Virology: Cultivation and Isolation of Viruses - PDF

Microbiology Virology Cultivation and Isolation of Viruses Microbiology| Cultivation and Isolation of Viruses Contents : Herpes B virus 10 Varicella-zoster virus 16 Cytomegalovirus 25 Epstein-Barr virus 32 Human herpesvirus 6,7,8 36 Poxviruses 40 Papillomaviruses 49 Hepatitis Viruses 53 Parvoviruses...

Microbiology Virology Cultivation and Isolation of Viruses Microbiology| Cultivation and Isolation of Viruses Contents : Herpes B virus 10 Varicella-zoster virus 16 Cytomegalovirus 25 Epstein-Barr virus 32 Human herpesvirus 6,7,8 36 Poxviruses 40 Papillomaviruses 49 Hepatitis Viruses 53 Parvoviruses B19 58 Microbiology| Cultivation and Isolation of Viruses Survey of DNA viruses : Herpesviruses that commonly infect humans include: herpes simplex virus types 1 and 2 (HSV-1, HSV-2) varicella zoster virus (HSV-3) cytomegalovirus (CMV) (HSV-5) Epstein-Barr virus (EBV) (HSV-4) Note: Herpes B virus of monkeys can also infect humans. There are nearly 100 viruses of the herpes group that infect many different animal species. herpesviruses 6 and 7 herpesvirus 8 (Kaposi sarcoma-associated herpesvirus [KSHV]). Microbiology| Cultivation and Isolation of Viruses Structure and Composition : Herpesviruses are large viruses. It is an enveloped virus. Different members of the group share architectural details and are indistinguishable by electron microscopy (EM). All herpesviruses have a core of double- stranded (ds) DNA, linear, in the form of a toroid, surrounded by a protein coat that exhibits icosahedral symmetry and has 162 capsomeres. Microbiology| Cultivation and Isolation of Viruses The nucleocapsid is surrounded by an envelope that is derived from the nuclear membrane of the infected cell and contains viral glycoprotein spikes about 8 nm long. An amorphous, sometimes asymmetric structure between the capsid and envelope is designated the tegument. The enveloped form measures 150–200 nm; the ―naked virion, 125 nm. It causes latent and recurring infection. It is frequent reactivate in immunosuppressed host. Some cause cancer. It replicate in nucleus, bud from nuclear membrane. Microbiology| Cultivation and Isolation of Viruses Microbiology| Cultivation and Isolation of Viruses Overview of Herpesvirus Diseases: HSV-1 and HSV-2 infect epithelial cells and establish latent infections in neurons, recurrences are common. HSV-1 is classically associated with oropharyngeal lesions and causes recurrent attacks of ―fever blisters. HSV-1 cause oro-facial (cold sore). HSV-1 is the leading cause of sporadic encephalitis in children (in United States). HSV-2 primarily infects the genital mucosa and is mainly responsible for genital herpes. Both viruses cause neurologic disease and also can cause neonatal infections that are often severe. It spread by direct contact. Diagnosis of HSV by culture, EM. Microbiology| Cultivation and Isolation of Viruses HSV1 HSV2 Gingostomatitis, and Keratoconjunctivitis sicca (KCS) Genital Herpes Latent infections in trigeminal nerve as an episomal (plasmid) form Latent infections in ganglia of sensory nerves that supply the site of primary infection Acute Necrotizing Encephalitis Considered Sexually transmitted diseases (STDs). Eruption last 14 days Herpes B virus Microbiology| Cultivation and Isolation of Viruses Herpes B virus : Herpes B virus of macaque monkeys can infect humans. Such infections are rare, but those that occur usually result in severe neurologic disease and are frequently fatal. Human herpesviruses (HHV) are frequently reactivated in stress, UV, fever, immunosuppressed patients (e.g., transplant recipients, cancer patients) and may cause severe disease, such as pneumonia or lymphomas. Microbiology| Cultivation and Isolation of Viruses Herpesvirus infections in humans : HSV are extremely widespread in the human population. HSV-1 is spread by contact, usually involving infected saliva. HSV-2 is transmitted sexually or from a maternal genital infection to a newborn. HSV growth cycle proceeds rapidly, requiring 8–16 hours for completion. Microbiology| Cultivation and Isolation of Viruses Clinical Findings : HSV-1 and HSV-2 may cause many clinical entities, and the infections may be primary or recurrent. Primary infections occur in persons without antibodies and in most individuals are clinically in apparent but result in antibody production and establishment of latent infections in sensory ganglia. Recurrent lesions are common. Microbiology| Cultivation and Isolation of Viruses Laboratory Diagnosis of HSV : A. Polymerase Chain Reaction (PCR). B. Isolation and Identification of Virus: HSV is easy to cultivate, and cytopathic effects usually occur in only 2–3 days. The agent is then identified by neutralization test or immunofluorescence staining with specific antiserum. Microbiology| Cultivation and Isolation of Viruses C. Cytopathology: A rapid cytologic method is to stain scrapings obtained from the base of a vesicle (e.g., with Giemsa’s stain); the presence of multinucleated giant cells indicates that herpesvirus (HSV-1, HSV-2, or varicella-zoster) is present. D. Serology: Antibodies appear in 4–7 days after infection and reach a peak in 2–4 weeks. Detection methods available include neutralization, immunofluorescence, and enzyme-linked immunosorbent assay. Treatment Several antiviral drugs have proved effective against HSV infections, including: Acyclovir, Valacyclovir, and Vidarabine. Varicella-zoster virus (HSV-3) Microbiology| Cultivation and Isolation of Viruses Varicella-zoster virus (HSV-3) (VZV) : Varicella (chickenpox) is a mild, highly contagious disease, chiefly of children, characterized clinically by a generalized malaise, fever, vesicular eruption of the skin and mucous membranes. The disease may be severe in adults and in immunocompromised individuals. It is viremia. Microbiology| Cultivation and Isolation of Viruses The incubation period of typical disease is 10–21 days. Cases of congenital varicella syndrome after maternal cases of chickenpox during pregnancy have been described. Varicella pneumonia is rare in healthy children but is the most common complication in neonates, adults, and immunocompromised patients. It is responsible for many varicella related deaths. Microbiology| Cultivation and Isolation of Viruses Zoster (shingles) is a sporadic, incapacitating disease of elderly or immunocompromised individuals, therapy, or aging that is characterized by pain and a rash limited in distribution to the skin innervated by a single sensory ganglion. The lesions are similar to those of varicella. The trunk, head, and neck are most commonly affected. Zoster (vaccine was licensed in the United States in 2006) Microbiology| Cultivation and Isolation of Viruses Both diseases are caused by the same virus. Whereas Varicella is the acute disease that follows primary contact with the virus (primary infection), Zoster is the response of the partially immune host to reactivation of varicella virus present in latent form in neurons in sensory ganglia. Adults who are infected for the first time with varicella-zoster virus are apt/able to develop serious viral pneumonia. Microbiology| Cultivation and Isolation of Viruses Properties of the Virus : Varicella-zoster virus is morphologically identical to HSV. It has no animal reservoir. The virus propagates in cultures of human embryonic tissue and produces typical intranuclear inclusion bodies. Cytopathic changes are more focal and spread much more slowly than those induced by HSV. Infectious virus remains strongly cell associated, and serial propagation is more easily accomplished by passage of infected cells than of tissue culture fluids. The same virus causes chickenpox and zoster. Microbiology| Cultivation and Isolation of Viruses Laboratory Diagnosis of (HSV-3) (VZV) : 1. PCR assays are preferred for sensitivity, specificity, and rapidity for rapid diagnostic procedures. 2. In stained smears of scrapings or swabs of the base of vesicles (Tzanck smear), multinucleated giant cells are seen. 3. A rise in specific antibody titer can be detected in the patient’s serum by various tests, including fluorescent antibody and enzyme immunoassay. Microbiology| Cultivation and Isolation of Viruses Treatment : Varicella-zoster virus (HSV-3) Several antiviral compounds provide effective therapy for varicella, including Acyclovir, Valacyclovir, Famciclovir, and Foscarnet. Note: The skin lesions of herpes zoster and chickenpox are histopathologically identical: both contain multinucleated giant cells with eosinophilic intranuclear inclusion bodies. Microbiology| Cultivation and Isolation of Viruses The rash of herpes zoster is similar to chickenpox, except that rash of herpes zoster is restricted to one area of the skin on one side of the body—namely, the dermatome innervated by the ganglion in which the latent virus reactivated. Also, the lesions of herpes zoster consist of closely grouped vesicles on an erythematous base, whereas those of chickenpox are individual and randomly distributed. These differences reflect intraneural spread of virus to the skin in herpes zoster, in contrast to viremic spread in chickenpox. Cytomegalovirus (HSV-5) Microbiology| Cultivation and Isolation of Viruses Cytomegalovirus (CMV) (HSV-5) : Cytomegalovirus replicates in epithelial cells of the respiratory tract, salivary glands, and kidneys and persists in lymphocytes. It causes an infectious mononucleosis (heterophil-negative). In newborns, cytomegalic inclusion disease may occur caused by intrauterine or early postnatal infection with the CMV. CMV is an important cause of congenital defects and mental retardation. Microbiology| Cultivation and Isolation of Viruses CMV are ubiquitous/diffuse herpesviruses that are common causes of human disease. In apparent infection is common during childhood and adolescence. Severe CMV infections are frequently found in adults who are immunosuppressed. CMV incubation period 4-8 weeks. Properties of the Virus: CMV has the largest genetic content of the human herpesviruses (HHV). Microbiology| Cultivation and Isolation of Viruses Pathogenesis and Pathology : A. Normal Hosts: CMV may be transmitted from person to person in several different ways, all requiring close contact with virus-bearing material. It is Usually asymptomatic but occasionally causes a spontaneous infectious mononucleosis syndrome. B. Immunosuppressed Hosts: Primary CMV infections in immunosuppressed hosts are much more severe than in normal hosts. Pneumonia is a frequent complication. Microbiology| Cultivation and Isolation of Viruses C. Congenital and Perinatal Infections: Fetal and newborn infections with CMV may be severe. The virus can be transmitted in utero with both primary and reactivated maternal infections. About one-third of pregnant women with primary infection transmit the virus. CMV can also be acquired by the infant from exposure to virus in the mother’s genital tract during delivery and from maternal breast milk. Congenital infection may result in death of the fetus in utero. Microbiology| Cultivation and Isolation of Viruses Laboratory Diagnosis : A. Polymerase Chain Reaction and Antigen Detection Assays. B. Isolation of Virus: The virus can be recovered most readily from throat washings and urine. In cultures, 2–3 weeks are usually needed for the appearance of cytologic changes. C. Serology: Detection of viral IgM antibodies suggests a current infection. Microbiology| Cultivation and Isolation of Viruses Treatment and Control : Ganciclovir, a nucleoside structurally related to acyclovir, has been used successfully to treat life threatening CMV infections in immunosuppressed patients. Foscarnet is recommended for treatment of CMV retinitis (is inflammation of the retina/eye). Acyclovir and valacyclovir have shown some benefits in bone marrow and renal transplant patients. Epstein-Barr virus (HSV-4) Microbiology| Cultivation and Isolation of Viruses Epstein-Barr virus (EBV) (HSV-4) : Epstein-Barr virus is a widespread herpesvirus that replicates in epithelial cells of the oropharynx and parotid gland and establishes latent infections in B lymphocytes. HSV-4 causes acute infectious mononucleosis (IM) and is associated with nasopharyngeal carcinoma and is the cause of human lymphoproliferative disorders, especially in immunocompromised patients. The major target cell for EBV is the B lymphocyte. EBV incubation period (30-50 days). EBV is also associated with several types of cancer. It spread by direct contact. Properties of the Virus: There are two major strains of EBV, types A and B. Microbiology| Cultivation and Isolation of Viruses Laboratory Diagnosis : A. Molecular assays for identification of virus. Paul–Bunnell test: The test is specific for heterophile antibodies (antibodies induced by external antigens) produced by immune system in response to EBV infection to screen for Infectious Mononucleosis (IM) by agglutination of the sheep RBCs by heterophile antibodies in patient's serum. Heterophile means it reacts with proteins across species lines. Microbiology| Cultivation and Isolation of Viruses B. Isolation of Virus: EBV can be isolated from saliva, peripheral blood, or lymphoid tissue by immortalization of normal human lymphocytes, usually obtained from umbilical cord blood. This assay is laborious/tired and time consuming (6–8 weeks), requires specialized facilities, and is seldom performed. C. Serology include enzyme-linked immunosorbent assays (ELIZA), immunoblot assays, and indirect immunofluorescence tests using EBV-positive lymphoid cells. Treatment Acyclovir. Human herpesvirus 6,7,8 Microbiology| Cultivation and Isolation of Viruses Human herpesvirus 6 (HHV-6) : Human herpesvirus 6 was first recognized in 1986. It infects T lymphocytes (lymphoproliferative). It is typically acquired in early infancy and childhood. This primary infection causes exanthem subitum (roseola infantum, or ―sixth disease), the mild common childhood disease characterized by a high fever and skin rash. The mode of transmission of HHV-6 is presumed to be via oral secretions. Infections persist for life. Reactivation appears to be common in transplant patients and during pregnancy. Microbiology| Cultivation and Isolation of Viruses Human Herpesvirus 7 (HHV-7) : Human Herpesvirus 7 is also T-lymphotropic human herpesvirus. HHV-7 appears to be a ubiquitous agent, with most infections occurring in childhood but later than the very early age of infection noted with HHV-6. Persistent infections are established in salivary glands, and the virus can be isolated from saliva of most individuals. Similar to HHV-6, primary infection with HHV-7 has been linked with roseola infantum in infants and young children. Herpesvirus 5, 6, 7 belong to beta subfamily. Microbiology| Cultivation and Isolation of Viruses Human Herpesvirus 8 (HHV-8) : Human Herpesvirus 8 (also called Kaposi sarcoma-associated herpesvirus [KSHV]). It was first detected in 1994 in Kaposi sarcoma specimens. KSHV is lymphotropic. It is the cause of Kaposi sarcomas, vascular tumors occurring in AIDS patients and of multicentric Castleman disease. HV-8 (closely related to EBV, HSV-4) and both related to gamma subfamily. Poxviruses Microbiology| Cultivation and Isolation of Viruses Poxviruses : Poxviruses are the largest and most complex of viruses infecting humans and very resistant to inactivation. Linear ds DNA and have own RNA polymerase. Infections with most poxviruses are characterized by a rash, although lesions induced by some members of the family are markedly proliferative. Microbiology| Cultivation and Isolation of Viruses The group includes vaccinia virus, variola virus, the etiologic agent of smallpox (the viral disease that has affected humans throughout recorded history). Most of the poxviruses that can cause disease in humans are contained in the genera Orthopoxvirus and Parapoxvirus. Edward Jenner introduced vaccination with live cowpox virus in 1798. Microbiology| Cultivation and Isolation of Viruses Clinical Findings : The incubation period of variola (smallpox) was 10–14 days. The onset was usually sudden. One to 5 days of fever and malaise preceded the appearance of the exanthems/rash, which began as macules, then papules, then vesicles, and finally pustules. Microbiology| Cultivation and Isolation of Viruses Laboratory Diagnosis : A. Isolation and Identification of Virus: 1. Skin lesions are the specimen of choice for viral detection and isolation. 2. Direct examination of clinical material in the electron microscope (EM) is used for rapid identification of virus particles. 3. Polymerase chain reaction (PCR) tests. 4. Viral antigen can be detected by immunohistochemistry in tissues and in material collected from skin lesions. 5. Cell cultures can be used for virus isolation. Microbiology| Cultivation and Isolation of Viruses B. Serology: Antibody assays can be used to confirm a diagnosis of poxvirus infection. Antibodies appear after the first week of infection that can be detected by hemagglutination-inhibition, neutralization, enzyme-linked immunosorbent assay, radioimmunoassay, or immunofluorescence tests. Treatment: Methisazone and Cidofovir. Poxviruses is eradicated by global vaccination. Adenoviruses Microbiology| Cultivation and Isolation of Viruses Adenovirus Infections In Humans : Pathogenesis Adenoviruses infect and replicate in epithelial cells of the respiratory tract, eye, gastrointestinal tract, and urinary tract. They usually do not spread beyond the regional lymph nodes. Group C viruses persist as latent infections for years in adenoids and tonsils and are shed in the feces for many months after the initial infection. In fact, the name adenovirus reflects the recovery of the initial isolate from explants of human adenoids. Most human adenoviruses replicate in intestinal epithelium after ingestion but usually produce subclinical infections rather than overt symptoms. It ds DNA, linear, naked icosahedral viruses. Microbiology| Cultivation and Isolation of Viruses Laboratory Diagnosis : A. Detection, Isolation, and Identification of Virus Samples should be collected from affected sites early in the illness to optimize virus isolation. Depending on the clinical disease, virus may be recovered from stool or urine or from a throat, conjunctival, or rectal swab. Cultures are incubated for 1–2 days. Polymerase chain reaction (PCR) assays are routinely used for diagnosis of adenovirus infect. B. Serology: Infection of humans with any adenovirus type stimulates a rise in complement-fixing antibodies to adenovirus group antigens shared by all types. Treatment: There is no specific treatment for adenovirus infections. Papillomaviruses Microbiology| Cultivation and Isolation of Viruses Papillomaviruses Classification : The Papillomaviridae family is a very large virus family currently divided into 16 genera, of which 5 contain members that infect humans (Alpha-, Beta-, Gamma-, Mupa-, and Nupapapillomavirus). The papillomaviruses are former members of the Papovaviridae family. It is ds DNA, circular, naked virus. It is stimulate DNA synthesis. It causes human cancer, especially cervical cancer. Microbiology| Cultivation and Isolation of Viruses Pathogenesis and Pathology : Transmission of viral infections occurs by close contact. Viral particles are released from the surface of papillomatous lesions. Papillomaviruses cause infections at cutaneous and mucosal sites, sometimes leading to the development of different kinds of warts, including skin warts, plantar (feet) warts, flat warts, anogenital warts, laryngeal papillomas, and several cancers, including those of the cervix, vulva, penis and anus, and a subset of head and neck cancers. Microbiology| Cultivation and Isolation of Viruses Human papillomavirus (HPV) genital infections are sexually transmitted. Based on the relative occurrence of viral DNA in certain cancers, HPV types 16 and 18 are considered to be high cancer risk; about 15 other less common types are also considered high risk. Many HPV types are considered benign. Hepatitis Viruses Microbiology| Cultivation and Isolation of Viruses Hepatitis Viruses : Viral hepatitis is a systemic disease primarily involving the liver (acute and chronic hepatitis) and cause hepatocellular carcinoma. Most cases of acute viral hepatitis in children and adults. Microbiology| Cultivation and Isolation of Viruses Hepatitis Viruses are caused by one of the following five agents: hepatitis A virus (HAV), the etiologic agent of viral hepatitis type A (infectious hepatitis). hepatitis B virus (HBV), which is associated with viral hepatitis B (serum hepatitis). hepatitis C virus (HCV), the agent of hepatitis C (posttransfusion hepatitis). hepatitis D (HDV), a Defective virus dependent on coinfection with HBV. hepatitis E virus (HEV), the agent of enterically transmitted hepatitis. Microbiology| Cultivation and Isolation of Viruses All Hepatitis viruses type are ss RNA, except hepatitis B virus is ds DNA. HAV and HEV is nonenveloped virus, while HBV, HCV and HDV are envelop virus. Hepatitis viruses produce acute inflammation of the liver, resulting in a clinical illness characterized by fever, gastrointestinal symptoms such as nausea and vomiting, and jaundice. Microbiology| Cultivation and Isolation of Viruses They have 2 major polypeptide: one glycosylated is present in HBsAg, one polypeptide is present in HBcAg. Hepatitis Type B HBV is classified as a hepadnavirus. HBV establishes chronic infections, especially in those infected as infants; it is a major factor in the eventual development of liver disease and hepatocellular carcinoma in those individuals. Parvoviruses B19 Microbiology| Cultivation and Isolation of Viruses Parvoviruses B19 : Parvoviruses B19 is pathogenic for humans and has a tropism for erythroid (RBC) progenitor cells. It is the cause of erythema infectious (fifth disease), a common childhood exanthem; of a polyarthralgia-arthritis syndrome in normal adults; of aplastic crisis in patients with hemolytic disorders; of chronic anemia in immunocompromised individuals; and of fetal death. It is very simple virus (ss DNA, linear). It is require helper virus. Incubation period 17 day. It is naked virus. Microbiology| Cultivation and Isolation of Viruses Hence, the major sites of virus replication in patients are assumed to be the adult marrow, some blood cells, and the fetal liver. Viral replication causes cell death, interrupting red blood cell production. B19 can be found in blood and respiratory secretions of infected patients. There is no evidence of virus excretion in feces or urine. The virus can be transmitted parenterally by blood transfusions or by infected blood products (clotting and immunoglobulin concentrates) and vertically from mother to fetus.