VIROLOGY - CHAPTER SIXTEEN
VIROLOGY - CHAPTER SIXTEEN
PARAINFLUENZA, RESPIRATORY SYNCYTIAL AND ADENO VIRUSES
Reading
Medical Microbiology, 5th Ed.
Paramyxiviruses
Mosby (Murray, Rosenthal, Pfaller)
PARAINFLUENZA VIRUS
Parainfluenza viruses are important viral pathogens causing upper and lower respiratory infections in adults and children. They are second to respiratory syncytial virus cause of lower respiratory tract disease in young children.
Classification
Family Paramyxoviridae
Genus Members
Paramyxovirus Parainfluenza [PIV types 1,2,3,4]
Mumps virus
Newcastle Disease Virus [birds]
Sendai virus [mice]
Morbillivirus Measles virus
Canine Distemper Virus
Pneumovirus Respiratory Syncytial Virus (RSV)
WEB RESOURCES
Big Picture Book of Viruses
Paramyxoviruses
CDC Parainfluenza information
[pic]Figure 1. Structure of a paramyxovirus
[pic]Figure 2. Paramyxovirus © Dr Linda Stannard, University of Cape Town, South Africa (used with permission)
Structure
Parainfluenza viruses are relatively large viruses of about 150-300 nm in diameter. They have a spherical or pleomorphic shape (figure 1 and 2).
The RNA is negative sense, unsegmented and single stranded (ss). The nucleocapsid core is filamentous or herringbone-like, has helical RNA tightly associated with Nucleoprotein (NP) Phosphoprotein (P) and Large protein (L)
These are enveloped viruses with a host-derived lipid bilayer associated with two virus-specific glycoproteins:
Hemagglutinin-Neuraminidase (HN). This is a viral attachment protein, that also causes hemadsorption and hemagglutination
Fusion protein (F). The F protein forms spikes out from the envelope. It promotes the fusion of host and viral cell membranes which is an initial step in infection. It is synthesized as a biologically inactive form (F0), which is activated by proteolytic cleavage to an active form that has 2 subunits, F1 and F2, linked by a disulfide bond.
Matrix (M) protein, located just within the envelope, is hydrophobic
[pic] Figure 3.
Transmission electron micrograph of parainfluenza virus. Two intact particles and free filamentous nucleocapsid. CDC/Dr. Erskine Palmer
|Structural Protein |Designation |Location |Function |
|Hemagglutinin-neuramini|HN |Envelope |Attachment to host cell receptors, |
|dase | | |hemagglutinin and neuraminidase activity|
|(glycoprotein) | | | |
|Fusion Protein |F |Envelope |Fusion, penetration, hemolysis |
|Matrix Protein |M |Inside the envelope |Assembly |
| | |Nucleocapsid | |
|Nucleoprotein |NP |Nucleocapsid |Complexed with RNA genome, |
|Phosphoprotein |P | |Part of the RNA polymerase complex |
| | |Nucleocapsid |Part of the RNA polymerase complex |
|Large Protein |L | | |
Isolation
Cell lines such as primary Rhesus monkey kidney epithelial Cells (PRMK), LLC-MK-2, and human embryonic kidney cells are used. Cytopathic effects occur such as rounding, bridging, cell lysis, and syncytium formation.
Hemadsorption (due to the interaction of viral hemagglutinin with specific erythrocyte receptors on guinea pig red cells) can be observed at 4° C. This may be seen even before the appearance of cytopathic effects and has been used for early diagnosis (especially PIV-1 and PIV-3).
Pathogenesis
The first step in the infection cycle involves attachment of the virus to host cell sialic acid receptors. This is mediated by viral attachment protein, a function served by the HN glycoprotein.
Next, the F protein catalyzes fusion of the viral envelope and host cell membrane, resulting in uncoating and release of the nucleocapsid structure into the host cell cytoplasm.
For transcription and protein synthesis to occur, first mRNA is formed with the help of RNA-dependent RNA polymerase which must be supplied by the virus. The polymerase function is carried out by the P and L proteins, and possibly also the NP. The genome is replicated by formation of a full-length positive sense RNA template onto which a negative sense RNA is then transcribed.
Assembly of the nucleocapsid occurs and M proteins are then associated with the viral glycoprotein modified cell membranes. Mature virions are released from host cell membranes by budding.
Epidemiology and Transmission
The virus is ubiquitous; infections occur as epidemics as well as sporadically. There can be repeated infections throughout life.
Parainfluenza viruses are sensitive to detergents and heat but can remain viable on surfaces for up to 10 hours.
Transmission occurs via the following routes:
Large droplets - person to person through close contact
Aerosols of respiratory secretions
Fomites (virus survives on surfaces)
[pic]Figure . Weekly reports of parainfluenza type 1 in the US. Seasonal variation. CDC
[pic]Figure . Weekly reports of parainfluenza type 2 in the US. Seasonal variation. CDC
[pic]Figure . Weekly reports of parainfluenza type 3 in the US. Seasonal variation. CDC
Clinical Features
Primary infections and re-infections occur but most infections are asymptomatic, especially in older children and adults. The incubation period is 2 to 6 days. Most persons have had primary infections before the age of 5 yrs.
Reinfections are clinically less severe, most commonly involve the upper respiratory tract and occur throughout life.
Fever and a spectrum of respiratory infections are caused by PIVs:
Rhinorrhea/rhinitis, pharyngitis, cough, croup (laryngotracheobronchitis), bronchiolitis, and pneumonia.
Croup - the subglottic region becomes narrower and results in difficulty with breathing, a seal bark-like cough and hoarseness.
PIV types 1 and 2 most often cause outbreaks of croup in autumn/early winter, with an alternate year pattern. PIV-1 tends to attack children ages 2-6 years.
PIV-3 can cause croup, though less commonly than PIV-1 and 2 and is sporadic. It often occurs in the spring and summer. Primary infection with PIV 3 in young infants and children of less than two years of age is a common cause of bronchiolitis (although RSV is a more common cause).
PIV-4 is associated with mild upper respiratory infections
Otitis media, parotitis, aseptic meningitis occur although they are rare.
Particularly severe and persistent infections are known to occur in immunocompromised children and adults; prolonged viral shedding is seen.
Clinical Diagnosis
Antigen detection
Radio-immunoasay, enzyme immunoassay, fluoro-immunoassay, and immunofluoresence methods are used for antigen detection.
Nasopharyngeal secretions are collected, from swabs or washings and transported in viral transport medium and on ice.
Shell vial assay is useful in detecting growth in 4-7 days. Hemadsorption can be noted before cytopathic effects. Immunofluoresence is confirmatory.
Antibody Detection
Serology uses hemagglutinin inhibition to demonstrate a difference between acute and convalescent levels. A 4-fold increase in antibody titers is considered positive. However, serologic diagnosis is of limited value because of the presence of nonspecific inhibitors and the antibody being heterotypic (antibody that is common to different PIV types as well as the mumps virus)
Treatment
There is no specific treatment. Supportive treatment for croup includes humidification of air and racemic epinephrine. Corticosteroids may be used in moderate to severe cases.
Immunity
Immunity following infection is short lived. The role of antibody is not clear since reinfection has been seen even with high levels of antibody.
Cell-mediated Immunity (CMI) is probably more important for limiting infection.
Infection control
Asymptomatic shedding is common, making it difficult to contain spread of infection. Hand washing and preventing contamination of surfaces with respiratory secretions are important for limiting nosocomial spread.
RESPIRATORY SYNCYTIAL VIRUS
Classification and structure
Family Paramyxoviridae, genus Pneumovirus. Infection of cells results in syncytium formation.
These are spherical or pleomorphic enveloped viruses (100-350 nm) with single-stranded, negative sense linear RNA.
The envelope has 2 glycoproteins:
F - fusion protein, is important for fusion of viral particles to target cells and fusing infected cells to neighboring cells to form syncytia.
G - which is highly glycosylated, is important for viral attachment to host cells
Antigenic variations in the type of G protein determine the subgroup (A or B).
RSV lacks H/N proteins unlike other members of the family Paramyxoviridae
Properties
These viruses survive on surfaces for up to 6 hours, on gloves for less than 2 hours. They rapidly lose viability with freeze-thaw cycles, in acidic conditions and with disinfectants.
Pathology and Pathogenesis
Virus attaches (via G protein) to cells of respiratory tract.
Infected cells undergo necrosis, also syncytia form through fusion.
Cell to cell transfer of virus leads to spread from upper to lower respiratory tract.
Smaller airways (bronchioles) become plugged with debris and mucin; bronchoconstriction also occurs. The host immune response also induces some of the pathological changes.
Epidemiology
RSV has a worldwide distribution and most children have had an RSV infection by age 4 years
Out breaks are seasonal occurring from late fall through spring (November to May)
The virus is transmitted via large droplets, through fomites and via hands
The virus enters through the eyes and nose
Viral shedding continues for less than 1 to 3 weeks but longer in immuno-compromised hosts
RSV is the most frequent cause of bronchiolitis but is an infrequent cause of croup
[pic] Figure .
Transmission electron micrograph of respiratory syncytial virus. Long
filamentous form. CDC/Dr. Erskine Palmer
[pic]
Morphologic traits of the Respiratory Syncytial Virus. The virion is variable in shape, and size (average diameter of between 120-300nm)
WEB RESOURCES
CDC RSV information
RSV in a child-care situation (CDC)
[pic]Figure . Weekly reports of RSV isolation in the US
[pic]Figure . Section of lung: acute pneumonia, epithelial syncytia formation in alveoli, respiratory syncytial virus
infection, calf pneumonia © Bristol Biomedical Image Archive. Used with permission
Clinical Features
Incubation Period: 4 - 6 days (range: 2 - 8 days)
Upper respiratory infection (‘bad cold’) in older children and adults:
Clinical features: fever, rhinitis, pharyngitis
Lower respiratory infection- Bronchiolitis and/or pneumonia may occur after the upper respiratory infection:
Clinical features: cough, tachypnea, respiratory distress, hypoxemia, cyanosis.
Cough can persist for 3 weeks.
In young infants one observes apnea, lethargy, irritability, poor feeding.
Radiological features: atelectasis, streaking, hyperinflation.
Severe infections occur in pre-term infants (especially less than 35 weeks gestation and those with chronic lung disease), children with cyanotic congenital heart disease, and immuno-compromised hosts.
Diagnosis
Nasal washings, nasal aspirates or swabs should be transported on ice.
Rapid Diagnosis: DFA, IFA, ELISA
Viral culture is carried out in cell lines such as HeLa, Hep-2, Monkey Kidney cells. Cytopathic effects are usually seen in 2-5 days. Shell vial technique is useful
Serology: neutralizing antibodies (by CF, immunofluorescence) is not very useful for young infants.
|What is a Shell |
|Vial? |
Treatment
Treatment is usually supportive by the provision of fluids, oxygen, humidification of air, respiratory support, bronchodilators
Ribavirin (see chemotherapy section) , a guanosine analogue (aerosol) has been used with some efficacy but is reserved for only persons at high risk for severe disease.
Immunity
Humoral immunity
Neutralizing antibody to F and G proteins, IgA is also produced.
Level of neutralizing antibody does not correspond to neutralizing activity
Immunity is short lived therefore reinfections are common.
Newborns may have some innate immunity
IgE response occurs in some individuals and may be a marker for future airway hyper-reactivity.
Cell mediated
T cells. Cytokine production also contributes to illness.
Prevention of spread
Handwashing
Isolation and cohort nursing
Protective gear: gowns, gloves, masks and goggles
Active immunization. The inactivated vaccine is no longer used because it was associated with an increase in severity of disease. Other vaccine candidates are in trial phases.
Passive immunoprophylaxis. There have been encouraging results from trials using pooled hyperimmune globulin (RespiGam) as monthly injections to susceptible infants during the RSV season. Now, a monoclonal antibody against F protein has been synthesized (Palivizumab- marketed as Synagis). It is used to prevent disease in children who are at risk for severe RSV infection.
WEB RESOURCES
Synagis information
Synagis package insert (pdf file)
Synagis Product monograph
(pdf file)
RespiGam package insert (pdf file)
[pic]
Negative-stain electron micrographs of human metapneumovirus.
(Photograph courtesy of Dr. Charles Humphrey of CDC/NCID/IDPA
Published in JID 2002;185:1660-3) HUMAN METAPNEUMOVIRUS
This virus (Pneumovirinae subfamily, Paramyxoviridae family) is closely related to RSV and was first recognized as a pathogen in the Netherlands in 2001. Its role in upper and lower respiratory tract infections is now being recognized world-wide. It is detected by PCR.
Metapneumovirus is ubiquitous and, by the age of five, most people are seropositive and have thus been infected by the virus. Many infections are asymptomatic but the virus can cause the symptoms of a cold, pneumonia or bronchitis. It may be responsible for about 15% of childhood common colds. There are distinct epidemics in the winter months. There are two main HMPV types (A and B), each with 2 subtypes (A1, A2; B1, B2).
ADENOVIRUS
READING
Medical Microbiology, 3rd Ed.-1998; Mosby (Murray et al)
Manual Of Clinical Microbiology, 6th Ed.-1995; ASM Press (Murray, Baron, Pfaller)
2000 Red Book; American Academy of Pediatrics
Textbook of Pediatric Infectious Diseases, 4th ed. 1998 (R.D. Feigin and J. D. Cherry)
These viruses were named "adenovirus" because they were first isolated in 1953 from tissue cultures of human adenoidal tissue.
Classification
They belong to family Adenoviridae, genus Mastadenovirus.
Adenoviruses are further classified into 6 subgroups (A through F), based on hemagglutinating properties and DNA homology.
About 47 serotypes have been isolated from humans.
Types 40, 41 belong to subgroup F and are enteric pathogens.
Common serotypes are 1 - 8, 11, 21, 35, 37, and 40.
[pic]Figure Structure of adenovirus
[pic]Adenovirus ©t Dr Stephen Fuller, 1998
Structure
These are non-enveloped viruses with a diameter of 70-90nm.
The genome is made of linear double-stranded (ds) DNA with 2 major proteins.
The capsid is icosahedral, comprised of 252 capsomeres. 240 are hexons; at the vertices are 12 pentons, from which a fiber with a terminal knob projects. This complex is toxic to cells - causing rounding and death of cells through inhibition of protein synthesis. The fiber proteins determine target cell specificity.
10 structural proteins are known.
[pic]Adenovirus © Dr Linda M Stannard, University of Cape Town, South Africa, 1995 (used with permission).
[pic]Figure . Transmission electron micrograph of adenovirus
CDC/Dr. G. William Gary, Jr.
Pathogenesis and Replication
Virus primarily attacks mucoepithelial cells of the conjunctiva, respiratory tract, gastrointestinal and genitourinary tracts. Attachment to host cell receptor occurs via the fiber protein. The virus replicates in the cytoplasm of host cells, but viral DNA replicates within the host cell nucleus. Early and late phases of replication occur, followed by assembly and release of virions.
Three types of infections occur in target cells:
Lytic - cell death occurs as a result of virus infection (mucoepithelial cells)
Latent / persistent / occult - virus remains in the host cell, which is not killed (lymphoid tissues such as tonsils, adenoids, Peyers patches)
Oncogenic transformation - cell growth and replication continue without cell death. This is seen in hamsters, most often with group A viruses (see oncogenic virus section).
Adenovirus also replicates in associated lymphoid tissues, and subsequent viremia can cause secondary infection in visceral organs.
Inefficient (error-prone) replication of the virus results in many excess antigenic components. These are liberated into the culture fluid in vitro as soluble antigens and lead to formation of basophilic staining intra-nuclear inclusion bodies in cells.
Properties
Adenoviruses are stable in the environment and to low pH, bile, and proteolytic enzymes - These properties make it possible for them to replicate to high titers in the GI tract.
WEB RESOURCES
CDC Adenovirus information
Clinical Syndromes
Almost half of adenoviral infections are subclinical
Most infections are self-limited and induce type-specific immunity
Incubation period is 2-14 days; for gastroenteritis usually 3-10 days
Different clinical syndromes have been described:
Eye
Epidemic Keratoconjunctivitis (EKC), acute follicular conjunctivitis, pharyngoconjunctival fever
Respiratory system
Common cold (rhinitis), pharyngitis (with or without fever), tonsillitis, bronchitis, pharyngoconjunctival fever, acute respiratory disease (LRI), pertussis-like syndrome, pneumonia- sometimes with sequelae
Genitourinary
Acute hemorrhagic cystitis, orchitis, nephritis, oculogenital syndrome
Gastrointestinal
Gastroenteritis, mesenteric adenitis, intussusception, hepatitis, appendicitis. Diarrhea tends to last longer than with other viral gastroenteritides
Rare results of adenovirus infections include- Meningitis, encephalitis, arthritis, skin rash, myocarditis, pericarditis, hepatitis. Fatal disease may occur in immunocompromised patients, as a result of a new infection or reactivation of latent virus
[pic]Figure . Weekly reports of respiratory adenovirus in the US. Seasonal variation. CDC
ADENOVIRUS- CLINICAL SYNDROMES (compiled)
|Clinical Syndrome |Features |Serotypes commonly Involved|Serotypes rarely |
| | | |Involved |
|URI |Coryza, pharyngitis, tonsillitis, fever |1, 2, 3, 5, 7 |4, 6, 11, 18, 21, |
| | | |29, 30 |
|Pharyngo-conjunctival fever |Fever, conjunctivits, pharyngitis, |3, 4, 7, 14 |1, 11, 16, 19, 37 |
| |headache, rash, lymphadenopathy | | |
|LRI |Bronchitis, pneumonia, fever, cough |3, 4, 7, 21 |14, 1, 2, 5, 35 |
|Pneumonia |Fever, respiratory distress, cough, |7 |1, 2, 3,4, 14, 21, |
| |severe in young children and infants | |7b |
|Pertussis-like Syndrome |Fever, paroxysmal cough, post-tussive |5 |1, 2, 3, 12, 14, 19,|
| |vomiting | |21, 35 |
|Acute Respiratory Disease |Tracheobronchitis, pneumonia, fever; |4, 7 |2, 3, 5, 8, 11, 14, |
| |epidemics in military recruits | |21 |
|Epidemic |Headache, conjunctivitis followed by |8, 19, 37 |2-7, 14, 15, 19, 37 |
|Keratoconjunctivitis |keratitis, preauricular lymphnodes | | |
|Acute follicular/ |Chemosis, follicles, subconjunctival |11 | |
|Hemorrhagic conjunctivitis |hemorrhage, preauricular lymph nodes | | |
|Acute Hemorrhagic cystitis |Blood in urine (macroscopic hematuria) |11, 4, 7, 1, 21 |34, 35 |
| |fever, dysuria | | |
|Gastro-enteritis |Diarrhea especially in children ................
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