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Lecture #2 (2).ppt

  1. RNA virus. Orthomyxoviridae. Paramyxoviridae. Kharkiv – 2020 V.N. Karazin Kharkov National University Faculty of medicine Department of general and сlinical immunology and allergology
  2. RNA viruses can be either double- stranded (dsRNA) or single-stranded (ssRNA) • Although relatively few RNA viruses have dsRNA genomes, dsRNA viruses are known to infect animals, plants, fungi, and at least one bacterial species. • More common are the viruses with ssRNA genomes.
  3. • Some ssRNA genomes have a base sequence that is identical to that of viral mRNA, in which case the genomic RNA strand is called the plus strand or positive strand. In fact, plus strand RNAs can direct protein synthesis immediately after entering the cell. • However, other viral RNA genomes are complementary rather than identical to viral mRNA, and are called minus or negative strands.
  4. Classification of RNA viruses – Reoviridae Orbivirus [Humans: encephalitis] – Rotavirus [Humans: diarrhea] – Cypovirus [Insects] – Togaviridae lphavirus [Humans: encephalitis] – Flavivirus [Humans: yellow » fever, dengue] – Rubivirus [Humans: rubella] – Pestivirus [Pigs: hog cholera]
  5. Classification of RNA viruses – Coronaviridae Infectious bronchitis virus [Humans: upper respiratory infection] – Picornaviridae Enterovirus [Humans: polio] – Rhinovirus » [Humans: common cold] – Hepatovirus » [Humans: hepatitis A]
  6. Classification of RNA viruses – Calciviridae Vesicular exanthema of swine – Norwalk virus – Hepatitis E virus – Retroviridae Oncornavirus C » [Birds, mice: sarcomas, leukemias] – Human T-cell leukemia virus – Human immunodeficiency virus
  7. Classification of RNA viruses – Orthomyxoviridae Influenza virus » [Humans, swine] – Rhabdoviridae Lyssavirus » [Warm-blooded animals: rabies] – Arenaviridae Lassa virus » [Humans: hemorrhagic fever]
  8. Classification of RNA viruses – Bunyaviridae California encephalitis virus [Humans] – Paramyxoviridae Paramyxovirus » [Humans: colds, respiratory infections, mumps] – Pneumovirus [Humans: pneumonia, common cold ] – Morbilliviris » [Humans: meesles]
  9. Orthomyxoviridae orthos, Greek for "straight"; myxa, Greek for "mucus") – Orthomyxoviridae - are a family of RNA viruses that includes five genera: – Influenzavirus A, – Influenzavirus B, – Influenzavirus C, – Isavirus – and Thogotovirus.
  10. Orthomyxoviridae – Influenzavirus A, В, С - cause influenza in vertebrates, including birds, humans, and other mammals. – Isaviruses infect salmon; – Thogotoviruses infect vertebrates and invertebrates, such as mosquitoes and sea lice.
  11. Infuenza –- is an acute infectious disease of the resperatory tract which occurs in sporadic, epidemic and pandemic forms.
  12. WHAT IS INFLUENZA? • Influenza, commonly called "the flu," is an illness caused by RNA viruses of the family Orthomyxo viridae
  13. Infuenza – Influenza viruses that infect the respiratory tract of many animals, birds, and humans.
  14. Infuenza – The modern history of the disease may be considered to date from the pandemic of 1889- 1990, during which Pfeiffer isolated Haemophilus influenzae and claimed that it was the causative agent.
  15. The three genera of Influenzavirus, which are identified by antigenic differences in their nucleoprotein and matrix protein infect vertebrates as follows: – Influenzavirus A infects humans, other mammals, and birds, and causes all flu pandemics – Influenzavirus B infects humans and seals – Influenzavirus C infects humans and pigs
  16. Morphology – The influenza virus is typically spherical, with a diameter of 80- 120 nm but pleomorphism is common.
  17. Morphology – Filamentous form, up to several micrometres in length and readily visible under the dark ground microscope, are frequent in freshly isolated strains.
  18. – The virus core consists of ribonucleoprotein in helical symmetry. – The negative sense single-stranded RNA genome is segmented and exist as eight pieces. – Also present is a viral RNA-dependent RNA polymerase which is essential for transcription of the viral RNA in infected host cells.
  19. – The nucleocapsid is surrounded by an envelope, which has an inner membrane protein layer and an outer lipid layer. – The membrane protein is also known as the matrix or «M-protein» composed of two components, M1 and M2. – The protein part of the envelope is virus coded but the lipid layer is derived from the modified host cell membrane, during the process of replication by budding.
  20. There are 2 distinct types of glycoprotein: one with • Hemagglutin (HA) • Neuramidase (NA) Anchoring this bases of each of these spikes on the inside of viral lipid bilayer are membrane proteins (M proteins and NP proteins)
  21. – Projecting from the envelope are two types of spikes (peplomers): hemagglutinin spikes which are triangular in cross-setion ant the mushroom-shaped neuraminidase peplomers which are less numerous. – Both surface antigen show a marked tendency for antigenic variation (drift).
  22. Structure
  23. Hemagglutinin • -have 16 subtypes and can attach to host sialic acid receptors are present on the surface of erythrocytes, so viruses with HA glycoproteins cause heme- glutination when mixed with red blood cells. • Host cell sialic acid receptors also exist on upper respiratory tract cell membranes and HA binding to theses receptors activates fusion of the host cell membrane with the virion membrane, resulting in dumping of the viral genome into the host cell. So HA is needed for adsorption.
  24. Neuraminidase (NA) • have 9 subtypes and is an important component of mucin the substance covering mucosal epithelial cells and forming an part of host upper respiratory defense barrier. Exposing the sialic acid and binding sites beneath.
  25. Antigenic drift • is a mechanism for variation in viruses that involves the accumulation of mutations within the genes that code for antibody binding sites. • This results in a new strain of virus particles which can’t be inhibited as effectively by the antibodies that were originally targeted against previous strains, making it easier for the virus to spread throughout a partially immune population.
  26. Antigenic drift • Antigenic drift occurs in both influenza A and influenza B viruses.
  27. Antigenic ‘shift’ • The segmented viral genome allows for formation of viral reassortants (‘recombinants’) between different strains or subtypes of virus. A doubly-infected host can thus give rise to a ‘new’ virus. Such a profound change in antigenic make-up (‘shift’)
  28. Laboratory diagnosis of influenza • Specimen: nasopharyngeal aspirate. • Detect virus antigen by indirect immunofluorescence: a very rapid method of diagnosis. • Serology: (widely used) Complement fixation test: with the “S” or soluble nucleoprotein antigen. • Isolation: monkey kidney tissue cultures.
  29. Laboratory diagnosis of influenza • Observe: for hemagglutination with human group O erythrocytes. • Type virus: by complement fixation, strain identification by haemagglutination- inhibition in a reference laboratory. A commercially available identification technique is Directigen FLU-A (an enzyme immunoassay [EIA] rapid test). This test can detect influenza A virus in clinical specimens in less than 15 minutes.
  30. Treatment • Treatment is mostly symptomatic. • Amantadine and rimantadine are useful • They reduce the average duration of the disease and cause symptomatic improvement, though virus shedding and antibody response are not affected. • Resistance to these drugs develops rapidly. • Zanamivir and oseltamivir, new drugs designed to block viral neuraminidase, have been found effective in the treatment and prevention of influenza, when admivistered as nasal spray Treatment is mostly symptomatic.
  31. Immunoprophylaxis • The main difficulty in the immunoprophylaxis of influenza is the frequent change in the antigenic make up of the virus • Vaccines can’t be made in bulk and stockpiled, as the appearance of a new variant will make the old vaccine obsolete
  32. Immunoprophylaxis • In cold countries, where it is necessery to protect old persons and other high-risk individuals, the practice is to immunise them with a vaccine containing the latest strains of type A and B viruses
  33. Immunoprophylaxis • The inactivated vaccines used throughout the world are purified egg- grown virus killed by formalin or bybpropriolactone. • The use of live attenuated vaccine is limited. Preparations containing detergent split virus or isolated surface subunits (H and N) are mostly used. The two latter types of vaccine are claimed to give fewer side-effects than whole virus vaccine.
  34. Immunoprophylaxis • Persons for whom clinical influenza would lead to further deterioration of their underlying condition are recommended as target groups for routine vaccination. • Generally, these are the elderly, those with chronic illnesses in the heart, lungs and airways, and those withmetabolic disorders or immune deficiencies. • Protective levels of anti-influenza antibody will ensue 1–3 weeks postvaccination. • Immunity to influenza will last for about a year as the ever-changing virus will outdate acquired immunity.
  35. Paramyxoviridae • from Greek para-, beyond, -myxo-,mucus or slime • are family of the negative-sense single stranded RNA viruses responsible for a number of human and animal diseases that includes following genera: Paramyxovirus (Humans: colds, respiratory infections, mumps), Pneumovirus (Humans: pneumonia, common cold), Morbillivirus (Humans: measles).
  36. General features on Paramyxoviridae family • Viruses replicate in the cytoplasm. • Viruses induce cell-cell fusion via F protein, causing multinucleated giant cells. • Paramyxoviridae are transmitted in the respiratory droplets and initiate infection in the respiratory tract. • Cell- medicated immunity causes many of the symptoms but is essential for control of the infection.
  37. Paramyxoviridae family • There are 4 paramyxoviridae that cause human disease: • parainfluenza virus, • respiratory syncytial virus, • mumps virus, • and measles virus.
  38. Parainfluenza virus • The parainfluenza virus causes upper respiratory infections in adults ranging from cold symptoms such as rhinitis, pharyngitis, and sinus congestion, to bronchitis and flu-like illness. • Children, elderly, and the immunocompromised also suffer from lower respiratory tract infections (pneumonia).
  39. Clinical features • Parainfluenza viruses are responsible for about 10 per cent of resperatory infections in children needing hospitalisation. • In adults cause milder respiratory infection in which sore throat and hoerseness of the voice are common. Rarely, they cause parotitis.
  40. Laboratory diagnosis • Speciment: throat and nasal swabs. • Virus isolation: Throat and nasal swabs are inoculated in primary monkey kidney cell cultures, or continuous monkey kidney cell lines with trypsin. Virus growth is detectrd by hemadsorption. Typing is by immunofluorescence, hemadsorption inhibition or hemagglutination inhibition.
  41. Laboratory diagnosis • Serology: serological diagnosis is hampered by wide antigenic cross- reactions. Paired sera can be tested by neutralisation, ELISA, HI (hemagglutination inhibition assay) or CF (complement fixation test) for rise in the titre of antibodies. • PCR: Molecular diagnosis using reverse transcriptase PCR is gaining more acceptance.
  42. Mumps virus • replicates in the upper respiratory tract and in the regional lymph nodes and spreads via the blood to distant organs. Infections can occur in many organs, but the most frequently involved is the parotid gland.
  43. Clinical features • Infection is acquired by inhalation, and probably also through the conjunctiva. The virus replicates in the upper respiratory tract and cervical lymph nodes and is disseminated through the boodstream to variouse organs. • Incubation period: 12-25 days. • Parotid swelling is usually the first sign of illness, though it may sometimes be preceded by prodromal malaise.
  44. Clinical features • Parotid swelling is unilateral to start with but may become bilateral. It is accompanied by fever, local pain and tenderness but the skin over the gland is not warm or erythematous. • Parotitis is non-suppurative ans ussually resolves within a week. • However, involvement of extraparotid sites may be seriouse and may sometimes occur even in the absence of paratitis.
  45. Complications • Epididymo-orchitis is a complication seen in about a third of postpubertal male patients. The testis becomes swollen and acutely painful, with accopanying fever and chills. • Orchitis is usually unilateral but when it is bilatrral and followed by testicular atrophy, sterility or low sperm counts may result. • 10 per cent show symptoms of meningitis • Mumps meningitis and meningoencephalitis ussually resolve without sequelae but deafness may sometimes result
  46. Laboratory diagnosis • A typical case of mumps needs no laboratory confirmation but it may be essential in atypical infection and where meningitis or other systemic involment is the sole manifestation. • Speciment: The virus may be isolated from salva (within 4-5 days), urine (up to two weeks) or CSF (8-9 days after onset of illness)
  47. Laboratory diagnosis • Virus isolation: The specimens must be inoculated soon after collection virus is labile. Virus growth can be detected by hemadsorption and identified by hemadsorption inhibition using specific antiserum. Cytopathic changes are not reliable. Isolation may take 1-2 weeks. • More rapid results can be obtained by immunofluorescence testing of infected cell cultures. This may become possitive as early as 2-3 days after inoculation.
  48. Laboratory diagnosis • Isolation can also be made by inoculation into six-to-eight-day-old chick embryos by the amniotic route and testing the amniotic fluid after 5-6 days for hemagglutinins. • The virus can be identified by hemagglutination inhibition using specific antisera. • Egg inoculation is less sensitive than call cultures for isolation. • Direct antigenic detection by IFA is helpful in early diagnostis.
  49. Laboratory diagnosis • Serology: Serological diagnosis depends on demonstration of a rise in the titre of antibodies in paired serum samples. • The CF (complement fixation test) and HI (hemagglutination inhibition assay) tests are commonly used but cross-reactions with perainfluenza viruses cause probleems.
  50. Laboratory diagnosis • IgM-ELISA is useful in this respect because cross-reacting antibodies are IgG and do not interfere with IgM-ELISA. • A positive CF test for antibody to the S antigen in the acute phase serum is presumptive evidence of current infection. • PCR: Molecular diagnosis using reverse transcriptase PCR is more rapid and sensitive.
  51. Vaccination • An effective live virus vaccine is available against mumps. • The vaccine is given as a single subcutaneouse injection, alone or in combination - MMR vaccine (measles, mumps and rubella vaccines). • It provides effective protection for at least ten years. • The vaccine may not prevent the disease if given after exposure to the infection but there are no contraindications for its use in this situation.
  52. Respiratory Syncytial Virus (RSV) • is so-named because it causes respiratory infections and contains an F- protein that causes formation of multinucleated giant cells (syncytial cells). This virus differs from the rest of its kin by lacking both the HA and NA glycoproteins.
  53. Clinical features • Most RSV infections are symptomatic. • The virus is hardly ever found in healthy persons. • Infection causes a broad range of respiratory illnesses. In infants, the disease may begin s febrile rhinorrhea, with cought and wheezing, progressing in 25-40 per cent to lower respiratory involvement, including tracheobronchitis, bronchiolitis and pneumonia.
  54. Laboratory diagnosis • Speciment: nasopheryngeal swabs or nasal washings. • Virus isolation: Samples should be inoculated in cell cultures immediately after collection. Freezing of clinical samples may destroy the virus. In cultured cells, RSV caused characteristic giant cell and syncytial formation but cytopathic effects take about 10 days to appear.
  55. Laboratory diagnosis • Earlier detection of viral growth in cell is possible by immunofluorescence tests. • Rapid diagnosis of RSV infection can be made by the immunofluorescence test on smears of nasapharyngeal swabs.
  56. Laboratory diagnosis • Serology: Serological diagnosis is by demonstration of rising antibody titres in paired serum samples by ELISA, CF (complement fixation test), neutralisation or immunofluorescence tests. • PCR: Molecular diagnosis by reverse transcriptase PCR is sensitive and rapid.
  57. Treatment and prophylaxis • Management is primarily by supportive care. • Administration of ribovirin by continuous aerosol has been found beneficial in hospitalised patients, decreasing the duration of illness and of virus shedding. • No effective vaccine is available.
  58. Measles (Rubeola) • is a highly contagious skin disease that is endemic throughout most of the world. It is a negative strand, enveloped RNA virus, in the genus Morbillivirus and the family Paramyxoviridae. • The measles virus is monotypic, but small variations at the epitope level have been described. The variations are based on genetic variability in the virus genes. • Such variations, however, have no effect on protective function since a measles infection still provides a lifelong immunity against reinfection.
  59. Clinical features • It takes about 9-11 days from the time of exposure to infection for the first signs of clinical disease to appear. These consist of prodromal malaise, fever, conjuctival infection, cought and nasal discharge. • After 3-4 days of prodromal illness, and rash appears. • A day or two before the rash begins, Koplik’s spots develop on the buccal mucosa and occasionally on the conjunctiva and intestinal mucosa.
  60. Clinical features • The prodromal illness subsides within a day or two of appearance of the rash. • The red maculopapular rash of measles typically appears on the forehead first and spreads downwards, to disappear in the same sequence 3-6 days later, leaving behind a brownish discolouration and finely granular desquamation.
  61. Laboratory diagnosis • In a typical case of measles, diagnosis is self-evident. In atypical cases, and for differentiation from rubella, laboratory tests are useful. • Speciment: nasal secretions, throat, conjunctiva and blood can be used. CSF (cerebrospinal fluid) is collected in SSPE (subacute sclerosing panencephalitis). • IFA: The measles virus antigen can be detected in cells on nasal secretions by immunofluorescence.
  62. Laboratory diagnosis • Virus isolation: The virus can be isolated from the nose, throat, conjunctiva and blood during the prodromal phase and up to about two days after the appearance of the rash. The virus may be obtained from urine for a few more days. • Primary human or monkey kidney and amnion cell are most useful. • Cytopathic changes may take up to a week to develop, but earlier diagnosis of viral growth is possible by immunofluorescence.
  63. Laboratory diagnosis • Serological diagnosis: Specific neutralisation, hemagglutination inhibition (HAI) and complement fixing antibodies (in CFT) develop early. • A fourfold rise in titre is looked for using paired sera collected during the acute phase and 10-21 days after.
  64. Laboratory diagnosis • Demonstration of measles-specific IGM in a single specimen of serum drawn between one and two weeks after the onset of the rash is confirmatory. • False negaives may occur if the serum is taken earlier than one week before or later than two weeks after onset of the rash.
  65. Laboratory diagnosis • Demonstration of high titre measles antibody in the CSF (cerebrospinal fluid) is diagnostic of SSPE (subacute sclerosing panencephalitis). • PCR: Reverse transcriptase PCR is a sensitive and specific method of diagnosis.
  66. Prophylaxis • Passive protection: • Normal human gammaglobulin given within six days of exposure can prevent or modify the disease, depending on the dose. • This is useful in children with immunodeficiency, pregnant women and others at special risk/
  67. Prophylaxis Active immunisation: • The recommended age for measles vaccination in developing countries is now nine month, while in the advanced nationss it remains 15 months. • A safe and effective live attenuated measles vaccine is available. • The vaccine given either by itself, or in combination, as the MMR vaccine (measles, mumps and rubella vaccines).
  68. Prophylaxis • A single subcutaneous injection of the measles vaccine provides protection beginning in about 12 days and lasting for over 20 years. • Contraindications are immunodeficiency, untreated tuberculosis and pregnancy.
  69. Prophylaxis • A live attenuated vaccine has been developed which can be given by intranasal aerosol to young babies and gives good protection irrespective of the presence of maternal antibodies. • Efforts are being made to eradicate measles by vaccination. • Considerable progress has been achieved in the USA and some other countries.