HERPES VIRUSES PART II, PARVO VIRUSES, PAPOVO VIRUSES

Cytomegalovirus Epstain barr virus HHV 6,7,8 Parvoviruses

Papovaviruses

Dr.C.Meenakshisundaram.,MD

CYTOMEGALOVIRUS

MICROBIOLOGY
Double-stranded

linear DNA

enveloped virus Member of Herpesviridae family Alpha-herpesvirus subfamily HSV-1 and 2, VZV Beta-herpesvirus subfamily CMV (HHV5), HHV 6, HHV 7 Gamma-herpesvirus subfamily EBV, HHV 8 (KSHV)

www.biosciences.bham.ac.uk

MICROBIOLOGY
CMV

150-200 nm in size Icosahedral nucleocapsid containing dsDNA 162 hexagonal protein capsomeres Additional layer of surrounding protein (tegument) Outer membrane envelope with glycoprotein complexes

www.biografix.de

MICROBIOLOGY
Largest

member of herpesviridae family 230-240 kilobase pairs Large cytomegalic cells with enlarged nuclei Violaceous intranuclear inclusions surrounded by a clear halo Basophilic stippling may be present in the cytoplasm Replication cycle Immediate early: 4 h Early: 4-24 h Late: 24 h

www.som.tulane.edu

PATHOGENESIS
Lytic virus with cytopathic effect Initial infection Epithelial cells of the salivary gland persistent infection and viral shedding Genitourinary system Proximal tubules near cortical areas Ultimately can be found in several tissues (salivary gland, lung, liver, kidney, intestine, adrenal gland and CNS) Other pathogenic mechanisms Granulomatous reaction, particularly in liver Immune complex formation Vasculitis Establishes a latent host infection May reactivate during a period of immunosuppression secondary to drugs or concurrent infection (eg. HIV)

PATHOGENESIS
Incubation

period: 28-60 days Primary infection symptoms: 9-60 days Viremia: 2-3 weeks IgM response: 30-60 days Peak viral titers: 4-7 weeks post infection

EPIDEMIOLOGY
CMV prevalence increases Transmission: transplanted with age organ, breast milk, urine, saliva, tears, stool, sexual Risk factors contact, blood, transplacental Work at day care/contact Seldom associated with with children mortality in immunocompetent Blood transfusion hosts (<1%) Multiple sexual partners Significant morbidity and Unprotected intercourse mortality in Abnormal cervical cytology immunocompromised Lower SES/underdeveloped nations Infection with STD

www.cdc.gov

CLINICAL MANIFESTATIONS

Immunocompetent

host disease
host disease
lbmi.org/pathologyimages

Immunocompromised

Congenital/Perinatal

disease

CLINICAL MANIFESTATIONS IMMUNOCOMPETENT

Usually

asymptomatic or mild flu-like symptoms www.crprc.ucdavis.edu Mononucleosis syndrome Fever/chills, malaise, myalgia Mild hepatitis, leukocytosis, atypical lymphocytes in blood x 6 weeks hepatomegaly, splenomegaly, pharyngitis Older patients, longer fever duration, Negative heterophile-agglutinin tests

CLINICAL MANIFESTATIONS IMMUNOCOMPETENT

Meningoencephalitis,

pericarditis, myocarditis, thrombocytopenia, hemolytic anemia, maculopapular rash, GI ulcers, pneumonia less common Reactivation possible Viremia Positive IgM in presence of IgG In setting of concurrent infections or stress

CLINICAL MANIFESTATIONS
IMMUNOCOMPROMISED
Significant

disease in the immunocompromised host Pneumonia Hepatitis Encephalitis Colitis/GI ulcerations Uveitis Retinitis Neuropathy CMV syndrome

Normal Retina

CMV Retinitis
www.5mcc.com

CLINICAL MANIFESTATIONS IMMUNOCOMPROMISED

Excretion

of CMV in saliva and urine common

Viremia

in organ transplant patients CXR: miliary/interstitial infiltrate, localized/nodular infiltrates less common

CMV Pneumonia

CLINICAL MANIFESTATIONS IMMUNOCOMPROMISED

Transplant

patients: Interstitial pneumonitis, GI disease, retinitis, hepatitis, encephalitis, and CMV syndrome HIV patients Retinitis and CNS involvement are common

CMV Cecal Ulcer

CMV Esophagitis www.vh.org/

CLINICAL MANIFESTATIONS CONGENITAL/PERINATAL

Clinical Findings Petechiae/Purpura(7176%) Jaundice (67%) Hepatosplenomegaly (60%) Microcephaly (53%) IUGR (50%) Retinitis Cerebral calcifications Hepatitis Non-immune hydrops Laboratory Findings www.med.nagoya-u.ac Elevated LFTs (83%) Hyperbilirubinemia (81%) Thrombocytopenia (77%) Elevated CSF protein (77%) Long Term Sequelae Hearing loss Mental retardation

CLINICAL MANIFESTATIONS
CONGENITAL/PERINATAL

CLINICAL MANIFESTATIONS
CONGENITAL/PERINATAL
Ventriculomegaly and Periventricular Calcifications

LABORATORY DIAGNOSIS
Demonstration

of cytomegalic cells in centrifuged deposits from urine or saliva Serology: CFT IHA IF ELISA

DIAGNOSTICS
Conventional

cell culture

Shell

vial culture

CMV

antigenemia (pp65)
methods

Cytomegalovirus-infected human diploid fibroblast cells in culture. Modified acridine orange staining

Molecular

(PCR))

DIAGNOSTIC STUDIES

Shell vial culture Early antigen detection with monoclonal antibodies Viremia has been shown to be a risk factor for CMV pneumonia in patients who have received allogenic marrow transplants Shell vial assay reduced identification time to 24-48 hours Monitoring of the shell vial assay prior to the onset of disease Practical method for starting early antiviral treatment Uses permissive cell line for CMV Centrifuged at a low speed and placed in an incubator After 24 and 48 hours, the tissue culture medium is removed and the cells are stained using a fluorescein-labeled anti-CMV antibody The cells are read using a fluorescent microscope Alternatively, the cells are stained with an antibody against CMV, followed by a fluorescein-labeled anti-Ig. Not as sensitive as traditional tissue culture

DIAGNOSTIC STUDIES

CMV antigenemia (pp65)

forums.gardenweb.com

Antigenemia: relatively new test developed in the late 1980s Recognition of CMV early antigen by a mixture of 2 mouse monoclonal antibodies, C-10 and C-1 The detector system is fluorescein-labeled anti-mouse Ig Cells are counted using a fluorescent microscope Any positive cell confirms the diagnosis of CMV viremia The literature has suggested that a higher cell count corresponds to risk of disease. Antigenemia has been used to predict CMV pneumonia in patients who have received transplants A positive antigenemia test result can be used as a trigger to institute ganciclovir therapy when a preemptive strategy is used for the prevention of CMV disease in transplant patients Available in 24 hours

DIAGNOSTIC STUDIES
Molecular

methods (PCR) Qualitative polymerase chain reaction PCR has been used to detect CMV in blood and tissue samples The PCR test depends on the multiplication of primers specific for a portion of a CMV gene Primers usually bind to the area of virus that codes for early antigen The test is extremely sensitive Positive before the antigenemia test in patients with viremia who have received transplants. The PCR test result is not usually positive in patients without CMV viremia. Qualitative PCR has also been used to detect CMV in histological sections

DIAGNOSTIC STUDIES

Molecular methods (PCR) Quantitative polymerase chain reaction Quantitative PCR has been used to detect plasma CMV Quantitative PCR is as sensitive as qualitative PCR and provides an estimate of the number of CMV genomes present in plasma Research Determine if the number of CMV genomes present in the plasma correlates with risk of disease in different at-risk populations Number of CMV genomes (ie, viral load) present would indicate whether therapy is necessary because patients below a certain cut-off would not develop CMV disease However, the level of viremia necessary for CMV disease to occur may vary depending on host factors and the type of organ transplant, and this may need to be determined empirically Literature from different organ transplant systems suggests that this method may be the test that discriminates between low-level viremia versus a higher level and CMV disease

MATERNAL DIAGNOSIS
CONGENITAL CMV
Serum

tested 2-4 weeks apart IgG seroconversion or fourfold increase (ie. 1:4 to 1:16) of IgG IgM useful but not always reliable sign of primary infection May persist for months Appears in reinfection False positive if RA >30% of IgG value may suggest active infection

FETAL DIAGNOSIS
CONGENITAL CMV Ultrasound findings Abdominal and liver calcifications Echogenic bowel Ascites Hepatosplenomegaly CNS involvement poorer prognosis CMV detected in amniotic fluid by culture or PCR Culture sensitivity: 50-69% PCR sensitivity: 77-100% Combined sensitivity: 80-100% Sensitivity of amniotic fluid testing markedly lower if performed before 21 weeks GA

TREATMENT
Ganciclovir

Targets UL54 protein Valganciclovir Foscarnet Cidofovir Nucleoside analogue Treatment not usually indicated in immunocompetent patients Indications Treatment of disease in immunocompromised: Retinitis, GI disease, pneumonitis, neurologic disease,

PREVENTION

Behavioral

Modification Avoidance of infectious saliva, urine, bodily fluids and high risk behaviour www.med.nagoya-cu.ac Careful hygiene practices

VACCINES
Live

attenuated vaccine developed (Plotkin 1991) Largest trial: Towne 125 strain Partial efficacy Economically beneficial Concerns Reactivation and infection of host Viral shedding from cervix or breast milk Possible oncogenic potential of vaccine virus

Glycoprotein

vaccine (Bourne 2001) Reduced in-utero CMV transmission Improved pregnancy outcome

EPSTEIN BARR VIRUS

DISCOVERY OF EBV
1958-

Burkitt's lymphoma described in the malaria belt of east Africa Epstein and Barr discover EBV through electron microscopy of cells cultured from Burkitt's lymphoma tissue EBV demonstrated as the etiological agent of infectious mononucleosis

1964-

1968-

EBV VIRAL STRUCTURE

A

core containing a linear, dsDNA molecule of about 175 kbp. An icosahedral capsid, approximately 100-110 nm in diameter, containing 162 capsomeres with a hole running down the long axis. An amorphous, sometimes asymmetric material that surrounds the capsid, designated as the tegument An envelope containing viral glycoprotein spikes on its surface.

PRIMARY INFECTION DISEASES

Infectious Mononucleosis (glandular fever) - fever, lymphadenopathy, and pharyngitis

Chronic active EBV infection - severe illness of more than six months, histologic evidence of organ disease, and demonstration of EBV antigens or EBV DNA in tissue (mimics chronic fatigue syndrome)
X-Linked Lymphoproliferative Disease inherited disease of males, absence of functional SAP gene impairs the normal interaction of T and B cells resulting in unregulated growth of EBV-infected B cells.

DISEASES RESULTING FROM EBV IN

REDUCED IMMUNITY PATIENTS
PTLD

(Post-transplant lymphoproliferative disease) -a tumor often found in organ transplant patients Oral Hairy Leukoplakia Nonmalignant hyperplastic lesion of epithelial cells
Oral Hairy Leukoplakia

CANCERS ASSOCIATED WITH EBV

Nasopharyngeal

Carcinoma Southern China, Northern Africa, and Alaskan Eskimos Elevated titers of IgA antibody to EBV structural proteins Burkitt's Lymphoma Found in equitorial Africa and associated with malaria which doesnt allow T-cells to control proliferation of EBV-infected B cells Tumors present in jaw Hodgkin's Disease EBV DNA detected in tumors Lymphoproliferative Disease Impaired T-cell immunity and cannot control proliferation of EBV-infected B cells

SITE OF INFECTION
Infection

of Epithelial Cells by EBV in vitro Active replication, production of virus, lysis of cell Infection of B cells by EBV in vitro Latent infection, with immortalization (proliferate indefinitely) of the virus-infected B cells Linear EBV genome becomes circular, forming an episome, and the genome usually remains latent in these B cells Viral replication is spontaneously activated in only a small percentage of latently infected B cells. Signal transduction pathways can reactivate EBV from the latent state

REPLICATION
EBV

replicates in the epithelial cells of the mouth, tongue, salivary glands, and oral cavity EBV infects B cells in the lymph nodes of the oral cavity Once inside B cells, EBV expresses proteins Nucleus: EBNA (Epstein-Barr Virus Nuclear Antigens) Plasma Membrane: LMP (Latent Membrane Proteins) Expression of these proteins stimulates B cell replication in lymph nodes producing clones Since many B cells are infected, polyclonal B-cell growth occurs which allows the disease to begin a long time after initial exposure to EBV

INFECTION AND REPLICATION

MODES OF TRANSMISSION

Intimate

Contact kissing, sharing food, coughing

IMMUNE SYSTEM TO THE RESCUE! (OR NOT)

Epithelial cells and polyclonal B cells express the viral-encoded LMP glycoprotein in their plasma membranes Killer T cells recognize the LMP glycoprotein and kill the EBV-infected cells While T cells are mounting an attack on B cells, the immune response of a person is abnormal producing atypical T cells and antibodies that can confirm diagnosis of infectious mononucleosis

IMMUNE SYSTEM TO THE RESCUE!

The ability of EBV to persist, despite potent immune effector responses against it, indicates that the virus has evolved strategies to elude the immune system.

SYMPTOMS
The classic triad of mononucleosis are Inflammation of the pharynx (or tonsils) usually severe Fever (higher in the evening) Lymphadenopathy (usually in the neck, groin or under the arms)

SYMPTOMS
Other symptoms include: Fatigue and malaise Rash (associated with the use of ampicillin) Headache Abdominal pain Occasional jaundice Enlargement of the spleen and liver

DIAGNOSIS OF EBV
Clinical

diagnosis- Classic triad of symptoms lasting 1-4 weeks Serologic test- Shows elevate white blood cell count, an increased number of lymphocytes, greater than 10% atypical lymphocytes Someone who appears to have infectious mononucleosis,a Paul-Bunnell test can be done Serology IF ELISA EBNA - Marker

COMPLICATIONS AND SYMPTOM ALLEVIATION

A

ruptured spleen (rare) Splenectomy anemia (steroid usage)

Hemolytic Airway

obstruction due to enlarged tonsils (steroid usage) platelet production, hypersplenism, or severe anemia (transfusions)

Decreased

TREATMENT OF EBV
Infectious

Mononucleosis No specific therapy just nonaspirins and rest Hairy Leukoplakia Acyclovir inhibits EBV replication Lymphoproliferative Disease reduce dose of immunosuppressive medication Surgical removal or irradiation of localized lymphoproliferative lesions

Oral

EBV

PREVENTION AND VACCINES

EBV

lymphoproliferative disease infusion of B-celldepleted marrow to offset the proliferation of donor B cells Removal of donor B cells along with T cells

No

vaccine found so far, though vaccine studies are underway

SUMMARY
As

many as 95% of adults between 35 and 40 years old have been infected. children become infected with EBV but do not usually show symptoms. EBV occurs during adulthood it causes infectious mononucleosis 35-50% of the time. lifelong, persistent infections - majority are benign

Many

When

Causes

HHV 6
Ubiquitous,spreads through saliva in early infancy Variants A and B Variant B is the cause of common childhood illness Exanthum subitum In old age group it is associated with infectious mononucleosis syndrome, focal encephalitis In immunodeficient it is associated with pneumonia and disseminated disease

HHV 7
Transmitted

through saliva

Shares CD4 receptor on T cells and thus contributes to furthur depletion of CD4 T cells in HIV infected patients

HHV 8
At

first Isolated from Kaposi sarcoma lesions in patients, of known HIV status
was later found in Kaposi sarcoma lesions in patients that were HIV negative

It

PARVO VIRUS

Parvovirus structure

From Medical Microbiology, 4th ed., Murray, Rosenthal, Kobayashi & Pfaller, Mosby Inc., 2002, Fig. 53-1.

Autonomous parvovirus replication

From Medical Microbiology, 5th ed., Murray, Rosenthal, Kobayashi & Pfaller, Mosby Inc., 2002, Fig. 56-2.

Helper dependent parvovirus (AAV) replication

Infection with adenovirus Infection without adenovirus

Lytic replication

AAV DNA integrates into chromosome 19

Superinfect with adenovirus

Parvovirus pathogenesis

From Medical Microbiology, 5th ed., Murray, Rosenthal & Pfaller, Mosby Inc., 2005, Fig. 56-5.

PARVOVIRUS B19
Structure

Small (5 kb) linear ssDNA genome, naked capsid,B19

Pathogenesis

respiratory transmission replication in nucleus, host dependent, needs helper virus Presents as respiratory infection with an erythematous maculopapular rash and arthralgia Erythema infectiosum starts with prominent erythema of cheeks spreading to trunk and limbs followed by lymphadenopathy and arthralgia

PARVOVIRUS B19
It occurs in young children and is called fifth disease Transient aplastic crisis in children with chronic hemolytic anaemias Infection during second or third trimester may result in hydrops fetalis

Diagnosis

serology, viral nucleic acid

Treatment/prevention

None

Parvovirus pathogenesis

From Medical Microbiology, 5th ed., Murray, Rosenthal & Pfaller, Mosby Inc., 2005, Fig. 56-3.

PAPOVA VIRUS
Pa papilloma virus of human beings Po - polyoma virus of mice Va vaculating virus of monkey Small , non enveloped ,DNA viruses

SIMIAN VACULATING VIRUS 40 (SV 40)

Isolated from uninoculated rhesus and cyenomologus monkey kidney tissue cultures Produces prominent cytoplastic vaculation when inoculated into kidney cell cultures Oncogenic for newborn hamsters Medical importance in preparation of live viral vaccines Live viral vaccines should be manufactured in monkey kidney tissue cultures tested and found free from SV 40 infection

PAPILLOMA VIRUS- SPECIES SPECIFIC

Human

papilloma virus infections only humans Grow only in organ cultures of human skin >70 types have been recognized based on genetic homology Serotypes 1,2,3,4 verrucus vulgaris (common warts ) children and adolescents on hands and feet

PAPILLOMA VIRUS
Serotypes

6, 11-Condylomata acuminata or genital warts moist soft, pedunculated found on external genitalia,Transmitted venereally,may occasionally turn malignant Serotypes 6,11-intraepithelial neoplasia Serotypes16,18-more severe invasive malignancies ca cervix and ca uterus

HUMAN PAPOVA VIRUSES

Poduce

malignancies in patients with impaired immunity JC virus- isolated in 1971 from a patient with hodgkins disease and progressive multifocal leucoencephalopathy(PML) Reported from USA ,UK , and FRANCE Grows only in human fetal glial cell cultures Highly oncogenic Malignant gliomas on inoculation into newborn hamsters .

BK VIRUS
Isolated

in 1971 from urine of a patient who underwent a kidney transplant Differs from JC virus Grows in a wide range of primary and continuous cell cultures Less oncogenic

BK AND JC VIRUSES
Primary

infection in child hood

Multiplies in brain and renal tract

Reactivation

from latent infection during immunodeficiency states