Monday, October 7, 2013

Simplified virology

Clinical virology Definition of virus Virus is a small infectious agent (10-30 nm) in diameter. It is metabolically inert, since it cannot replicate outside of a living cell I,e needs a living media (tissue culture) for isolation. Because viruses are non-motile, they are entirely dependent on external physical factors for chance movement and spread to infect other susceptible cellsIt is composed of nucleic acid either DNA or RNA and a capsid. Some viruses acquire envelope from the host. CLASSIFICATION Viruses are broadly classified primarily upon the type of genomic nucleic acid, eg. DNA or RNA , and then further by the number of strands of nucleic acid (eg. double-stranded DNA, double-stranded RNA or single-stranded RNA, with a positive or negative "sense" of that single strand). Retroviruses are a special category of RNA viruses that require reverse transcription of their RNA to DNA and then integration of that DNA into the host cell genome before replication can take place. They carry a reverse transcriptase enzyme as part of the virion. A) DNA viruses  -Adenovirus, Upper and lower respiratory infections, hemorrhagic cystitis, keratoconjunctivitis.  -Herpes Viruses • Human herpesvirus 1 (herpes simplex virus type 1) • Human herpesvirus 2 (herpes simplex virus type 2) These cause: gingiva stomatitis - vesicles and ulcers in the mouth herpes genitalis - vesicles and ulcers on genitalia herpes labialis (cold sores, fever blisters) - vesicles and ulcers of lips herpes gladiatorum - clusters of vesicles and ulcers on skin encephalitis keratoconjunctivitis whitlow (felon) a purulent infection involving the pulp of the distal phalanx of a finger • Human herpesvirus 3 (varicella-zoster virus) - this virus causes:  chickenpox (varicella) - epithelial cell infection resulting in an exanthem of macules, papules, pustules, vesicles and shallow ulcers  shingles (zoster) - peripheral nerve cell infection with an eruption in the overlying epidermis • Human herpesvirus 4 (Epstein-Barr virus) - This virus causes:  infectious mononucleosis (b-lymphocyte infection)  Burkitt's lymphoma  oropharyngeal carcinoma • Human herpesvirus 5 (cytomegalovirus) - this is the agent of:  cytomegalovirus mononucleosis - similar to, but milder than, infectious mononucleosis  cytomegalic inclusion disease (salivary gland disease) - a generalized and often fatal disease of the newborn • Human herpesvirus 6 (human b-lymphotrophic virus) - causes exanthem subitum (fourth disease, Duke disease or roseola infantum and possibly multiple sclerosis) • Human herpesvirus 8 - causes Kaposi's sarcoma in AIDS patients • Human herpesvirus 7 - causes a cryptic infection of the T-helper cell  -EBV---Mononucleosis, associated with Burrkitt s lymphoma and nasopharyngeal carcinoma.  -HBV—Hepatitis B  -B19 virus - causes fifth disease (erythema infectiosum), bone marrow aplasia and polyarthralgia. Target tissue is the erythroid cell B) RNA viruses  Influenza virus---Influenza  Measles virus---measles  Mumps virus---mumps and orcitis  Rubella virus---Rubella and congenital malformation as cardio vascular and neurological.  Parainfluenza-----Bronchiolitis in infants and croups inyoung children  RSV------Bronchiolitis and pneumonia in infants  HIV---AIDS  HCV----Hepatitis C and complications  HDV----hepatitis  HAV---Hepatitis A in children  Coxsachie viruses------aseptic meningitis, myocarditis and pericarditis  Rota virus----Diarrhea in young children  Rhinovirus------common cold Laborator Diagnosis OF VIRUSES Collection of specimens for viral disease diagnosis: -Swabs of the lesion sites and transport in viral transport media -Aspiration of secretions or exudates -Excreta such as urine or stool. -Biopsy samples obtained by needle aspirations, open exploration or endoscopy. -Blood samples for antigen detection of some viruses, for serological tests, and for PCR. Diagnosis of viral infections Viruses can be studied in a number of direct and indirect ways and all these methods can be applied in a diagnostic situation, ie. is this patient infected with a particular virus? There are two approaches: Detection and demonstration of the virus itself; and The study of the host's response to that virus Detection of virus : 1-Culture. II-Detection of viral antigen III-Detection of viral DNA or RNA. 1-Culture. The types of cell culture are -Monolayer cultures Primary Semiconscious Continuous line - Tissue culture -Organ culture Detection of viral growth by 1-Characteristic cytopathic effect. 2-Haemadsorption (attachment of RBCS to the surface of virus infected cells) Some viruses (eg. myxo- and paramyxoviruses, including influenza) have the property of haemagglutination (causing red blood cells to stick together ) which can be used to detect and quantitate the virus (by haemagglutination). 3-Interference with the formation of cytopathic effect e.g neutralisation of viral infectivity by antibodies can be used to detect and quantify either virus or specific antibody to that virus. 4-Adecrease in acid production by infected and dying cells. 5- Recent method depend on use of fluorescent antibodies to the infective virus antigen, or by use of enzyme linked immunosorbant assay (ELISA). II-Viral antigens can be detected by a wide range of serological techniques utilizing polyclonal or monoclonal antibodies. Techniques include precipitation, agglutination, immunofluorescence, ELISA, complement fixation and radio immuno assays. III-Molecular techniques of both protein chemistry and nucleic acid biochemistry have greatly improved the specificity of virus diagnostic procedures. Methods include:- • polyacrylamide gel electrophoresis (PAGE) of protein fragments • western blotting, and identification of specific proteins with labelled probes • polymerase chain reaction (PCR), to amplify specific segments of viral nucleic acid • Southern blotting, and DNA hybridisation with labelled probes • sequencing of portions of the viral genome • restriction fragment length polymorphisms of viral nucleic acid VIRAL HEPATITIS The term VIRAL HEPATITIS is usually used to describe infections caused by agents whose primary tissue tropism is the liver. To date, at least five hepatitis viruses have been recognised, and these have been named:- Hepatitis A, B, C, D and E. Acute hepatitis may also occur as part of the clinical course of a number of viral infections, including human cytomegalovirus, Epstein-Barr virus, herpes simplex virus, yellow fever virus and rubella. Viral hepatitis can be classified according to mode of transmission ENTERICALLY TRANSMITTED HEPATITIS: A and E PARENTERALLY TRANSMITTED HEPATITIS B , C , D and G Hepatitis A Hepatitis B Hepatitis C Virus HAV(RNA) member of picornavirus HBV(DNA) from Hepadna virus HCV(RNA) from Togavirus related to the Flavi and Pesti viruses Incubation peroid 2-6 weeks 6 weeks-6 months 1-3 months Mode of transmission Faeco-oral Parenreral,sexual,vertical Parenteral Clinical course Short-mild Prolonged and more severe than A Mild, chronic in 50% of cases Carrier & Chronicity No Yes Yes Laboratory diagnosis Elevated ALT,AST up to 10 folds Virus cannot be cultured in vitro from clinical material, and diagnosis is made on the presence of HAV-specific IgM in the patient's blood. Elevated ALT,AST from 10-100 folds Acute infection with resolution Viral antigens: 1) Surface antigen (HBsAg) is secreted in excess into the blood as 22 nm spheres and tubules. Its presence in serum indicates that virus replication is occurring in the liver 2) 'e' antigen (HBeAg) secreted protein is shed in small amounts into the blood. Its presence in serum indicates that a high level of viral replication is occurring in the liver 3) core antigen (HBcAg) core protein is not found in blood Antibody response: 1) Surface antibody (anti-HBs) becomes detectable late in convalescence, and indicates immunity following infection. It remains detectable for life and is not found in chronic carriers (see below). 2) e antibody (anti-HBe) becomes detectable as viral replication falls. It indicates low infectivity in a carrier. 3) Core IgM rises early in infection and indicates recent infection 4) Core IgG rises soon after IgM, and remains present for life in both chronic carriers as well as those who clear the infection. Its presence indicates exposure to HBV. Chronic hepatitis B Persistance of surface antigen and prolonged persitance of e antigen. Mild elevation of ALT,AST With fluctuation in AST(surrogate Marker of chronic hepatitis C). 1) Serology . 1-HCV-specific IgG indicates exposure, not infectivity 2) PCR detects viral genome in patient's serum 3) Quantitative PCR to detect viral load response to therapy. Hepatitis E Recently identified cause of enterically transmitted non-A, non-B (NANB) hepatitis Clinical Features Incubation period 30-40 days Acute, self limiting hepatitis, no chronic carrier state Age: predominantly young adults, 15-40 years Complications Fulminant hepatitis in pregnant women. Mortality rate is high (up to 40%). Virus cannot be cultured in vitro. 1) Calicivirus-like particles in the stool, by electron microscopy 2) Specific IgM in serum 3) PCR HEV-specific sequences in stool Delta Agent Defective virus which requires Hepatitis B as a helper virus in order to replicate. Infection therefore only occurs in patients who are already infected with Hepatitis B. Clinial Features Increased severity of liver disease in Hepatitis B carriers. Hepatitis G (HGV) A virus originally cloned from the serum of a surgeon with non-A, non-B, non-C hepatitis, has been called Hepatitis G virus. It was implicated as a cause of parenterally transmitted hepatitis, but is no longer believed to be a major agent of liver disease. It has been classified as a Flavivirus and is distantly related to HCV. HIV 1 and 2 Human Immunodeficiency Viruses Transmission Infection is transmitted in a manner identical to that of hepatitis B. 1.) Blood products • Blood transfusions • Intra-venous drug abusers - sharing of needles • Health care workers: needlestick injuries - risk approximately 0.36% (depends on extent of the injury) muco-cutaneous exposure - no sero-conversion incidents have been reported 2.) Organ transplants 3.) Sexual intercourse Both homosexual and hetero-sexual exposure Increased risk of transmission if partners have other sexually transmitted diseases 4.) Vertical Transmission 10-40% of babies born of HIV-infected mothers will be infected. Infection may occur in utero during birth post-natally, through breast feeding Primary infection About 90% of patients develop a flu-like illness which co-incides with seroconversion, between 2 and 4 weeks post exposure. Symptoms include, fever, night sweats, sore throat, lymphadenopathy, diarrhoea. The illness is self limiting. Asymptomatic phase Of variable duration, from 2 to 10 years. Patients are clinically well, but infectious. Prodromal phase This period is heralded by the insidious onset of a variety of prodromal disorders, including: weight loss, fever, persistant lymphadenopathy, oral candidiasis and diarrhoea. These symptoms precede the progression to AIDS. Acquired Immunodeficiency Syndrome (AIDS) Syndrome with the following features: 1) Constitutional disease: fever, diarrhoea, weight loss, skin rashes 2) Neurological disease: dementia, myelopathy, peripheral neuropathy 3) Immunodeficiency: Increased susceptibility to opportunistic infections: (see table 1) 4) Rare malignancies: Kaposi sarcoma, oral hairy leukoplakia, lymphomas. Kaposi sarcoma - is a tumour of endothelial cells. Prior to the AIDS epidemic, this tumuor was rare and only found in middle aged African and Mediterranean Jewish men, in whom it was an indolent condition. AIDS patients develop a disseminated highly aggressive form of the disease. Paediatric Infection Following infection in the perinatal period, babies may develop a progressive illness in the first few months of life (No latent period). Clinical features include: Failure to thrive, diarrhoea, lymphadenopathy, susceptibility to opportunistic infections hepato-splenomegaly, lymphoid interstitial pneumonia and parotitis. Pathogenesis HIV infects CD4 cells Disseminated infection Specific immune Response Antibody Cell mediated immunity Clearance of most virus Some persistence a) Gradual loss of CD4 cells b) Destruction of microenvironment of lymphoid tissue IMMUNO-DEFICIENCY Cell tropism: CD4+ T cells, Macrophages Destruction of CD4 cells occurs through the following mechanisms: direct lysis of infected cells syncytium formation Infection of precursor cells and destruction of microenvironment Impairment of CD4 cell function LABORATORY DIAGNOSIS Serology IgG develops 4-6 weeks post exposure and remains detectable for life. Its presence in serum therefore indicates infection. Exception: Uninfected infants of HIV positive mothers Direct detection of virus p24 antigen ELISA culture from PBMC's PCR VACCINE PROSPECTS There is no effective vaccine available for HIV. Attempts have been made to develop a vaccine, using: • purified viral envelope glycoproteins, gp120 or 160 • whole inactivated virus • live attenuated HIV strains (lacking certain genes) • live recombinant virus vectors, expressing HIV proteins. A major difficulty is the fact that neutralizing antibody in the serum does not protect the host from infection with HIV: Possible reasons for this include: • Antibody enhancement of infection • Rapid virus mutation may result in variation of envelope antigens (escape mutants) • HIV can infect cells in sites that are sequestered from antibody • Host may be infected by whole virus-infected cells

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