HIV from a person’s body, or permanently control the virus and render it unable to cause disease. A ‘sterilising’ cure would completely eliminate the virus. A ‘functional’ cure would suppress HIV viral load, keeping it below the level of detection without the use of ART. The virus would not be eliminated from the body but would be effectively controlled and prevented from causing any illness.

gene therapy

A type of experimental treatment in which foreign genetic material (DNA or RNA) is inserted into a person's cells to prevent or fight disease.

deoxyribonucleic acid (DNA)

The material in the nucleus of a cell where genetic information is stored.

herpes simplex virus (HSV)

A viral infection which may cause sores around the mouth or genitals.

But those data are out now, and the news is generally unfavourable. EBT-101 did not maintain HIV viral suppression when used alone at the initial dose tested, though it may have delayed viral rebound in one participant. Also, its good safety profile suggests that similar CRISPR approaches may be feasible for other latent viral infections such as herpes simplex and hepatitis B.

“We know that many people were hopeful that a first trial could provide evidence of a possible cure for HIV because the field has been waiting over 20 years for a cure,” Excision BioTherapeutics senior vice president Dr William Kennedy said in a news release . “However, it was essential that this clinical trial establish safety for EBT-101 as a gene therapy product as well as safety related to the use of CRISPR for the field.”

CRISPR for HIV

Antiretroviral therapy can keep HIV replication suppressed indefinitely, but the virus inserts its genetic blueprints into the DNA of human cells and establishes a long-lasting reservoir that the drugs can’t reach. This integrated HIV DNA lies dormant in resting T cells during treatment, but it can start producing new virus when antiretrovirals are stopped, making a cure nearly impossible. The only way to determine whether an experimental intervention leads to long-term remission is to discontinue antiretroviral therapy with careful monitoring in an analytic treatment interruption.

Professor Kamel Khalili of Temple University in Philadelphia and colleagues have been studying gene therapy with the aim of curing HIV for more than a decade. Their work employs CRISPR/Cas9 – sometimes referred to as ‘molecular scissors’ – a technology that combines guide RNAs that home in on specific segments of DNA and a nuclease enzyme that cuts the genetic material at the desired site.

In 2014 and 2016, the researchers reported that a CRISPR/Cas9 tool could cut out HIV genes from CD4 cells in laboratory studies. A study published in 2019 showed that this approach could remove integrated HIV genes and clear latent viral reservoirs in mice. And at the 2019 Conference on Retroviruses and Opportunistic Infections, the Temple University team reported that CRISPR/Cas9 therapy successfully removed segments of an HIV-like virus from viral reservoirs in monkeys.

This research led to the development of EBT-101, a CRISPR-based therapy delivered by an adeno-associated virus vector that uses two guide RNAs to target three sites on the integrated HIV genome. Making cuts at these locations prevents the production of intact new virus. Last August, researchers reported that a single dose of a simian version of the therapy safely and effectively removed integrated SIV in monkeys on antiretroviral therapy. But this study did not include a treatment interruption, so it could not show whether the animals were functionally cured.

The first human clinical trial of EBT-101 ( NCT05144386 ) started in 2022, testing the therapy in people on antiretroviral treatment with a stable undetectable viral load. Excision announced that the first participant in the phase I/II trial received EBT-101 that July, and the study protocol called for participants who maintained viral suppression at 12 weeks after receiving the gene therapy to undergo an analytic treatment interruption.

At last week’s conference, Dr Rachel Presti of Washington University St. Louis School of Medicine provided updated study results. Of the five participants who received a single infusion of the initial dose of EBT-101, three stopped antiretroviral therapy. All three experienced viral rebound and had to restart antiretrovirals. This likely occurred because the gene therapy did not reach all cells harbouring latent HIV, and even a very small number of cells containing residual HIV DNA is enough to re-establish viral replication.

One EBT-101 recipient was able to maintain viral suppression for 16 weeks after treatment discontinuation, considerably longer than it typically takes for the virus to rebound after stopping antiretrovirals. This suggests that EBT-101 or similar CRISPR therapies might one day play a role in a combination cure strategy.

“Initial data from the EBT-101-001 trial provides important clinical evidence that a gene editing treatment modality can be safely delivered for targeting the HIV DNA reservoirs in human cells,” Presti said. “This study provides researchers with invaluable insights for how CRISPR technology can be applied for addressing infectious disease and was an important first step towards additional programs designed to optimize this treatment modality for treating the millions of individuals who are impacted by HIV and other infectious disease.”

Excision is now testing a higher dose of EBT-101 in a second cohort and is exploring new CRISPR delivery methods that might be more efficient than the adeno-associated virus vector. One possibility is lipid nanoparticles like the ones used to deliver messenger RNA in COVID-19 vaccines.

“Viral rebound likely occurred because the gene therapy did not reach all cells harbouring latent HIV”

The company is also exploring CRISPR-based approaches for other latent infections. In other presentations at last week’s meeting, researchers reported promising preclinical results for experimental therapies for herpes simplex (EBT-104) and hepatitis B (EBT-107). Herpes simplex virus (HSV) persists in nerve cells, from which it can reactivate to cause cold sores, genital herpes and keratitis (eye inflammation). Hepatitis B virus (HBV) establishes chronic infection in the liver, where it can lead to cirrhosis and liver cancer. Unlike HIV and other retroviruses, however, HSV and HBV do not integrate their genetic blueprints into the chromosomes of host cells, so they may be easier to remove.

Many lessons have been learned from the small number of people who naturally control HIV, the somewhat larger group of post-treatment controllers and the handful of people who have been cured after stem cell transplants. But for now, a broadly applicable functional cure remains a long-term prospect.

Presti R. First-in-human clinical trial of systemic CRISPR-CA9 multiplex targeting of latent HIV. American Society of Gene & Cell Therapy, Baltimore, 2024. Excision BioTherapeutics. Excision BioTherapeutics announces data from the phase 1/2 trial of EBT-101 in hiv and in vivo efficacy data in herpes virus and hepatitis B. Press release, 13 May 2024.

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CRISPR gene therapy EBT-101 does not prevent HIV viral rebound
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