Learning objectives

1) Learn about the eight human herpesviruses, and specifically learn (a) what disease(s) each virus causes, (b) route of transmission, (c) methods of diagnosis, and (d) antiviral drugs that are used against each virus.

2) Learn the basics of the productive cycle of herpes simplex virus replication (i.e. the paradigm that is most often used to discuss aspects of herpesvirus biology). Understand the concept of viral latency, reactivation, and recurrence and their fundamental importance in the life cycle of the herpesviruses.

3) Understand the fundamental distinction between the neurotropic herpes viruses discussed this hour and the blood-borne herpesviruses discussed next hour by Dr. Levy.

4) Understand that herpes simplex virus is not the flu!! As a medical practitioner, it is easy to categorize this as an enveloped DNA virus that causes X, Y, and Z, and is treated with acyclovir. When you diagnose a 17-year old girl with genital herpes, try and remember that this is not the flu. There is a lot of stigma and misinformation that surrounds herpes simplex virus. Patients (& their significant others) need to understand the nature of the infection as much as they need the antivirals.

Keywords Reading (Parham)

infectious virus Chapter 41

latent viral genomes Chapter 43 (p.408-410)

latency vs. reactivation vs. recurrence

1. Herpesviruses.

A. Host range. Herpesviruses are believed to be present in all vertebrates and have been isolated from catfish, frogs, cows, dogs, cats, seals, squirrel monkeys, horses, kangaroos, goats, turkeys, birds, cheetahs, sea turtles, pigs, etc. .....oh, and humans too.

B. Physical properties. Of the 100s of herpesviruses, their virions are physically indistinguishable by electron microscopy and all appear as large enveloped particles (~200nm). Clinically, an envelope means two things: (1) virus is exquisitely sensitive to soap and (2) is transmitted by contact with blood, saliva, secretions (i.e. not by inanimate objects!!).

C. Genome. Virions of all herpesviruses contain linear, double-stranded DNA. Herpesviruses are clearly distinguished from one another based on analysis of their DNA genomes which range in size from 150 – 230 kbp and can contain from 80-200 genes.

D. Tropism. Broadly, herpesviruses can be split into 2 groups based on their use of neurons or leukocytes as host cells that are critical to the completion of their life cycle.

2. Human herpesviruses.

Virus name Abbreviation Tropism Disease Antiviral
*Herpes simplex virus 1 HSV-1 neurons many acyclovir
*Herpes simplex virus 2 HSV-2 neurons many acyclovir
*Varicella-zoster Virus VZV neurons chickenpox, shingles acyclovir
Cytomegalovirus CMV monocytes many ganciclovir
Epstein-Barr Virus EBV B cells mononucleosis none
Human herpesvirus 6 HHV-6 leukocytes roseola none
Human herpesvirus 7 HHV-7 leukocytes ? none
Human herpesvirus 8 HHV-8 leukocytes Kaposi’s sarcoma none

* Viruses covered in this hour.

3. General rules about neurotropic herpesviruses (of humans).

1. The viruses. Varicella-zoster Virus (VZV)

Herpes simplex virus type 1 (HSV-1) and type 2 (HSV-2)

2. The similarities.

A. ACUTE INFECTION. Biologically, these three neurotropic herpesviruses are all quite similar in the big picture of their life cycles and survival strategies. Neurotropic herpesviruses (1) gain access to the body via a mucosal surface (mouth, nose, eyes, lungs, genitals) or a break in the skin, (2) replicate to very high levels in the epithelium at the site of infection (buckets of infectious virus), (3) infect the nerve endings that innervate the site of infection, and (4) travel back to the neuronal cell bodies in the innervating ganglia and either (a) replicate and produce more virus, or (b) establish latency in the neuron.

B. LATENCY (bummer!). These viruses have a dual life cycle. Upon entry into neurons, the virus establishes latent infections in many neurons. Latency is established by the following steps: (1) the viral nucleocapsid is shipped to the nucleus of the neuron, (2) the nucleocapsid is unpackaged and the DNA genome is converted from a linear form to a circular form (i.e. "Look Ma, no ends!!- important for stability), and for reasons that are unclear (3) lytic phase viral gene expression fails to initiate and gene expression from most of the ~80 viral genes is repressed.

In the absence of viral protein expression (i.e. antigens), there is no way for the immune system to distinguish a latently infected neuron from an uninfected neuron. Thus, 3 weeks after infection the immune system mops up all the sites of productive infection at the skin (i.e. appear as scabbed-over lesions), and what the host is left with is a reservoir of neurons that harbor latent viral genomes (i.e. transcriptionally quiet circular DNA). As a biologist, this is fascinating because it means that the virus has evolved a strategy that allows it to permanently colonize its host. As a human being, this sucks!

C. REACTIVATION (the kicker!). Not only do these viruses permanently colonize their hosts, but they have evolved means to periodically re-awaken (or REACTIVATE) from the latent state. Once a latent virus reactivates in a particular neuron, this serves as a source of infectious virus that can be shipped back out to the skin where it can (a) spread to another person, or (b) replicate in the epithelium and cause some type of recurrent herpetic disease.

D. SUCCESSFUL STRATEGY?

Precise estimates vary with population surveyed, but seroepidemiology (i.e. checking people for the presence of antibodies to a given infectious agent) indicates that in the United States:

~70% are seropositive for HSV-1 (~175 million Americans)

~20% are seropositive for HSV-2 (~50 million Americans)

>90% are seropositive for VZV (~230+ million Americans)

3. Time of acquisition.

A. HSV-1. HSV-1 is most commonly associated with cold sores (symptomatic) but asymptomatic shedding of infectious virus at the oral mucosa is quite common. Over 50% of all people acquire HSV-1 before the age of 6. BOTTOM LINE: It is likely that Johnny will get a little something extra in one of those kisses from Mom, Dad, or the family before the age of 6.

B. HSV-2. HSV-2 is most commonly associated with genital herpes (symptomatic) but asymptomatic shedding of infectious virus is more the rule than the exception. HSV-2 seroprevalence increases sharply from <1% to 20% between the ages of 15 and 25 (onset of sexual activity). BOTTOM LINE: Every time you do the dance with a new partner, you roll the dice.

C. VZV. VZV usually causes an obvious symptomatic primary infection (i.e. chickenpox). Thus, life experience readily corroborates the VZV seroprevalence data which show that 70% of people acquire VZV by the age of 9. Unlike HSV-1 and HSV-2, VZV spreads in epidemic waves because the virus is often shed in the lungs and coughed up in aerosolized droplets. Thus, like the plague, once aerosolized it spreads like wildfire through susceptible populations (e.g. grade schools).

4. Molecular biology of herpes simplex virus replication.

1. Productive cycle of HSV replication.

The cascade of herpes simplex virus gene expression proceeds as follows:

Virion enters cell ® nucleocapsid shipped to nucleus

¯

VP16

¯

Immediate-Early (IE) genes (5 of these)

¯

Early genes (~20 of these)

¯

Replication of viral DNA ® Late genes (~50 of these) ® new infectious virus produced

Functionally, the genes of HSV can be grouped and defined as follows:

VP16: a virion-associated transactivator that is abundant in the tegument (space between the nucleocapsid and the envelope) and which binds and transactivates the promoters of the five immediate-early (IE) genes.

IE: Five proteins named in such a way as to clearly highlight their biological function- these are ICP0, ICP4, ICP22, ICP27, and ICP47. Collectively, these proteins irreversibly transform the cell into a factory that will produce copious quantities of virus before it dies.

Early: Expression of IE genes creates a permissive environment for expression of mRNAs and protein from the early genes. The early genes encode for 7 essential DNA replication proteins (e.g. HSV DNA polymerase, helicase) as well as nucleotide-generating enzymes that allow HSV to replicate in terminally differentiated neurons (e.g. thymidine kinase, ribonucleotide reductase, dUTPase).

Late: Mostly virion structural and DNA packaging proteins. All late genes have weak promoters, so late gene expression does not occur until after viral DNA replication is under way (i.e. ssDNA intermediates are better templates for RNA transcription). Aside from the standard complement of capsid proteins, HSV encodes 13 glycoproteins- WOOF!! Will discuss the immunoevasion properties of some of these in Tuesday’s lecture.

2. Latency and reactivation.

At the moment, HSV latency is not "understood" in a scientifically satisfying way. The following is a hypothesis about how HSV latency may be regulated, and serves primarily to give you a mental picture of how the process may work.

A. ICP0. ICP0 is one of the 5 IE genes of HSV and is very important for the progression of virus replication. In its absence, HSV replication is easily repressed by the innate interferon response.

B. LATs. The LATs, or latency-associated transcripts, are the only region of the HSV genome that remains transcriptionally active during latency (i.e. latently infected neurons contain high levels of LATs), and the LATs do not encode for any known protein.

C. The LAT-ICP0 locus. All the action surrounding regulation of HSV latency appears to reside in the LAT-ICP0 region of the HSV genome. ICP0 is a key player in initiating HSV reactivation. LATs appear to be important for establishment and maintenance of latency. Functionally, these two gene products clearly have a ying-yang relationship.

Coincidentally (NOT!!), these two genes lie antisense of one another such that their RNAs can hybridize via a 1kb+ overlap (highly unusual arrangement for viral genes). One hypothesis is that LATs are antisense RNAs that serve as a sink for ICP0 mRNA such that latency is maintained. Under this hypothesis, reactivation would involve a coordinated downregulation of LAT expression and upregulation of ICP0 mRNA. Functionally, ICP0 can substitute for VP16 and re-initiate viral replication by the same cascade of gene expression as outlined above.

5. Clinical relevance and specifics about human disease.

1. Herpes simplex virus:

A. Most HSV infections are asymptomatic (75% of seropositive do not know that they are infected).

B. Causes the following diseases:

C. Differences between HSV-1 and HSV-2:

- The rule of thumb is HSV-1 above the waist, HSV-2 below the waist, but there are no hard and fast boundaries. All bets are off when oral sex enters the picture (HSV can be transmitted in both directions).

- Biologically (and at level of DNA sequence), it is hard to tell -1 from -2. Most labs distinguish HSV-1 from HSV-2 based on distinct epitopes that are present in HSV-2 proteins that are not present in HSV-1, and vice versa (e.g. some epitopes of glycoprotein G are unique to HSV-2).

- HSV-2 generally cause more problem with recurrent herpetic disease.

 

2. Varicella Zoster Virus

A. Most VZV infections are symptomatic (~95%). However, a third of adult individuals that say they were never infected with chickenpox are seropositive for VZV.

B. This virus causes the following diseases:

- chickenpox (primary infection) = varicella

- shingles (reactivation) = zoster; generally occurs in older people (i.e. 60 years+)

- Common sequelae of shingles is post-herpetic neuralgia. This is chronic pain that lasts for months after the shingles resolve, and which does not respond to antiviral drugs.

 

SUMMARY:

1. Know the spectrum of diseases caused by HSV and VZV.

2. Know the mechanism of action of acyclovir.

3. Know the biological and clinical features that are common to HSV and VZV.

4. Know the basic mechanism that differentiates viruses that cause persistent and latent viral infections versus acute viral infection.

5. Know how viruses interfere with antigen presentation to CTLs (CD8+ T cell effectors)

Herpes Simplex Virus

HSV-1 usually associated with oro-facial lesions. HSV-2 usually associated with genital lesions.

Both viruses cause painful vesicles on the skin at the site of inoculation.

EPIDEMIOLOGY: Infection with HSV1 is almost universal. This is known because, although many infections are sub-clinical, virtually 100% of adults have antibodies in their serum. Most individuals become infected in first few years of life. Virus is shed from the infected area and spread occurs as a result of direct contact with lesions. For example, through kissing (HSV-1) or sexual intercourse (HSV-2). Virus may also, however, be shed in saliva and genital secretions and can be transmitted in absence of clinical lesions.

CLINICAL FEATURES. Two patterns of disease (1) Primary infection and (2) Recurrent disease.

1. PRIMARY INFECTION. Most primary infections are silent. In clinically apparent cases, vesicles usually develop at between 1-3 days post exposure and remain localized to the site of inoculation. However, in immunocompromised individuals the virus may disseminate. The nature of the disease is determined by the site of inoculation:

Gingivo-stomatitis: Most common form of primary infection; inoculation is usually through kissing. There is a wide spectrum of severity, from trivial to extensive disease. Painful vesicles develop inside the mouth on the bucchal mucosa and gums, on the lips and skin around the mouth. The vesicles inside the mouth ulcerate and become covered with a greyish slough. Lesions may occur at other sites on the head and neck as well. The primary eruption is often associated with fever and cervical lymphadenopathy. The illness is self limiting and lesions usually heal within 14 days.

Kaposi’s Varicelliform eruption: Super-infection of eczematous skin with HSV.

Herpetic Whitlow: Inoculation of virus into the fingers - an occupational hazard of doctors, nurses and dentists. May be mistaken for a paronychia and incised .

Conjunctivitis, Keratitis: Herpetic lesion on the cornea - is called a dendritic ulcer because of its branching appearance. Pain and photophobia are prominant features. Conjunctivitis and oedema of the lids commonly accompany primary infection. Lesions usually heal within 3 weeks.

Genital Herpes: Sexually transmitted herpetic lesions. Usually due to HSV2 but 20-30% of cases are due to type HSV1. Primary eruption lasts ±10-14 days.

Acute necrotizing encephalitis: Infection of the brain by HSV. Neurons of the temporal lobe are most commonly involved. Infection is severe and necrotising. Clinical features include: sudden onset of fever, headache, confusion and alteration in personality. Mortality is high and neurological impairment in the survivors is invariable. Encephalitis may be due to primary infection or reactivation.

Neonatal Infection: This is a relatively rare condition. Neonates have poor cell mediated immunity and are at increased risk of disseminated infection if exposed to HSV in perinatal period. Exposure may occur:

1) if mother has genital herpes at the time of delivery. (only significant risk if it is a primary infection).

2) In the post-natal period, if the infant is handled by people with herpetic lesions.

The disease may take one of three forms:

Cutaneous lesions: These are confined to the skin, and the prognosis is good.

Generalized infection: This is a serious condition, with a high fatality rate. Virus disseminates throughout the organs. Cinical features include jaundice, hepatosplenomegaly, thrombocytopenia, pneumonia and encephalitis.

Encephalitis: Direct infection of brain tissue.

2. LATENCY AND RECURRENT DISEASE

HSV-1 and HSV-2 can establish a latent infection in the ganglia of the nerves that supply the site of the primary infection (Genital area - sacral ganglia; Oro-facial - trigeminal ganglion). Following primary oro-facial infection, the virus enters sensory nerve endings and travels up the axon and establishes a latent infection in the trigeminal ganglion. The viral genome persists in an episomal form (plasmid) in the nucleus of the neurone. No viral genes are expressed. This state of latency may persist for many years. In a percentage of people, the virus will reactivate: - A cycle of viral replication occurs in the neurone and virus particles travel down the axon to reinfect the skin or mucous membrane in the area supplied by the nerve. Reactivation may be provoked by a number of stimuli: including sunlight, stress, febrile illnesses, menstruation or immunosuppression.

CLINICAL MANIFESTATIONS OF REACTIVATION

1. Cold sores (follows gingivo-stomatitis): Following one of a variety of stimuli, vesicles erupt on the muco-cutaneous junctions of the nose or mouth. These are more localized than the primary infection and heal more rapidly (7-10 days). The eruption is often preceeded by paraesthesia of the involved area.

2. Recurrent genital herpes: Recurrence with HSV 2 infections is more common than with HSV 1. Lesions are less extensive and heal more rapidly than the primary infection.

3. Keratitis: The virus reaches the cornea via the ophthalmic branch of the trigeminal nerve; the clinical lesion is termed a dendritic ulcer. It heals more rapidly than the primary infection.

LABORATORY DIAGNOSIS

Direct detection by electron microscopy of herpesvirus particles in vesicle fluid

Culture: Isolation from clinical material from skin lesions may be inoculated onto cell monolayers which are monitored for the development of characteristic cytopathic effect. This is usually detected within three days

Serology is not very useful because there is a high prevalence of antibody in the normal population.

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Varicella-Zoster Virus

CLINICAL FEATURES

There are two clinical entities: (1) Varicella or chicken pox, and (2) herpes zoster or shingles

VARICELLA (CHICKEN POX) (PRIMARY INFECTION)

Common childhood infection that presents as a mild febrile illness associated with a generalized vesicular rash. The incubation period is long, about 21 days. Vesicles appear as successive "crops", so that lesions of different ages present at same time. The lesions progress from macule to papule to vesicle to pustule to scab.

In children the disease is usually trivial and complications are rare. If infection is delayed until adulthood the disease may be more severe and complications more frequent. Infection is transmitted either by respiratory droplets or by direct contact with skin lesions. Varicella is followed by long lasting immunity. Complications:

Post Infectious Encephalomyelitis - self limiting condition, with a good prognosis.

Haemorrhagic varicella - fulminating infection in immunocompromised patients: high fatality rate

Pneumonia - common complication of varicella in adults.

Congenital varicella syndrome: Infants born to mothers who have varicella in early pregnancy may develop a syndrome associated with; limb hypoplasia, muscular atrophy, mental retardation and skin scarring. This condition is extremely rare.

Perinatal varicella: If the mother acquires varicella more than 14 days before delivery, the disease in the infant is mild. This is because the severity of the disease is ameliorated by the presence of passively acquired maternal antibody. If the mother acquires infection less than 14 days before delivery the neonate is likely to develop severe disease. An injection of special hyperimmune globulin should be administered to the baby.

ZOSTER (SHINGLES)- REACTIVATION LESION OF VZV

Like HSV, the virus (VZV) establishes a latent infection in sensory ganglia. Reactivation usually occurs many years after primary infection and is often associated with immunosuppression of the host. The virus travels down the axon and re-infects the dermatome supplied by the sensory ganglion to produce painful vesicles on the skin. Common sites include the thoracic dermatomes and those supplied by the trigeminal nerve. Post herpetic neuralgia is a common complication.

LABORATORY DIAGNOSIS

Direct detection by electron microscopy can be used to detect herpes virus particles in vesicle fluid.

Culture: Isolation from clinical material (lesions) used to inoculate cell monolayers. The virus grows slowly with the production of typical cytopathic effect usually evident in seven to ten days.

Serology can detect specific IgM; present in both primary varicella as well as zoster.