HUMAN HERPES VIRUSES

Dr. R. Someshwaran, MD., Assistant professor, Department of Microbiology, KFMS&R

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Objectives of today’s class To know and understand ‘What is a

Virus?’ To revise Baltimore classification of

virus based on replication mechanism

To name atleast 4 DNA and 4 RNA virus

To know Structure, Mode of transmission, Pathogenesis, Clinical spectrum, Lab diagnosis, Complications of CMV, EBV, HHV-6, 7 & 8.

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WHAT IS A VIRUS?

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VIRUS – General propertiesObligate intracellular parasiteNeither a Prokaryote nor an EukaryoteNo cellular organisationContain either DNA or RNA but never bothLack enzymes for protein and nucleic acid synthesisNeeds synthetic machinery of host for replication Resistant to antibacterial antibiotics

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VIRUS – General propertiesOccupies a twilight zone b/w living and non-livingCan be crystallized – Filterable virusesInfectious nucleic acid can be extractedLiving chemicals - Smallest living units Ultra microscopic - Can cause cancerVirion – extracellular Infectious particleViroid – No extra cellular dormant phase (Virion) + Genome smaller to viruses

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Elementary bodies – Pox virusLargest virus – 300 nm Pox virus ~ MycoplasmaSmallest virus – 20 nm Parvo virus ~ Hemocyanin Viruses multiply by Complex process but not by binary fission Do not grow on inanimate media Ribosomes are absent Sensitive to Interferon +

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Morphology Size: Electron microscope - Ultra

microscopic Structure and shape: Capsid - protein

coat - Symmetrically arranged Capsomeres (Penton @ vertices or Hexon @ Facets - Polypeptides) - impenetrable shell aroun nucleic acid core.

All virus have 12 Pentons but number of Hexons may vary

Symmetry: Icosahedral (Cubical), Helical & Complex

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Morphology Envelope: Enveloped / Non-enveloped or

Naked Peplomers - Projecting spikes on envelope 2 types 1. Hemagglutinin 2.

Neuraminidase (Receptor Destroying Enzyme) - Mushroom shaped

Envelopes confer chemical, antigenic and biological properties on viruses

Fibrils protrude from vertices (As in Adeno virus)

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Refer Page No: 429, General properties of virus

Ananthanarayan & Paniker’s 9th Edition for Diagram

Icosahedral naked virus Helical naked virus

Icosahedral enveloped virus Helical enveloped virus

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General properties of Virus All viruses are disrupted in Alkaline pH, Entero

virus very resistant to acidic pH. Most active antiviral disinfectants: Oxidising

agents like Hydrogen peroxide, potassium permanganate, Hypochlorites

All enveloped viruses are lipid soluble with ether, Chloroform, Bile salts

Viruses are inactivated by Sunlight, UV rays, Ionising radiation (X-rays, Gamma rays, Cosmic rays)

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General properties of virus Hemagglutination – Elution -

Hemagglutination inhibition Viral multiplication: (Add a diagram)1. Attachment (Adsorption), 2. Penetration, 3. Uncoating, 4. Biosynthesis, 5. Maturation, 6. Release - Transcription of mRNA into early and late

protein is the rate limiting and important step in viral replication

- Eclipse phase: Virus cannot be demonstrated in the host cell from the stage of penentration till the appearance of daughter virions

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BALTIMORE CLASSIFICATION

(Based on Replication mechanism)

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VIRUS

REPLICATION MECHANISM

EXAMPLES

CLASS I Fully Double Stranded DNA viruses

Adeno, Herpes, Papova virus

CLASS II

Single stranded DNA virus Parvo virusCLASS III

Double stranded RNA virus ReovirusCLASS IV

Single stranded mRNA – Positive sense, Positive strand or Plus sense (Based on mRNA transcription)

Picorna, Togavirus

CLASS V

Single stranded mRNA – Negative strand or Minus sense or Anti-sense Where RNA polarity is opposite to mRNA (Based on mRNA transcription)

Rhabdo, Ortho-myxo, Para-myxo

CLASS VI

Single stranded RNA genome – RNA-DNA hybrid – DNA hybrid (Pro virus)

Retrovirus

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DNA Viruses

Members causing disease in Humans

Herpesviridae

Herpes simplex 1&2, CMv, EBV, Varicella zoster, HSV 6,7,8

Hepadnaviridae

Hepatitis B virus

Adenovirus Human Adenoviruses A-FPoxviridae Variola, Vaccinia, Cow pox,

Monkey poxPapovaviridae

Papilloma, Polyoma, Vacuolating viruses

Parvoviridae Parvovirus

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RNA Viruses Members causing disease

in HumansPicornaviridae Enteroviruses – Poliovirus,

ECHO virus, Coxsackie virus

Orthomyxoviridae

Influenza virus A, B, C

Paramyxoviridae

Paramyxoviruses – Mumps virus, Parainfluenza virusesMorbilivirus – Measles virusPneumoviruses – Respiratory syncytial virus (RSV)

Togaviridae Alpha (Group A) - Chikungunya virusRubivirus – Rubella virus

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RNA Viruses

Members causing disease in Humans

Flaviviridae Flaviviruses – Yellow fever, dengue viruses, Japanese encephalitis virus, Hepatitis C virus

Bunyaviridae

Hantaviruses, Sand fly fever

Rhabdoviridae

Vesiculovirus – Chandipura virus, Lyssavirus – Rabies virus

Arenaviridae

LCM, Lassa fever

Reoviridae Human Rota VirusCoronaviridae

Human Coronavirus, SARS virus

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RNA Viruses

Members causing disease in Humans

Retroviridae HIV 1&2, HTLV 1&2Caliciviridae

Norwalk virus, Hepatitis E virus

Filoviridae Marburg virus, Ebola virusAstroviridae Human astrovirusDeltaviruses

Hepatitis D (delta virus)

Prions CJD, Kuru,

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HERPES VIRUS

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Introduction to HERPES VIRUS Herpes virus comprises of over 100

Enveloped DNA viruses affecting Humans and animals

Characterised by their ability to cause Latent infections – enabling virus to persist within infected hosts and to undergo periodic reactivation.

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Herpes virus morphology Capsid is Icosahedral - Composed of

162 capsomers - Enclosing the core containing Double stranded DNA genome

Nucleocapsid covered by a lipid envelope (derived from modified host cell membrane through which naked virions bud during replication)

Envelope carries surface spikes about 8 nm long, Between envelope and capsid is the tegument (amorphous)

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Herpes virus morphology Enveloped virion measures

about 200 nm in diameter Naked virion measures about

100 nm in diameter Replication of Herpes virus

takes place in the host cell nucleus

Herpes viruses forms “Cowdry type A inclusion bodies which is also called Lipschutz bodies”

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CLASSIFICATION Alphaherpesviruses: Short replicative cycles (12-18 hours), Variable host range Tendency to cause Latent infection in

sensory ganglia (Neurons) In culture: Rapidly cytopathic Infectious viruses released from cells Ex: HHV 1, HHV 2 & HHV 3

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CLASSIFICATION Betaherpesviruses: Replicate slowly (>24 hours), Narrow host range Grows best in fibroblast Tendency to cause enlargement of infected cells

(Cytomegaly) Latent infection of salivary glands and other organs In culture: Cytopathic effect is slow Virus remains cell-associated Ex: HHV 4, HHV 8

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CLASSIFICATION Gammaherpesviruses: Replicate in Lymphoblastoid cells Narrow host range Frequently causes Latent infection Specific for B or T lymphocytes Ex: HHV 5, HHV 6, HHV 7

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CLASSIFICATION

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HHV 1 & HHV 2 - Morphology

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HHV 1- Clinical manifestations

EM of Herpes virus 1

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HHV 1 & HHV 2 - Pathogenesis

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HHV 1& 2 Diagnosis Multinucleate Giant cells – Tzanck’s

smear

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HHV 3 Varicella zoster cirus – Varicella

Herpein – To creep, Zoster – ‘Girdle’ Tzanck’s smear showing Multinucleate

giant cells

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Clinical case scenarion: A 21 years old female presented with a

complaint of malaise and loss of appetite X 1 month and sore throat and fever (especially in afternoon) since 5 days. O/E: Cervical lymphadenopathy+, Mild hepatosplenomegaly+. CBC showed Absolute Mononuclear lymphocytosis (mostly atypical lymphocytes). Her serum was tested for Paul Bunnell test where IgM was positive.

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Diagnosis Human Herpes Virus 4 (HHV-4)

Epstein Barr virus infection Infectious mononucleosis Glandular fever Kissing’s disease

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HHV 4 Ebstein Barr virus - EBV

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Epstein Barr Virus 1958 – Burkitt – Described an unusual

lymphoma among children in Africa - Suggested the cause to be Mosquito Borne virus

Many other ‘Passenger viruses isolated from such culture lymphoma cell tumors’ by Epstein.

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EBV or HHV-4 1964 – Epstein, Barr and Achong –

identified a new type of herpes virus EB virus specifically affecting B lymphocyte lineage. CD 21 receptors necessary for acquiring EBV infection is found on Humans and some subhuman primates

EBV infected cells are transformed & immortalized (grow continuously in vitro)

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EBV EPIDEMIOLOGY Ubiquitous in all human populations EB virus infection leads to latency,

periodic reactivation and life long persistence.

EBV infection occur in infancy and childhood, usually asymptomatic

EBV primary infection often delayed and leads to infectious mononucleosis and the EBV antibodies present in 95% of adults

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EBV EPIDEMIOLOGY Source of infection: Saliva of infected persons –

shed virus in oro-pharyngeal secretions for months following 1º infection and intermittently thereafter.

Not highly contagious, droplets and aerosols not effective in disease transmission

Intimate oral contact (as in kissing) – predominant mode of transmission – hence the name ‘Kissing disease’

Transmitted rarely after blood or bone marrow transfusion

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EBV – Clinical spectrum Infectious mononucleosis EBV associated malignancies: Burkitt’s lymphoma Lymphomas in immunodeficient persons

such as with AIDS & transplant recipients Nasopharyngeal carcinoma in persons of

Chinese origin

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EBV - PathogenicityVirus enters the Pharyngeal

epithelial cells

Through CR 2 or CD 21 receptors present on B lymphocytes

Multiplies locally Invades blood stream

Infects B lymphocytesA. Latent infection – Cells

transformed & Immortalised – indefinite

growth in vitro

Polyclonal activation – produce many types of

ImmunoglobulinEx: Heterophile sheep

erythrocyte agglutination

B. Lytic infection – Cell death and release of

progeny virions

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EBV Pathogenicity Mononucleosis represents a polyclonal

transformation of B lymphocytes EBV antigens expressed on surface of the

infected B cells. Atypical lymphocytes – seen in blood smear are

nothing but T lymphocytes undergoing Blast transformation in response to neoantigens.

Intermittent reactivation leads to clonal proliferation of infected B cells, which is kept in check by activated T cells

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EBV Pathogenicity In immunocompromised – B cell clones may

replicate unchecked resulting in Lymphomas Hyperendemic malaria (Prevalent in Africa) –

believed to cause Immune impairment in children with Burkitt Lymphoma

Nearly half of the lymphomas seen in Immunodeficient persons contains EV virus DNA sequences.

Genetic influence is best illustrated in XLP (X linked Lymphoproliferative) or Duncan syndrome, where extreme susceptibility to EBV+

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Infectious mononucleosis Glandular fever Acute self-limited illness Usually seen in non-immune young

adults following primary infection with EBV

Incubation period: 4-8 weeks. Characterised by Fever, Sore throat,

Anorexia, Lethargy, Lymphadenopathy and presence of abnormal lymphocytes in peripheral blood smears, mild transient rash+

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HHV 4 Ebstein Barr virus - EBV

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Infectious mononucleosis Patients treated with Ampicillin develop

maculopapular rash due to immune complex reaction to drug

Often associated Sub-clinical Hepatitis+ Complications: Hematological,

Neurological, Cardiac & Pulmonary conditions and Splenic rupture

Spontaneous resolution occurs in 2-4 weeks

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Lab diagnosis of EBV Blood examination: Leukopenia followed

by leukocytosis in later stages with atypical lymphocytosis (Abnormal mononuclear cells) characterized by deep basophilic vacuolated cytoplasm and kidney shaped nuclei showing a lattice of fenestrated chromatin. DD: Lymphocytic leukemia

Paul – Bunnell test: Standard diagnostic test done with heat inactivated sera @ 56ºC for 30 minutes

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Lab diagnosis of EBV cont.. Differential agglutination test: Differential

absorption of Agglutinins with Ox red cells or Guinea pig kidney necessary to remove Forssman antigen (induced by injection of horse serum)

Normal serum: Ox RBC not absorbed, Guinea pig kidney absorbed

Infectious mononucleosis: Guinea pig kidney not absorbed, Ox RBC absorbed.

Immunofluorescence and ELISA available

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Lab diagnosis of EBV cont… IgM antibody to VCA – Viral Capsid Antigen appears

soon after primary infection and disappears in 1-2 weeks. (Reliable indication of primary infection)

IgG anti-VCA persists throughout life and indicates past infection

New appearance of antibody to EB Nuclear Antigen (EBNA) is also a useful marker of primary infection

Antibodies to Early antigens (EA) high in EB-associated lymphomas (Limited availablity)

CMV, Toxoplasmosis can be a risk factor for EBV infection.

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Case scenarion A 27 years old male a K/C/O HIV/AIDS

presented to Ophthalmology OPD with Progressive diminution of vision X 3 months. CD4 counts<100 cells/mm3. Fundus examination: Fluffy retinal infiltrates+ Multiple hemorrhages+ Serological tests positive for IgG antibodies and PCR were positive for CMV DNA.

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DIAGNOSIS CMV retinitis made Patient was started on Ganciclovir. Note: Patient did not take Anti-HIV

medications regularly.

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Cytomegalovirus (CMV) – HHV 5

Salivary gland viruses (Former name) Enveloped DNA viruses Characterised by enlargement of

infected cells (Cytomegaly) and prominent intranuclear inclusions

Owl’s eye inclusion body Prolonged Latency - Disseminated

disease

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HHV 5 Cytomegalovirus - CMV

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HHV 5 Cytomegalovirus - CMV

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Cytomegalovirus (CMV) – HHV 5

Largest virus in Herpes virus family (150-200 nm)

Strict host specificity CMV identified in Humans, Monkeys, Guinea pigs Cytopathic effect in culture delayed (Takes upto

50 days) Human CMV – antigenically unrelated to other

Herpes viruses CMV Disease is rare, but infection is extremely

common

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CMV – Clinical spectrum Congenital infection: Following Intra-

uterine infection – Fetal death or Cytomegalic Inclusion disease of the new born – often fatal.

Generalised infection: Hepatosplenomegaly, jaundice, thrombocytopenic purpura and hemolytic anemia. Cytomegalic Inclusion disease is the Most common cause of Microcephaly.

Others: Chorioretinitis, Cerebral calcification resembling congenital toxoplasmosis

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CMV – Clinical spectrum Infection in infants: Primary CMV

infection followed by reactivation in mothers during pregnancy – Cause. Perinatal infection through genital secretions can occur.

Infection in children: Usually asymptomatic, Heterophile antibody negative, Infectious mononucleosis following transfusion of CMV infected blood (Post-transfusion mononucleosis)

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CMV – Epidemiology Spreads slowly and by close contact Transmission is by saliva or by sexual contact. Special mode of transmission by blood

transfusion or organ transplantation Virus detected in saliva, urine, blood, semen,

milk, cervical secretions Viruria in congenitally infected persists for 4-5

years 1% neonates in Usa infected, 80% adults have

antibodies to CMV

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CMV Lab diagnosis Specimens: Urine, Saliva, Semen and

cervical secretions Isolation: Human fibroblast cultures Growth detected in shell vial cultures

and by staining the cells with fluorescent tagged antibody to Early antigens (EA) against CMV.

Simpler but less reliable – Cytomegalic cells in centrifuge deposits where owl eye Inclusion bodies are seen.

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CMV Lab diagnosis Serology: ELISA, CFT, IF, IHA Screening for antibody among blood

donors and transplant or organ donors.

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CMV Prevention and treatment

Indicated in high risk cases like organ transplants, immunodeficient persons and in premature infants.

Acyclovir, Ganciclovir, Foscarnet No vaccines available Experimentally: Live attenuated vaccine

(Towne 125 and AD 169 strains) and a purified CMV polypeptide vaccine found to be immunogenic but not effective in protecting immuno deficient individuals

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HHV 5 Cytomegalovirus - CMV

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HHV 5 Cytomegalovirus - CMV

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HHV-6 HHV 6 – Human B

lymphotropic virus. Spreads through saliva in

Early infancy. Two variants A & B Where B causes mild

childhood illness “Exanthum subitum or Roseola infantum or Sixth disease”

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HHV-7 or R K virus HHV 7 Isolated in 1990

from Peripheral CD4 cells of a healthy person. Like HHV6, HHV7 is also widely distributed and transmitted through saliva.

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HHV-8 or KSHV 1994 – A new Herpes

virus identified from Kaposi’ Sarcoma tissue from AIDS patient

But also seen in Non- HIV Kaposi’s Sarcoma patients

HHV 8 (Kaposi Sarcoma-associated Herpes virus or KSHV)

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QUIZOPHILIA

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Summary 1 All major Herpes viruses are found Worldwide Exposure is common – As most adults have

antibodies to these viruses Serious disease is relatively infrequent Severity of disease depends on Age and

immune status HSV 1(Above waist) & HSV2 (Below waist)

cause mucocutaneous vesicular and ulcerative lesions due to intimate contact b/w infected and non-infected host

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Summary 2 Replication of HSV 1 & HSV 2 occurs in Local

epithelial cells until eruption occurs on the surface. Involves CMI and Humoral immunity. To prevent – avoid contact with infected – Acyclovir for severe infection – No vaccine available,.

VZV causes Chicken pox or Varicella – vesicular rash in children – starts in head & trunk – moves to extremities. Vesicles pustular, crusted & scabbed prior to healing. VSV usually manifests as a Zoster or Shingles (Painful vesicular lesion on a nerve course).

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Summary 3 VZV infection – Aerosol transmission – Initial

infection of epithelial cells in nasopharynx – followed by Viral replication, Viremia, Skin rash and Latency in Dorsal root Ganglion. Later in life VZV may emerge to cause Zoster. Involves CMI and Humoral immunity.

A live attenuated vaccine available for Childhood VZV and a High - titre human immunoglobulin may be given for immunocompromised. Fpr VZV encephalitis - Acyclovir

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Summary 4 Cytomegalovirus (CMV) causes Congenital

infection of the liver, retina and CNS with rash, Mononucleosis syndrome in older children and adults.

Life-threatening disseminated infection affecting lungs, CNS, liver, retina and GIT in patients with Acquired Immuno Deficiency Syndrome (AIDS) & Transplant patients.

Infection transmitted by intimate contact with secretions (Breast milk, Saliva & Urine of infected patients)

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Summary 5 Cytomegalovirus (CMV) replicates in Salivary

glands and Kidney cells – cells fuse to form Multinucleated giant cells (cytomegalic inclusions as CPE) – virus persists in tissues with Chronic shedding. CMI & Humoral immunity important.

Prevent CMV transmission by screening blood and organs before transplant.

In Bone transplant patients, CMV pneumonia can be prevented by Specific Immunoglobulin and Ganciclovir.

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Summary 6 Epstein-Barr virus (EBV) causes Infectious

mononucleosis and Burkitt’s lymphoma. Also attributed in pathogenesis of Nasopharyngeal carcinoma.

EBV transmitted by intimate contact, EBV grows in B lymphocytes and other lymphatic tissues. Both CMI & Humoral immunity important for outcome of infection.

Paul Bunnel test – Heterophile agglutination test to detect antibodies in serum of infected patients.

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Summary 7 HHV 6 – Human B lymphotropic virus.

Spreads through saliva in Early infancy. Two variants A & B, where B causes mild childhood illness “Exanthum subitum or Roseola infantum or Sixth disease”

HHV 7 Isolated in 1990 from Peripheral CD4 cells of a healthy person. Like HHV6, HHV7 is also widely distributed and transmitted through saliva.

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Summary 8 1994 – A new Herpes virus identified

from Kaposi’ Sarcoma tissue from AIDS patient but also seen in Non- HIV Kaposi’s Sarcoma patients – HHV 8 (Kaposi Sarcoma-associated Herpes virus or KSHV)

Herpes Simian virus causes ‘Cold sores in monkeys’ and Severe CNS disease in Humans. Risk group 4 pathogen.

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Take home message Herpes viruses – Enveloped DNA virus Spread by Intimate contact Hallmark of Herpes viruses are Latency,

periodic reactivation and life long persistence

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QUIZ 1 A 23 years old female presented with

maculopapular rash over the upper limbs and trunk for 7 days duration. Exudate from papule sent for this stain. Identify the causative organism and pick up the diagnosis.

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Slapped cheek disease – Parvo B19 virus

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Thank you