Unit 7 - DNA Viruses

Herpes viridae: genome/envelope, representative human diseases

dsDNA; enveloped

HSV, VZV, EBV, CMV, HHV-6/7/8

Adenoviridae: genome/envelope, representative human diseases

dsDNA; non-enveloped

respiratory, conjuctival, and GI disease

Papillomaviridae - genome/envelope, representative human diseases

dsDNA; non-enveloped

warts and HPV-associated cancers

Poxviridae - genome/envelope, representative human diseases

dsDNA; complex virion

smallpox, mpox, molluscum contagiosum

parvoviridae

ssDNA; non-enveloped

parvovirus B19/fifth disease

what is the structure of herpesviridae?

Four layers:

• dsDNA genome

• Icosahedral capsid

  • protein shell around dsDNA

• Tegument

  • protein layer between capsid and envelope

• Envelope

  • lipid membrane enables attachment and fusion

  • virus replicates inside nucleus of host cell

what are the 8 human herpesviruses (HHV)/ herpes viridae?

HSV-1/ HSV-2

VZV/ HHV-3

EBV/ HHV-4

CMV/ HHV-5

HHV-6/ HHV-7

HHV-8/ KSHV

-all can establish latency and reactivate later

HSV-1/ HSV-2 - major syndromes, latent reservoir

oral/genital lesions (cold sores), keratitis, encephalitis, neonatal herpes

latent reservoir in sensory neurons liek trigeminal nerve root ganglion or sacral root ganglia

VZV/ HHV-3 - major syndromes, latent reservoir

Varicella (chicken pox); shingles

latent reservoir in sensory nerve ganglia like sacral nerve root ganglia

EBV/ HHV-4 - major syndromes, latent reservoir

infectious mononucleosis; lymphoid/epithelial tumors

latent reservoir in B lymphocytes

CMV/ HHV-5 - major syndromes, latent reservoir

asymptomatic or mono-like infection; especially important for transplants

latent reservoir in leukocytes and endothelial cells

HHV-6/ HHV-7 - major syndromes, latent reservoir

roseola (sixth disease) in children

latent reservoir in T cells and other cells

HHV-8/ KSHV - major syndromes, latent reservoir

Kaposi sarcoma; primary effusion lymphoma

latent reservoir in B cells/ endothelial lineage

process of latency and reactivation

primary infection

• viral replication and shedding occurs after exposure; symptoms may be absent or present

latent phase

• viral genome persists within defined host cells with limited gene expression

reactivation

• immune changes may allow renewed replication, shedding or symptomatic disease

how is herpes simplex virus (HSV) transmitted?

through direct contact with active vesicles or body fluids

• cannot penetrate intact skin

• travels through breaks in skin or mucous membranes in mouth or genital areas

• asymptomatic shedding makes transmission possible with hard to notice symptoms

what are the two types of herpes simplex virus (HSV) and what do they primarily effect

HSV1

• primarily oral, ocular, and CNS infections

• ~60-80% of Americans infected

HSV2

• primarily genital and neonatal herpes

clinical manifestations of oral herpes (typically HSV1)

mucosal vesicles inside or around mouth

• tingling pain precedes formation

• vesicles break open, crust over, and heal

• two types:

  • herpes labialis → cold sore

  • herpetic gingivostomatitis

clinical manifestations of genital herpes (typically HSV2)

vesicles appear around genitals, buttocks, or rectum

symptoms include fever, body aches, swollen lymph nodes, or headache

recurrent outbreaks due to latency

estimated 2-4x increased risk of HIV transmission

clinical manifestations of ocular herpes (typically HSV1)

swelling around eyelids

keratitis can result in ulceration

may lead to blindness

clinical manifestations of HSV - herpetic whitlow

infection of fingers

common among healthcare workers exposed to oral secretions

often observed in thumb-sucking children with HSV-1 oral infection

clinical manifestations of neonatal herpes (HSV2)

transmission from mother to fetus

highest risk (~50%) in mothers with unrecognized primary infection

85-90% of cases occur during birth

• provide antiviral therapy during delivery to reduce risk

Clinical manifestations of HSV - HSV encephalitis

HSV-2 in neonates

HSV-1 in older children and adults

may result in fever, seizures, altered mental status, confusion

what is the preferred method of diagnosis of HSV

nucleic acid amplification test (NAAT) such as PCR

• amplify viral DNA from lesion

• PCR is recommended when HSV encephalitis is suspected

what are additional methods (other than NAAT) for diagnosis of HSV

Viral culture

• MRC-5 cells used to grow virus

• identification of viral strain with monoclonal Abs or molecular

Serology

• positive results (type-specific IgG) cannot distinguish recent or past infection (due to reactivation of virus)

Direct fluorescent antibody (DFA)

• swab of vesicle incubated with type-specific Ab

  • less sensitive

how is HSV treated?

Antiviral drugs → help reduce shedding and transmission during childbirth

• nucleoside analogs

  • Acyclovir

    • Acyclovir monophosphate converted by host into acyclovir triphosphate (ACV-TP)

    • ACV-TP inhibits viral DNA replication

      • incorporates in synthesized DNA, but lacks 3’ -OH group resulting in premature chain termination

  • Valacyclovir

    • Famicyclovir

how can an HSV infection be prevented?

no vaccine

condoms as a barrier

• reduce risk, but don’t eliminate it

gloves

• healthcare workers to prevent herpetic whitlow

how is varicella-zoster virus (VZV) transmitted

through direct contact or inhalation of infectious vesicles

• virions produced at high levels, making them very infectious

what are the two distinct diseases of varicella-zoster virus (VZV)

Varicella: chickenpox

Herpes-zoster: shingles

VZV - Varicella: chickenpox clinical manifestations in children vs. adults

Children: blister-like rash with intense itching and fever

• vesicular lesions on face and trunk

• secondary bacterial infections due to scratching

Adults, neonates, or pregnancy: more severe

• pneumonia, premature labor, congenital malformations

VZV - herpes zoster: shingles clinical manifestations

after chickenpox, virus travels to sensory nerve endings in dorsal ganglia cells → latency

reactivation

• 15-30% → usually older individuals

• postherpetic neuralgia

  • >1 year of pain

• immune suppression

what is the typical and preferred diagnosis for varicella-zoster virus (VZV)

typically diagnosed based on clnical presentation

preferred: NAAT (PCR) from infected vesicle material are best diagnostic material

what are other methods (other than NAAT) to diagnose VZV

Viral culture

• more difficult than HSV

Direct immunofluorescent assay (DFA)

• vesicle swab or fluid

serology

• IgM is not reliable due to reactivation

• IgG detectable through ELISA (after pt. has formed Abs to virus)

  • ELISA is most common serology test

treatment of VZV

airborne isolation to stop inhalation of respiratory droplets

antivirals

• acyclovir or valacyclovir → adults and immunocompromised individuals

prevention of VZV

Vaccine:

1995: live, attenuated varicella virus

• 12 months or older, adolescents, adults

• problematic breakthrough cases

  • mild illness, contagious

  • recommended booster

2005: combined w/ MMR as MMRV (called Proquad)

2017: Shingrix as recombinant virus

• recommended for person aged 50 years or older to combat shingles

• 2 doses 2-6 months apart

Epstein-Barr Virus (EBV) transmission

~40-100% of adults exposed

intimate contact with salivary secretions (kissing; sharing drinks, cups, utensils; children’s toys)

blood transfusions

bone marrow/solid organ transplants

sexual contact

perinatal contact

how does EBV infect cells of the body?

infects B lymphocytes and epithelial cells

• complement receptor 2 (CR2/CD21) used for viral attachment and entry into B cells

after entry into cell, virus replicates

lyses host cell

releases infectious virions

Clinical manifestations of EBV

Children: typically asymptomatic to mild infection

Adolescents: infectious mononucleosis

• classical triad: fever, cervical lymphadenopathy, sore throat (pharyngitis)

• other symptoms: splenomegaly, hepatomegaly, periorbital edema

Cancers

• Burkitt’s lymphoma

• Nasopharyngeal carcinoma

• Hodgkin lymphoma

EBV cancers - Burkitt’s lymphoma

malignant neoplasm of B cells in germinal center

90% of lymphomas in transplant patients at EBV+

overexpression of c-myc leads to increased cellular proliferation

• often presents as tumors in jaw or face region

EBV cancers - nasopharyngeal carcinoma

nearly all cases are linked to EBV

no clear oncogene

hyperendemic in certain regions suggesting additional factors (China, Taiwan, Malaysia, Indonesia)

EBV cancers - Hodgkin lymphoma

~50% of cases linked to EBV

Reed-Sternberg cells present in lymph nodes

• derived from B lymphocytes

• multinucleated

• sometimes looks like an “owl’s eye”

• presence of cell is necessary for diagnosis

how is EBV diagnosed?

Classical symptoms

absolute lymphocytosis

• 50% of total leukocytes are lymphocytes

  • 10-20% are reactive

  • mostly T cells responding to virus

elevated liver enzymes (ALT, AST)

• seen in 50% of patients

Serology

• infection of B cell causes polyclonal activation

• B cells proliferate and secrete Abs

  • heterophile Abs

  • EBV-specific Abs

EBV diagnosis - heterophile Abs

Abs that may cross-react with Ags from a different species → Ags on animal erythrocytes

IgM class

good screening tool (Mono-spot)

• ~40% of pts. will have detectable amounts in 1st week; 90% by week 4

• Paul Bunnel presumptive test

  • test for _____ _____

  • Pt. serum added to sheep RBCs → look for agglutination

    • pos. titer > 56

    • refer to Davidson differential test (confirmatory test)

what can cause false positives when testing for heterophile Abs in EBV diagnosis?

Forsmann Abs

• produced after bacterial infections

• found in 98% of the population

Serum sickness

• Abs made after exposure to animal proteins in vaccines

• not common anymore

use EBV-specific Abs to confirm suspected cases

EBV diagnosis - EBV-specific Abs

ELISA detection method

• high sensitivity (95-99%)

• detect Abs to Ags at different stages of infection

  • initial viral replication

    • Early Antigen (EA)-D: diffuse distribution in nucleus and cytoplasm

    • EA-R: restricted to cytoplasm only

  • late Ags appearing during lytic cycle

    • viral capsid Ags (VCAs)

  • latent Ags

    • Epstein-Barr Nuclear Ag (EBNA): 1, 2, 3A, 3B, 3C

    • Latent Membrane Protein (LMP): 1, 2A, B

EBV specific Ags - initial viral replication

early antigen (EA)-D: diffuse distribution in nucleus and cytoplasm

EA-R: restricted to cytoplasm only

EBV specific Ags - late antigens appearing during lytic cycle

Viral Capsid Antigens (VCAs)

EBV specific Ags - latent Ags

Epstein-Barr Nuclear Ag (EBNA): 1, 2, 3A, 3B, 3C

Latent Membrane protein (LMP): 1, 2A, B

EBV-specific Abs - what Abs indicate acute infectious mononucleosis (IM)

IgM anti-VCA

anti-EA-D

EBV-specific Abs - what Abs indicate convalescence

IgG anti-VCA

Anti-EBNA

Anti-EA-R

EBV-specific Abs - what Abs indicate past infection

IgG anti-VCA

Anti-EBNA

what do these panel results for EBV specific Abs indicate:

VCA IgM: neg

VCA IgG: neg

EA: neg

EBNA: neg

negative for EBV

what do these panel results for EBV specific Abs indicate:

VCA IgM: pos

VCA IgG: pos

EA: pos

EBNA: neg

acute EBV infection

what do these panel results for EBV specific Abs indicate:

VCA IgM: neg

VCA IgG: pos

EA: pos (neg)

EBNA: pos

convalescent EBV infection

what do these panel results for EBV specific Abs indicate:

VCA IgM: neg

VCA IgG: pos

EA: neg

EBNA: pos

past EBV infection

what do these panel results for EBV specific Abs indicate:

VCA IgM: neg

VCA IgG: pos

EA: pos

EBNA: pos (neg)

chronic EBV infection

how is EBV diagnosed?

NAAT (PCR)

• viral load assays help monitor treatment

• important for transplants and malignancies

viral culture

• not practical in most cases

how is EBV treated?

self-limiting

• rest plus medication for fever/pain relief

antivirals

• for severe infection including encephalitis or hepatitis

• acyclovir or valacyclovir

how is cytomegalovirus transmitted?

close, prolonged contact with infectious body secretions (intimate sexual contact, blood transfusions, solid organ transplants, perinatally)

virus can be isolated in saliva, urine, stool, vaginal, and cervical secretions, semen, breast milk, blood

50-80% of adults in US are CMV+ by age 40

• 30-50% of children are CMV+ by 5

how is cytomegalovirus (CMV) transmitted in the body

Infects a wide variety of cells

• endothelial and epitelial cells

• PMNs (neutrophils)

• monocytes and macrophages

• lymphocytes

persists in WBCs in latent stage

• monocytes, DCs, myeloid progenitors

• may reactivate later due to autoimmunity, stress, etc.

Cytomegalovirus (CMV) - congenital infections

most common congenital infection in US

• 1 in 200 babies born with it → most severe cases can result in mother losing baby

• 1 in 5 babies with congenital _____ will have long-term health problems

  • low birth weight

  • hepatosplenomegaly

  • Reactive lymphocytosis

  • petechiae in 50% of cases

  • hearing loss/deafness

• lower risk is mom is already seropositive at time of conception

CMV clinical manifestations - primary infection

asymptomatic in immunocompetent host

may present as self-limiting, mild IM-like illness

• sore throat lymphadenopathy, fatigue

major concern for immunocompromised individuals

• organ transplant recipients

• HIV+ patients

  • in both populations, infection can result in hepatitis, pneumonia, colitis, encephalitis, retinitis

  • increased rate of graft rejection or graft vs. host

CMV clinical manifestations - infection after transplant

due to large percentage of CMV+ patients, blood products are screened for ____

• primary infection may happen if ____- patient transfused/transplanted with ____+ blood product

• transplant pts may go through reactivation of their own infection

  • ____+ patient is purposefully immunocompromised before transplant → virus reactivates

what is the preferred method of diagnosis for CMV?

NAAT (PCR)

• congenital infections can be screened using amniotic fluid before birth or saliva after birth

• transplants can be screened using plasma or whole blood

  • both donor and recipient

  • after transplant, quantitative PCR can help track if infection happens

what are other methods to diagnose CMV (other than NAAT)

serology via ELISA

• IgM will not indicate recent infection due to reactivation

• IgG is more helpful

  • 4 fold increase in titer can suggest recent infection

  • otherwise, its likely a past infection

histology

• biopsy tissue and stain

viral culture

• MRC-5 cells

• historical method, not frequently used

treatment and prevention of CMV

screening of blood products of transplants

leukoreduce products

• filter WBCs to remove before transfusion/transplantation

• leukoreduced products are considered safe for most patients

CMV immune globulin

• IgG Abs against CMV

• in combination with antivirals helps to reduce CMV death/disease

Barriers to infection

• condoms

• handwashing → especially after handling children or toys

antiviral drugs

• nucleoside analogs (valganciclovir and ganciclovir)

• cidofovir and foscarnet → target DNA polymerase in drug resistant CMV

Human Herpesvirus 6 (HHV-6)

transmitted via respiratory droplets

two species:

• 6A and 6B

• infects children early in life (6-18 months)

• causes Roseola

  • sixth disease - soft pink rash on torso and extends to face and extremities

    • five other diseases cause rash during childhood: measles, scarlet fever, Rubella, parvovirus B19, additional one that doesn’t exist

  • common complication is febrile seizures

  • diagnosis with PCR and viral load testing

Human Herpesvirus 7 (HHV-7)

>90% of adults are infected

• shed in saliva of ~80% of healthy adults

infects T cells (via CD4) as well as other host cells

• downregulates CD4 expression, which can interfere with HIV infection

primarily asymptomatic

• may cause Roseola

• clinically identical to HHV-6

• rarely causes severe infection

testing rarely required

Human Herpesvirus 8 (HHV-8)

<1% of general population is a carrier → most US cases are found in HIV+ individuals

linked to cancerous condition called Kaposi sarcoma

• cancer cells from the lymphatic endothelium are found on the skin or mucous membranes

• tumor lesions appear purple, red, or brown patches

• commonly seen on feet, legs, and face

  • may be seen in mouth, GI tract, lungs, and lymph nodes

• diagnosis is made by biopsy of lesion

  • PCR performed on tissue

• treatment is typically antiretroviral therapy for HIV along with chemotherapy

what family of viruses are able to encode its own machinery for genome transcription

poxviridae

features of the poxviridae family of viruses

large dsDNA virus, roughly the same size as Chlamydia

“Brick-like” shape with inner membrane and outer envelope

no conventional icosahedral capsid

cytoplasmic replication → encodes its own machinery for genome transcription

what are the species of poxviridae viruses?

83 total

Variola virus (smallpox)

cowpox virus

Mpox virus

vaccinia virus

molluscum contagiosum virus

what are general clinical manifestations of the poxviridae family of viruses?

causes fluid-filled vesicles (pustules) to form on the skin

damage and craters formed called poxes

Variola virus (smallpox) transmission

exclusive to humans

• no animal reservoir or vector

transmitted through:

• respiratory droplets

• direct contact with vesicles

• indirect contact with contaminated items

no asymptomatic carriers

highly infectious

~30% mortality

clinical manifestations of variola virus (smallpox)

incubation period: 1-3 weeks

prodromal: 2-4 days

• fever, headache, body aches

• may be infectious at this stage

early rash: ~4 days

• oral lesions - small red spots on tongue and mouth

  • change into sores, break open, virus spreads to all parts of the body

pustular rash and scabbing: ~5-7 days later

• rash usually more concentrated on face and extremities

• ~6 days: pustules form crust and scab over

• scabs fall off within 2-3 weeks

  • no longer contagious

  • may leave pox scars

how is variola virus (smallpox) diagnosed

mainly through signs and symptoms

immediately contact public health authorities before specimen collection or transport (it has been functionally eradicated)

Confirmation via NAAT (PCR)

• collect specimens for each disease stage

  • Prodromal: nasopharyngeal swabs, whole blood

  • Early rash: tonsillar swab, lesion biopsy, whole blood

  • pustules: lesion fluid, whole blood

  • scabs: lesion scab, whole blood

treatment and prevention of smallpox (variola virus)

Vaccine

• first vaccine developed by Edward Jenner in 1796

  • Milkmaids exposed to cowpox (Vaccinia virus) were immune to smallpox

  • Jenner inoculated 8 year old neighbor with fluid from cowpox sore from milkmaid

  • exposed neighbor to smallpox → immune to infection

• 1939: Vaccinia virus in smallpox vaccine was not cowpox, but likely horsepox

  • provide immunity against small pox

  • still used today

• small pox has been declared eradicated since 1980

  • last US case 1949, last global case 1977

• ACAM2000

  • Live vaccinia virus

    • Bifurcated needle with two prong

    • pustule should appear within 1 week

• JYNNEOS

  • live-attenuated vaccinia virus

    • 2 doses administered via subcutaneous injection

why did global smallpox eradication succeed?

favorable biology

• humans were the only natural reservoir; no silent animal reservoir maintained variola transmission

recognizable and traceable

• characteristic rash and transmission mainly after illness begins supported detection, isolation, and contact tracing

effective public health tools

• potent, stable vaccinia vaccine; bifurcated-needle delivery, global surveillance and ring vaccination around cases

Mpox (Monkeypox) transmission

may spread to anyone through close, personal, skin-to-skin contact including direct contact with rash, especially sexual contact or through saliva or upper respiratory secretions

low risk from indirect contact through fomites

clinical presentation of Mpox

symptoms start within 3 week of exposure

after rash, rash develops within 1-4 days

Mpox risk and disease severity

less severe and lower mortality compared to smallpox

Molluscum contagiosum transmission

skin-to-skin contact and fomites

common in children and also occurs with sexual contact

Molluscum contagiosum clinical manifestations

firm, dome-shaped papules with central umbilication

benign, but may appear anywhere on body

may become itchy, sore, or swollen

typically resolves without scarring

diagnosis of molluscum contagiosum

usually clinical (signs/symptoms)

biopsy/histology can reveal intracytoplasmic Henderson-Patterson inclusion bodies in atypical cases

Parvovirus B19 (fifth disease) - virus features

non-enveloped, ssDNA virus

smallest DNA virus (22-26 nm)

typically associated with veterinary pathogens in dogs and cats

what are clinical manifestations of parvovirus B19 (fifth disease)

erythema infectiosum (fifth disease)

• develops classic “slapped-cheek” facial rash

• can be followed by a lacy body rash in children

adults:

• arthralgia or arthritis can be more prominent than rash

transmission of parvovirus B19

respiratory droplets

blood and blood products

vertical transmission

pathogenesis of parvovirus B19

infects erythroid precursors

• may temporarily suppress erythropoiesis

  • P Ag as RBC receptor for entry

• Individuals with sickle cell disease or chronic hemolytic anemias are high risk → transient aplastic crisis (RBC cell production isn’t keeping up with need of the body)

• severe fetal anemia

  • <5% of pregnant women

  • may lead to miscarriage or stillbirth (hydrops fetalis)

Erythema infectiosum (fifth disease)

• most infections occur during childhood (80% adults seropositive by 65, 20% of cases asymptomatic)

• incubation period: 14-18 days

  • prodromal phase includes fever, headache, runny nose

    • adults may have painful, swollen joints

  • classic “slapped cheek” rash appears

    • secondary rash may appear later on chest, rash, trunk, etc.

    • adults may only present with secondary rash

Parvovirus B19 - laboratory diagnosis

serology

• B19-specific IgM can support recent infection

• IgG usually indicates past exposure

• interpret with symptom timing

Molecular testing

• B19 DNA PCR (whole blood, amniotic fluid, tissue/biopsy) is especially useful in aplastic crisis, persistent infection, or immunocompromised patients

pregnancy

• maternal testing and fetal evaluation depend on timing and clinical risk; coordinate with obstetric specialists

what are the non-herpes DNA viruses?

Adenovirus

HPV

Variola

Mpox

Molluscum

Parvovirus B19

Adenovirus - shedding/transmission and treatment/prevention

respiratory, ocular, or fecal shedding; prolonged after illness

usually supportive treatment; hand hygiene/ disinfection; type 4/7 vaccine only select military

HPV - shedding/transmission and treatment/prevention

transmission through skin/mucosal contact, often silent infection for months/years

treat lesions/dysplasia; vaccination and cervical screening

Variola - shedding/transmission and treatment/prevention

transmission after symptoms; infectious until scabs separate

eradicated; vaccination/public-health response if suspected

Mpox -shedding/transmission and treatment/prevention

close contact with lesions/material until all scabs shed and new skin forms

supportive treatment; vaccines for eligible groups, specialist for severe disease

Molluscum - shedding/transmission and treatment/prevention

transmission through skin contact, fomites, autoinoculation while lesions present

cover lesions, avoid scratching; often resolves without therapy

parvovirus B19 - shedding/transmission and treatment/prevention

supportive care; special management for aplastic crisis, pregnancy, or chronic anemia

where do most DNA viruses replicate?

in the nucleus

poxviruses are the exception

Herpesviruses establish _____; PCR positivity or seropositivity requires clinical interpretation

latency

PCR is central for identifying DNA viruses, but specimen and timing determine ____

usefulness

skin lesion, CSF, plasma, cervical specimen and neonatal urine answer different questions

prevention is ______-specific

virus

VZV, HPV and orthopox vaccines exist; HSV vaccination is not currently available

Molecular testing - clinical presentation: fresh oral/genital vesicles

what test/specimen should be used and what are key caveats

HSV NAAT from lesion

caveat: Type HSV-1 vs HSV-2