Cytomegalovirus Presentation

CYTOMEGALOVIRUS Introduction History Classification Structure Replication Epidemiology • Pathogenesis • Immune responses • Clinical manifestations • Diagnosis • Treatment • Prevention Introduction • Cytomegalovirus is a double-stranded DNA virus belonging to the β herpes virus & is the largest member of the human herpesvirus. • Infection with CMV is common in all human population, reaching 60 – 70 % in U.S & nearly 100% in some parts of Africa. • Causes asymptomatic illness in normal hosts & multi-organ disease in immunocompromised & transplant recipients. Introduction • • • CMV has the ability to establish latent infection. Primary or secondary infection can occur. May directly or indirectly contribute to chronic inflammatory disease & cancer. History • Cytomegalovirus was originally called human salivary gland virus or cytomegalic inclusion disease virus. • Human CMV was isolated independently in 1956, by Rowe, Smith, Weller & their collegues. – Rowe – Adenoids of normal children – Smith – Salivary gland of a child with disseminated disease. – Weller – Liver biopsy specimen from a child with a congenital infection syndrome. Classification • CMV belongs to the family Herpesviridae, subfamily Betaherpesvirinae. • Minor genetic & antigenic differences exist among human CMV isolates, but many evidences suggest that all human CMV strains are closely related. Structure • Human CMV is the largest member of human herpesvirus measuring 130 nm in diameter. • The virion consists of an icosahedral capsid encasing a 235 kbp linear genome, surrounded by a tegument & enveloped in a lipid bilayer carrying a number of virus-encoded glycoproteins. • The genome consists of linear DNA with inverted repeats. Structure Structure • Three types of viral particles are produced in CMV infected cells: – Virions – Non-infectious enveloped particles: Lack electron dense DNA core. – Dense bodies: Composed of several tegument proteins enclosed in an envelope. Lack both nucleocapsid & viral DNA. Capsid • Three capsid forms: – Type A: Lack DNA. – Type B: Precursors of mature capsids that lack DNA, but contain viral assembly protein. – Type C: Mature nucleocapsids. Capsid proteins • The capsid is composed of 7 proteins: – Major capsid protein (MCP) – Minor capsid protein (mCP) – Minor capsid binding protein (mC-BP) – Smallest capsid protein (SCP) – 3 Assembly proteins (AP) Envelope • Envelope glycoproteins: • Glycoprotein B (gB) – Major heparan sulfate proteoglycan-binding glycoprotein • Glycoprotein H, L & O (gH/gL/gO) – Involved in membrane fusion. • Integral membrane protein • Lipid bilayer : derived from nuclear & cytoplasmic membrane. Tegument • Tegument proteins are conserved among Betaherpesviruses. • At least 25 proteins are located in the tegument. • All are phosphorylated & highly immunogenic. – pp65 or lower matrix protein: Associated with dense bodies & used in antigenemia assays. – pp150 or basic phosphoprotein: Incorporated preferentially into virions & used in recombinant protein based serological assays. Genome • The genome consists of linear double-stranded DNA with inverted repeats. • The human CMV genome has two unique components, UL & US, flanked by inverted repeats. Replication • Host range: – Human CMV replicates only in human cells. – A laboratory-attenuated strain (Towne) has been grown in primary chimpanzee skin fibroblasts. • Host cells: – Human fibroblast cells (MRC-5) – Fibroblasts, epithelial cells, macrophages, smooth muscle & endothelial cells support replication. Replication Attachment to heparan sulfate proteoglycans via envelope glycoproteins. Conformational change resulting in high affinity binding. Penetration mediated by gH/gL/gO complex. Transportation to the nucleus. Replication Transcription & viral replication Assembly of the progeny viral particles. Incorporation of the viral glycoproteins in the nuclear membrane. Progeny nucleocapsids bud through the nuclear membrane. Final envelopment may occur at cytoplasmic membrane when the progeny viruses are released from the cell. Replication Epidemiology • CMV has a worldwide distribution. • In developed countries 40% of adolescents are infected and ultimately 70% of the population is infected. • In developing countries, over 90% of people are infected. • CMV infection occurs in 50 – 70 % of organ transplant recipients. Epidemiology • Source of infection is virus-infected body fluids (urine, genital secretions, saliva & blood) • Virus is readily transmitted by blood products & organs. • Transmission occurs in 30 – 70 % of infants breast fed by seropositive mothers. • Sexual route is the most common route of transmission in adolescents & adults. Epidemiology • Patients with immunosuppression are at increased risk. • Congenital CMV infection can occur in neonates. – Primary gestational infection: 35-50% transmission – Reactivation or reinfection: 1-3% transmission • Multiple infection can occur with genetically different strains. Pathogenesis • CMV is efficiently transmitted by direct contact. • Primary infection typically starts with replication in mucosa. • A systemic phase disseminates virus in the host via a leukocyte associated viraemia lasting for several months accompanied by persistent viral shedding in various body fluids. Pathogenesis • Primary infection is controlled by a combination of innate & adaptive immune responses, after which latent infection continues for life. • Myeloid-lineage cells become targets for lifelong latency. • The pathogenesis is due to lytic viral shedding resulting from a failure of host immunity to control effectively viral replication & spread. Pathogenesis • CMV also exerts its pathogenic potential through non-lytic mechanisms. These include: – Down regulation of of MHC – I protein expression reducing the impact of CTL immunity. – Modulation of normal host cell function. – Impairement of neighboring cellular functions through induction of chemokines & cytokines. – Host derived immunopathological responses resulting in significant organ damage. • CMV infection induces immunosuppression, with low CD4+ cells. Pathogenesis • Pathological findings include evidence of virus infection in almost all organ systems. • Histological findings include large refractile cells (cytomegalic) with so called owl’s eye intranuclear inclusions. Immune responses Cell mediated immunity • Individual with deficiency of cellular immunity exhibit highest risk for CMV disease. • Both NK cells and cytotoxic T lymphocytes play prominent role in immune clearance. • Clearance from particular organs, such as salivary glands, is dependent on a CD4+ T helper cell response. Cell mediated immunity • Cellular immune responses appear to be directed primarily at the protein component of virion tegument (pp65 & pp150) • Role of cytokines in cellular responses against CMV is unknown. Humoral immunity • Humoral antibody response is critical in some settings such as in neonatal disease and solid organ transplantation. • Virus neutralising antibody assays correlate well with patient outcome. • Virus neutralising antibodies are directed against gB, gH, gL and viral tegument (pp65 & pp150). Clinical manifestations • Normal hosts: Infectious mononucleosis syndrome • At risk groups: Invasive CMV disease. – Newborn infant infected in utero – Immunocompromised allograft recipients – Patients with AIDS. Cytomegalovirus mononucleosis • 20 – 50% of heterophile negative infectious mononucleosis is due to CMV. • Clinical syndrome: – Fever – Hepatitis – Atypical lymphocytosis • Fever is predominant, tonsillitis & pharyngitis are rare, enlargement of lymph nodes & spleen is not a prominent feature. • Mild hepatitis is an important clue. Cytomegalovirus mononucleosis • Complications: – – – – – – – Interstitial pneumonia Hepatitis Guillain-Barre syndrome Meningoencephalitis Myocarditis Thrombocytopenia & hemolytic anaemia Skin eruptions Complications Congenital CMV infection • Intrauterine CMV infection occurs in 0.5 – 22% of all live births. • Symptoms: Cytomegalic inclusion disease – – – – – Jaundice Hepatosplenomegaly Petechial rash Multiple organ involvement CNS: microcephaly, chorioretinitis, motor disability and cerebral calcification. • Neurological sequelae & hearing disorders persist. Congenital CMV infection • Mortality rate ranges from 11 – 20 %. • Perinatal infection is more common during late stages of pregnancy and during breast feeding. Usually asymptomatic or may present with subtle hearing loss. Allograft recipients • Infection can develop following reactivation of latent infection in the recipient or infection by the virus present in the donor organ. • Use of steroids associated with increased risk. • CMV pneumonia in transplant recipients is fatal. • Transplant vasculopathy/ vascular sclerosis can occur. CMV infection in AIDS patients • CMV is the most common viral opportunistic infection in AIDS patients. • Gastrointestinal tract, retina and brain are the common sites involved. • CMV retinitis occur when CD4 cell count falls to less than 50 cell/ mm3. CMV retinitis Laboratory diagnosis • Diagnosis almost always depends on laboratory confirmation & clinical diagnosis cannot be made. Specimens • Blood, bronchoalveolar lavage, urine or other body fluids and/ or biopsy tissues Microscopy 1. Cytomegalic cells – Demonstration of enlarged infected cells with prominent intranuclear inclusions in centrifuged urine or saliva. 2. Histopathology and immunochemistry in biopsy materials are commonly used but the sensitivity & specificity are not well documented. Histopathology Antigen detection • CMV antigenemia test – Direct detection of antigens in neutrophils by the use of monoclonal antibodies against the CMV matrix protein pp65. – It can be used to predict the likelihood of invasive disease in transplant & AIDS patients. – It can detect CMV antigen in spinal fluid & in blood. – It is less sensitive than PCR. CMV pp65 antigenemia test Viral isolation • Conventional cell culture: Human diploid fibroblasts (MRC-5 cells). – Recovery may be augmented by blind passage. – Cytopathic effect consists of foci of enlarged, refractile, oval to round cells, that contain ‘ground glass’ appearing inclusions in the cytoplasm – Require upto 4 weeks to reveal cytopathology. Cytopathic Effect of CMV Viral isolation • Shell vial culture: – – Uses centrifugation to enhance infectivity followed by immunofluorescent staining for major immediate early nuclear antigen. Yield results within 24 hours. DEAFF test for CMV Serology • CFT, IHA, ELISA & latex agglutination test. • Detection of IgG is an evidence of infection, but does not indicate active viral shedding. • Measurement of CMV-specific IgM responses could distinguish between acute & past infection. – It is less sensitive & less specific. – Upto 40% with past infection have IgM antiCMV antibodies. • Measurement of anti-CMV IgG avidity can help distinguish between recent & past infection. Serology • Antibodies against non-structural proteins, particularly p52, is more characteristic of acute infection, whereas those against basic matrix phosphoprotein p150 is seen in individuals with long term seropositivity. Detection of viral nucleic acid • PCR is a highly sensitive method for screening. • PCR is more sensitive than culture for demonstration of CMV in CSF. • Presence of CMV DNA in AIDS patients could predict development of CMV retinitis. • Real-time PCR permit quantitation of viral burden, which helps prediction of invasive disease. Treatment • Drugs in current clinical use: – Ganciclovir – Foscarnet • Recently approved drugs: – Cidofovir – Fomivirsen Ganciclovir • It is a nucleoside analogue that inhibits viral DNA polymerase. • Ganciclovir is phosphorylated to ganciclovir monophosphate by viral enzyme, which is later converted to triphosphate by cellular enzymes. Ganciclovir • Uses: – Invasive infections in transplant recipients. – Retinitis & colitis in AIDS patients. – Congenital CMV infection. – Prophylaxis in bone marrow transplant recipients. • Resistance to ganciclovir is due to selection of mutants that are unable to phosphorylate ganciclovir. Foscarnet • It is a pyrophosphate analogue that directly inhibits viral DNA polymerase. • Useful in treatment of ganciclovir resistant strains in AIDS patients with CMV retinitis. • Foscarnet is associated with significant renal toxicity. Cidofovir • It is a nucleoside analogue. • It contains a phosphonate group & does not require phosphorylation by viral enzyme. • Causes significant nephrotoxicity. Fomivirsen • It is an antisense inhibitor. • Used for direct intravitreal injection for treatment of CMV retinitis. Preemptive Vs prophylactic therapy • Preemptive therapy: Detection of CMV DNA in plasma using PCR and initiating antiviral therapy to lower CMV DNA levels to prevent development CMV end-organ disease. • Prophylactic therapy: – AIDS patients with CD4 count less than 100 cells/ mm3. – First 100 days after transplantation. Prevention • Prevention by avoidance of exposure is generally not practical. • Donor selection or filtration of blood to remove contaminated leucocytes is highly effective in case of transfusion. • CMV immunoglobulin & prophylactic antiviral agents are the major approaches for prevention. • Serious CMV disease can be prevented in transplant recipients with antiviral therapy. Vaccines • Live attenuated vaccine (Towne strain) – 50% recipients develop humoral & cellular immunity – Less protective than natural infection. – Can’t be used in pregnant women & immunocompromised. • Subunit vaccine: – Envelope glycoprotein complex, gB (gpUL55), is being studied as a possible candidate. Indian studies • Seroprevalence of Cytomegalovirus among Voluntary Blood Donors in Delhi, India. J health popul nutr 2002 dec;20(4):348-351. – 95% of blood donors in India are seropositive for CMV. • Sheevani, *N Jindal, A Aggarwal. A pilot seroepidemiological study of cytomegalovirus infection in women of child bearing age. Indian Journal of Medical Microbiology, (2005) 23 (1):34-36 – Prevalence rate of 87.4%. Indian studies • C.T. Shoby et al. Human Cytomegalovirus Perinatal Infections in a Tertiary Care Setting. Indian Pediatrics 2002; 39:561-564 – The frequency of detection of HCMV infection was 11.7% by nested PCR among congenitally sick neonates. – To establish the HCMV association in suspected congenital infection, they recommend both nested PCR on urine and blood IgM testing of the baby taking any one parameter as definitive evidence. Indian studies • Cytomegalovirus co-infection in patients with HIV/AIDS in north India. Indian Journal of Medical Research, Mar 2003 by Mujtaba, S, Varma, S, Sehgal, S – 32.4% showed a positive reaction by PCR – CMV appears to be an important co-infection in patients with AIDS in India and PCR is a powerful tool for detection of CMV in blood • M. Rao et al. Cytomegalovirus Infection in a Seroendemic Renal Transplant Population: A Longitudinal Study of Virological Markers. Nephron 2000;84:367-373 – Quantitation of CMV DNAemia may play an important role in diagnosis. Indian studies • Pal SR, Chitkara NK, Krech V. Sero-epidemiology of cytomegalovirus infection in and around Chandigarh. Indian J Med Res 1972;60:973-8. • Madhavan HN, Prakash K, Agarwal SC. Cytomegalovirus infections in Pondicherry: a serological survey. Indian J Med Res 1974;62:297-300. • Mukundan P, Jadhav M, Jacob John T. Prevalence of cytomegalovirus antibody in young children in Vellore. Indian J Med Res 1977;65:589-92. References • Infectious diseases & their etiological agents – Mandell • Topley & Wilson • Gorbach’s Infectious diseases • Cohen’s Infectious diseases • Field’s virology • Textbook of microbiology – Ananthanarayanan & Paniker • Internet sources. Thank you