Replication

Viral replication has nuclear and cytoplasmic phases. The initial steps of replication include attachment and fusion between the viral envelope and cell membrane to liberate the nucleocapsid into the cytoplasm of the cell. Several cellular receptors and viral envelope glycoproteins are required for viral attachment. The initial attachment to the cell membrane involves the interactions of viral glycoproteins C and B with cellular heparin sulfate.¹⁷ Subsequently, viral glycoprotein D binds to cellular co-receptors that belong to the tumor necrosis factor receptor family of proteins, the immunoglobulin superfamily (nectin family), or both.^18,19 The ubiquity of these receptors underscores the wide host range of herpesviruses, and their presence on sensory neurons implicates their role in the development of neuronal infection, and therefore latency.^20–22

After attachment, the de-enveloped tegument capsid structure is transported to nuclear pores where viral DNA is released into the nucleus. After fusion of the virion envelope with the host cell membrane, the virions release several functional proteins. The virion host shutoff protein shuts off synthesis (by increasing cellular RNA degradation), whereas VP16 turns on transcription of immediate early genes of HSV replication.²³ Some of these immediate early gene products (designated α-genes), are important determinants of neurovirulence in animal models, whereas others are required for synthesis of a subsequent polypeptide group, the β or early polypeptides. Many β-proteins are regulatory proteins and enzymes required for DNA replication. Most antiviral drugs in current use or development interfere with β-proteins, such as the viral DNA polymerase enzyme or DNA helicase.^24,25 Transcription of the viral genome, replication of viral DNA, and assembly of capsids take place in the nucleus.²⁶ Moreover, the late (γ) class of HSV genes requires viral DNA replication for expression. These late proteins are structural and assist with viral egress. DNA replication takes place in a “rolling circle” pattern similar to a roll of toilet paper. Specific viral genes “clip” the end of the viral DNA into the procapsid.

After nucleocapsids are assembled in the nucleus, envelopment occurs as the nucleocapsids bud through the inner nuclear membrane into the perinuclear space. In some cells, viral replication in the nucleus forms two types of inclusion bodies: (1) type A basophilic Feulgen-positive bodies that contain viral DNA and (2) eosinophilic inclusion bodies that are devoid of viral nucleic acid or protein and represent a “scar” of infection. Virions are then transported through the endoplasmic reticulum and Golgi apparatus to the cell surface. The entire viral production cycle takes 4 to 6 hours, and an infected cell survives for 16 to 20 hours. HSV is cytopathic to cells that harbor the full cycle of HSV replication.²⁷

Molecular Features of Latency

HSV infection of some neuronal cells does not result in cell death. Instead, viral genomes are maintained by the cell in a repressed state compatible with survival and normal activities of the cell, called latency.^28,29 Latency is associated with transcription of only a limited number of virus-encoded RNAs.^30–32 Subsequent activation of the viral genome may occur, resulting in the normal pattern of regulated viral gene expression, replication, and release of HSV, although without apparent damage to the infected neuron. The release of virions from the neuron follows a complex process of anterograde transport down the length of neuronal axons.^33,34 Subsequent viral entry into epithelial cells can result in viral replication; this process is termed reactivation.^35,36

Although infectious virus typically cannot be cultured from sensory or autonomic nervous system ganglia dissected from cadavers, maintenance and growth of the neural cells in tissue culture results in production of infectious virions (explantation) and in subsequent permissive infection of susceptible cells (cocultivation).³⁷ The fact that HSV replication was first detected in neurons during reactivation in vitro suggested that the neuron harbors latent virus in vivo.³⁰ Viral DNA and RNA have since been found in neural tissue at times when infectious virus cannot be isolated.^32,38 Documentation of individual neurons infected with multiple strains of drug susceptible and resistant virus has been shown in severely immunosuppressed persons.³⁹ More recently, this phenomenon has been reported in immunocompetent subjects,⁴⁰ suggesting that the ganglia is probably continually reseeded with HSV throughout the course of chronic infection, although drug resistance is typically not of clinical importance in immunocompetent patients.

Three noncoding RNA “latency-associated” transcripts (LATs) are the only transcripts in abundance in the nuclei of latently infected neurons.^30,41,42 Deletion mutants of the genomic region that can become latent have been made, and the efficiency of their later reactivation is reduced.^41,43 In addition, substitution of HSV-1 LAT for HSV-2 LAT induces an HSV-1 reactivation pattern. In both trigeminal and dorsal root ganglia, LATs appear to maintain, rather than establish, latency.⁴⁴ HSV-1 LATs promote the survival of acutely infected neurons perhaps by inhibiting apoptotic pathways.^45,46 LAT-derived microRNA is highly expressed during latency and appears to silence expression of the key neurovirulence factor ICP34.5⁴⁷ and prevent expression of ICP0, an immediate early protein that is vital to HSV reactivation, through antisense binding of mRNA. However, exit from latency appears to correlate with complete derepression of HSV replication genes rather than just a small cluster of viral genes.⁴⁸

Although latency is the predominant state of virus on a per neuron basis, the high frequency of oral and genital tract reactivation for HSV-1 and HSV-2 suggests that the virus is rarely quiescent within the entire biomass of ganglionic tissue.^49,50 Recent experimental data suggest that HSV-2 antigen is frequently and nearly continually shed into genital mucosa. Mathematical models of HSV-2 reactivation predict that viral release from the ganglia occurs in a nearly continuous drip,⁵⁰ whereas only a single HSV particle from neurons is necessary to spark viral replication in genital epithelial cells.⁵⁰ Recent work employing microdissection plus real-time polymerase chain reaction (PCR) assay of individual neurons from cadaveric trigeminal ganglia explants revealed that many more neurons (2% to 10%) harbor HSV than would be predicted by in situ hybridization studies for LAT.^51,52 Moreover, viral copy number is highly variable between neurons with extremely high levels in certain neurons.^51,52 HSV DNA copy numbers are similar in LAT-positive and LAT-negative neurons, adding uncertainty to the role that LATs play in preventing reactivation.

LAT transcript abundance and low genome copy number correlate with subnuclear positioning of HSV genomes around the centromere.⁵³ Indeed, chromatization of HSV DNA appears to play a vital role in silencing expression of lytic replication genes.^54,55 Although certain viral transcripts are known to be necessary for reactivation from latency, the molecular mechanisms of HSV latency are not fully understood, and strategies to interrupt or maintain latency in neurons are in developmental stages.⁵⁶

HSV-1

Infection with HSV-1 is acquired more frequently and earlier than infection with HSV-2. More than 90% of adults have antibodies to HSV-1 by the fifth decade of life. Prevalence of antibody to HSV-1 increases with age and demonstrates an inverse correlation with socioeconomic status. In much of Asia and Africa, HSV-1 infection is nearly universal and is acquired early in childhood. However, in post–World War II era Western populations, 80% to 100% of middle-aged adults of lower socioeconomic status had antibodies to HSV-1, compared with only 30% to 50% of adults in higher socioeconomic groups.^58,59 Serosurveys continue to show a decline in the age-specific prevalence rates for HSV-1 infection in both the United States and most of Europe, although socioeconomic class distinctions remain.⁶⁰ In developed countries, a decrease in HSV-1 acquisition in childhood accounts for the increased frequency of sexually acquired HSV-1 infections in adolescents, as well as the increased proportion of neonatal HSV cases that are due to HSV-1.^59,61–63 Although primary HSV-1 and HSV-2 are clinically indistinguishable, in the United States, genital HSV-1 is now more common in white patients whereas primary genital HSV-2 remains more common in black patients.⁶⁴

HSV-2

Antibodies to HSV-2 start to appear during puberty and correlate with initiation of sexual activity.^60,65 Widespread use of serologic testing has provided a detailed characterization of a worldwide HSV-2 pandemic over the past 2 decades (Fig. 138-1).^60,66,67,68 Most African surveys indicate very high levels of infection, although risk varies within each country according to gender and region.⁶⁹ Seroprevalence is lower in Europe, Australia, Latin America, and Asia, although it remains highly dependent on the risk of the group being evaluated. In the United States, nationwide surveys showed an increase in HSV-2 seroprevalence from 16.4% to 21.7% of adults between 1979 and 1991, with a decrease to 17% from 1999 to 2004.^60,66,70 The cumulative lifetime incidence of HSV-2 reaches 25% in white women, 20% in white men, 80% in black women, and 60% in black men. The higher rates of HSV-2 among African Americans may reflect patterns of sexual networking rather than high-risk individual behavior.⁷¹

HSV-2 prevalence in a population is defined by geographic region, gender, sexual habits, and study population. There is a consistently higher prevalence of HSV-2 in women than in men,^58,59,63,67 and HSV-2 infection is also prevalent among HIV-infected persons, persons recruited from sexually transmitted disease clinics, and men who have sex with men.^67,72 HSV-2 antibody levels are closely related to the lifetime number of sexual partners, age of sexual debut, and a history of other sexually transmitted diseases.^73,74

The global incidence of HSV-2 infection has been estimated at 23 million new cases per year.⁷⁵ Local incidence rates of HSV-2 infection are often difficult to estimate owing to the common nature of asymptomatic seroconversion, attenuation of symptoms due to prior HSV-1 infection, location of lesions in nonvisible locations (e.g., perianal), and differential access to health care and diagnostics. For instance, among HSV-seronegative women in the control arm of an HSV-2 vaccine trial, only half of seroconversions were clinically symptomatic.⁷⁶ Nevertheless, data are accumulating and vary between populations based on risk characteristics and geography. Vaccine and condom prevention studies conducted in serodiscordant American couples document HSV-2 seroincidence levels between 6.7 and 8.6 infections per 100 person-years for women and 1.5 to 3.7 per 100 person-years for men.^77–79 Seroincidence in several high-risk urban youth cohorts was 11.7 cases per 100 person-years.⁸⁰ In African populations characterized by high preexisting HSV-2 seroprevalence, seroincidence was 1.8 to 12.9 per 100 person-years, with higher acquisition rates among persons infected with HIV-1 and among seronegative women in a monogamous relationship with a seropositive man.^81–83 Among adolescent cohorts in sub-Saharan Africa, incidence exceeding 20 per 100 person-years has been reported.^84,85

HSV-2 Risk Factors

Cofactors for risk for genital HSV-2 acquisition in an individual are well defined from prospective trials. Despite equivalent shedding rates according to gender,⁸⁶ women are consistently at higher risk for HSV-2 acquisition than men. Possible explanations include greater mucosal surface area, higher likelihood of asymptomatic ulcers in men that may facilitate transmission, or positioning within social networks. It is uncertain whether past HSV-1 infection reduces the risk for infection with HSV-2. However, persons with prior HSV-1 infection are three times as likely to acquire HSV-2 subclinically.⁷⁶ In contrast to bacterial sexually transmitted diseases, HSV-2 is commonly transmitted within long-term couples rather than casual sexual relationships. Longitudinal studies of such couples showed transmission rates varying from 3% to 12% per year.^79,87 The median time to transmission within discordant couples is 3 months, with a median number of only 40 sex acts prior to transmission, or an approximate 3.5% per-coital risk.⁷⁹ Moreover, one third of source partners within serodiscordant couples deny a history of genital lesions.⁸⁸

Knowledge of a long-term partner’s HSV-2–positive status decreases transmission incidence by 50%, highlighting the importance of formal diagnosis and disclosure of infection.^79,89 Consistent condom use decreases HSV acquisition among women, and chemoprophylaxis of the source partner also decreases transmission.^79,87 Circumcision decreases both HIV-1 and HSV-2 acquisition risk in men.^90–92 However, male circumcision does not impact transmission rates of HSV-2 among female partners.⁹³

Ganglionic Immunity

After resolution of primary disease, viral DNA is present in 2% to 11% of ganglion cells in the anatomic region of initial infection.⁵¹ Therefore, many neurons may contribute to reactivation. Aside from the previously described molecular features of latency, host T-cell responses at the ganglion level may influence the frequency and severity of HSV reactivation. Although it is not known whether reactivating stimuli transiently suppress these immune cells or independently upregulate transcription of lytic genes, or both, it is well documented that CD8⁺ T cells juxtapose to HSV-1 latently infected neurons in human trigeminal ganglia.¹¹⁷ These activated cells are reactive to a broad array of HSV antigens.¹¹⁸ In murine models, resident CD8⁺ lymphocytes and lymphocyte-derived cytokines appear to be important in preventing infectious virions from being transported down the length of the axon for release in the basal layer of the epidermis^119–121 and can block reactivation both with interferon-γ release¹²² and granzyme B degradation of immediate early protein ICP4.¹²³ In addition, there appears to be a latent viral load in the entire biomass of ganglia that correlates positively with number of neurons infected and rate of reactivation but inversely with number of CD8⁺ cells present.^124,125

Mucosal Immunity

Recent studies mostly involving genital mucosal responses to HSV-2 infection indicate that viral-host interactions in the mucosa dictate clinical expression of disease. A distinct subset of HSV-2 specific CD8+ T cells that persist in genital skin conduct immune surveillance directed at containing virus at the point of release into the epithelium and are first responders capable of controlling HSV-2 and aborting clinical lesions.¹²⁶ Once virus reaches the dermal-epidermal junction, there are two possible outcomes: subclinical shedding or recurrence defined clinically by a skin blister and ulceration (Fig. 138-2). Histologically, herpetic lesions involve a thin-walled vesicle or ulceration in the basal region, multinucleated cells that may include intranuclear inclusion, necrosis, and an acute inflammatory infection, including neutrophils, natural killer (NK) cells, B cells, and T cells.¹²⁷ Reepithelialization occurs once viral replication is restricted, almost always in the absence of a scar.