INFECTIOUS DISEASE

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VIROLOGY - CHAPTER  SIXTEEN

PARAINFLUENZA, RESPIRATORY SYNCYTIAL AND ADENO VIRUSES

Dr Margaret Hunt
Professor Emerita
University of South Carolina School of Medicine

TEACHING OBJECTIVES
Brief review of paramyxovirus virus structure, properties and classification.
Discussion of human parainfluenza virus infections, disease, epidemiology, prevention and treatment.
Discussion of respiratory syncytial virus infections, disease, epidemiology, prevention and treatment.
Discussion of human metapneumovirus infections, disease, epidemiology, prevention and treatment.
Brief review of adenovirus structure, properties and classification
Discussion of adenovirus infections, disease, epidemiology, prevention and treatment.
 

The common cold
The common cold is as an acute, self-limited catarrhal (Latin catarrhus, to flow down) syndrome limited to the mucosal membranes of the upper respiratory tract. Rhinoviruses, of which there are more than 100 serotypes, cause an estimated 30% to 50% of colds. Coronaviruses account for perhaps 10% of cases.

The viruses covered in this chapter are additional frequent causes of the common cold, However, they can also cause more serious disease.
 

PARAMYXOVIRUSES GENERAL

Classification

Family Paramyxoviridae

Genus Members

Paramyxovirus - Parainfluenza [PIV types 1,2,3,4]

Rubulavirus - Mumps virus

Newcastle Disease Virus [birds]

Sendai virus [mice]

Morbillivirus - Measles virus

Canine Distemper Virus

Pneumovirus - Respiratory Syncytial Virus (RSV)

Metapneumovirus

Structure

Paramyxoviruses are enveloped RNA viruses. Their RNA is negative-sense and is non-segmented. The nucleocapsid has helical symmetry

The vriral envelope contains two virally coded glycoproteins (table 1):

• The F protein which has fusion activity.

• The attachment protein - H, HN or G according to whether is has hemagglutination activity (H), hemagglutination plus neuraminidase activity (HN) or neither (G)

The general structure of paramyxovirsues is shown in figure 1

Table 1 Paramyxovirus family surface glycoproteins
GENUS	GLYCOPROTEINS	TYPICAL MEMBERS
PARAMYXOVIRUS SUB-FAMILY
Paramyxovirus	HN, F	HPIV1, HP1V3
Rubulavirus	HN, F	HPIV2, HPIV4, Mumps virus
Morbillivirus	H, F	Measles virus
PNEUMOVIRUS SUB-FAMILY
Pneumovirus	G, F	Respiratory syncytial virus
Metapneumovirus	G, F	Metapneumoviruses

 

Parainfluenza viruses are important viral pathogens causing upper and lower respiratory infections in adults and children. They are second to respiratory syncytial virus as a cause of lower respiratory tract disease in young children.

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

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 from 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, restricted to humans and antigenically stable. Most people have had a primary infection by all four serotypes by the age of five; infections occur as epidemics as well as sporadically. There can be repeated infections throughout life.

People usually shed virus for about 1 week but immunocompromised individuals may shed for much longer.

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): The virus is relatively unstable, but can survive on surfaces for a few hours.

Parainfluenza virus is highly contagious.

Weekly reports of parainfluenza type 2 in the US. Seasonal variation. CDC

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. 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. It is associated with fever, cough, hoarseness, stridor on inspiration and expiration.This is predominantly a  disease of children under six years of age (because they have narrower airways). Many viruses can cause croup but HPIV is the most common cause (HPIV1>HPIV2>HPIV3).

HPIV 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.

HPIV-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).

HPIV-4 is associated with mild upper respiratory infections. The upper airway is more often involved than the lower airway. There are two types of PIV4: A and B

Otitis media, parotitis, aseptic meningitis occur although they are rare.

Particularly severe and persistent infections are known to occur in immunocompromized children and adults in whom 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. These techniques are 70-90% sensitive.

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

Most HPIV infections are mild and self-limiting. There is no specific treatment and no anti-virals are available. Supportive treatment for croup includes humidification of air and racemic epinephrine (Racemic epinephrine is a 1:1 mixture of the dextrorotatory and levorotatory isomers of epinephrine. The L form is the active component) . 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 is probably more important for limiting infection.

Breastfeeding may protect babies from HPIVs during their first few months of life because mothers may have protective antibodies in their breast milk.

 

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.

Classification and structure

Family Paramyxoviridae, genus Pneumovirus.

Infection of cells by RSV often results in syncytium formation, hence the name. The virus was first discovered in chimpanzees (Chimpanzee Coryza Virus) and accidental infection in humans led to its recognition as a human pathogen.

These are spherical or pleomorphic enveloped viruses (100-350 nm) with single-stranded, negative sense linear RNA. There are two non-structural and eight structural proteins.

The envelope has two glycoproteins:

• F protein, the fusion protein, is important for fusion of viral particles to target cells and fusing infected cells to neighboring cells to form syncytia.
• G protein, 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 six hours, on gloves for  less than two hours. They rapidly lose viability with freeze-thaw cycles, in acidic conditions and with disinfectants.

Pathology and Pathogenesis

RSV attaches (via the G protein) to cells of nasal mucosa and upper respiratory tract. The F protein allows fusion of the viral envelope with the host cell plasma membrane. The virus can also infect the eye. Infected cells may undergo necrosis and syncytia form through cell-cell fusion (which is often seen with cultured cells). Cell to cell transfer of virus leads to spread from upper to lower respiratory tract.

Mucosal edema occurs and there is increased mucin secretion. There is also cell necrosis that leads to sloughing of debris. Smaller airways (bronchioles) become plugged with debris and mucin; bronchoconstriction also occurs. Peribronchial lymphocytes may infiltrate the tissue.

The host immune response also induces some of the pathological changes. IgE response in some individuals is linked to airway hyper-reactivity. Cell-mediated immunity and humoral response limits the severity of the infection.

Epidemiology

RSV has a worldwide distribution and and is an important cause of lower respiratory tract disease in young infants. Most children have had an RSV infection by age 4 years. RSV is the most frequent cause of bronchiolitis but is an infrequent cause of croup.

Outbreaks are seasonal occurring from late fall through spring (November to May), the virus being  transmitted via large droplets, through fomites and via the hands. RSV can survive on surfaces for up to six hours. Viral shedding continues for  less than 1 to 3 weeks but longer in immuno-compromised hosts.

Nosocomial spread is common. Viral shedding can last for up to three weeks and infants can show a high titer of shed virus, especially initially (10⁷ viral particles per ml). Asymptomatic viral shedding is also observed. There is prolonged shedding in immunocompromized individuals.

75,000 to 125,000 infants are hospitalized each year in the US because of RSV infections. These account for 50 to 90% of hospitalizations for bronchiolitis.

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)

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

The incubation period is 4 to 6 days (range: 2 to 8 days). First, there is an upper respiratory infection (‘bad cold’) in older children and adults with clinical features of fever, rhinitis, pharyngitis. Lower respiratory infection (bronchiolitis and/or pneumonia) may occur after  the upper respiratory infection and results in the clinical features of cough, tachypnea, respiratory distress, hypoxemia, cyanosis. The cough can persist for 3 weeks.

In young infants one may observe  apnea, lethargy, irritability, poor feeding, otitis media and croup.

Radiological examination may show atelectasis, streaking, hyperinflation and perihilar infiltrates, especially in the right middle and upper lobes.

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 immunocompromized hosts. There are up to 3000 deaths per year in the United States.
 

Diagnosis

Nasal washings, nasal aspirates or swabs should be transported on ice and processed immediately. Rapid diagnosis is carried out using  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 with immunofluorescence is useful. 

What is a Shell Vial?

Treatment

Treatment is usually supportive by the provision of fluids, oxygen, humidification of air, respiratory support. Steroids and bronchodilators have not proved useful.

Chemotherapy
Ribavirin (Virazole) (see chemotherapy) , a guanosine analogue (aerosol) has been used with some efficacy but is used  only in persons at high risk for severe disease (premature and immunocompromized infants).

An experimental nucleoside-analog drug taken orally, ALS-008176, has shown promise in decreasing viral load and increasing clearance. The time until the virus was undetectable in the experimental protocol was 1.3 to 2.3 days, depending on the dosage, compared to 7.2 days for patients given a placebo. This drug appears to inhibit replication of RSV in already infected cells as well as protecting epithelial cells.

 

Immunity

Humoral immunity 
Neutralizing antibodies are against F and G proteins. IgA is also produced during an infection. The level of neutralizing antibody does not correspond to neutralizing activity. Immunity is short lived, therefore reinfections are common. Newborns may have some innate immunity

An IgE response occurs in some individuals and may be a marker for future airway hyper-reactivity.

Cell mediated immunity
 This is carried out by T cells. Cytokine production contributes to illness.

Prevention of spread

Hand washing is important as is isolation and cohort nursing. Health care providers should wear protective gear, i.e.  gowns, gloves, masks and goggles

Immunization

Vaccine
The inactivated vaccine is no longer used because it was associated with an increase in severity of disease. Other subunit vaccine candidates are in trial phases but no vaccine is available yet.

Passive immunity by Palivizumab
Palivizumab (Synagis) is a humanized monoclonal antibody that gives passive immunity against RSV. It is made by recombinant DNA technology. The constituent antibodies bind an epitope in the A antigenic site of the RSV envelope fusion (F) protein on the surface of the virus thus blocking membrane fusion. It also prevents cell-cell fusion of RSV-infected cells. Certain RSV variants are resistant to Palivizumab in laboratory experiments as a result of mutation in the F protein at the antibody binding site. No known sequence variations outside the A antigenic site on RSV F have been demonstrated to render RSV resistant to neutralization by Palivizumab.

Palivizumab injections are recommended for infants that are high-risk for serious lower respiratory tract disease caused by RSV because of prematurity or other medical problems such as congenital heart disease. In Phase III clinical trials, Palivizumab reduced the risk of hospitalization as a result of RSV infection by about 50%. It is given once a month via intramuscular injection during the RSV season. It is also very expensive.
 

 

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 both upper and lower respiratory tract infections with symptoms of a cold, otitis media, pneumonia or bronchitis. There can also be pneumonia in marrow recipients that can possibly be fatal.

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) and allfour of these circulate in the population with the dominant strain varying.

Metapneumovirus can be detected by PCR but there is no commercially available testing.

Classification

Adenoviruses belong to family Adenoviridae, genus Mastadenovirus.

They 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.

 Structure of adenovirus

Adenovirus © Dr Stephen Fuller, 1998 

Structure

Adenoviruses 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.

Adenovirus
© Dr Linda M Stannard, University of Cape Town, South Africa, 1995 (used with permission).

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 viruses).

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.

Clinical Syndromes

Almost half of adenoviral infections are subclinical. Most infections are self-limited and induce type-specific immunity.

The 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

Epidemiology

Endemic, epidemic and sporadic infections occur. Outbreaks have been noted in military recruits, swimming pool users, residential institutions, hospitals, day care centers etc.

Transmission is by droplets, the fecal-oral route (direct and through poorly chlorinated water) or fomites

Many infections are subclinical

Infections are most communicable in the first few days of illness, however infective period continues since clinical infection may be followed by intermittent and prolonged rectal shedding

Secondary attack rate within families is up to 50%;

Adenovrius outbreaks are seasonal: Respiratory disease mainly occurs in late winter through early summer. Pharyngoconjunctival and EKC infections occur in the summer months while GI disease does not seem to be seasonal

Diagnosis

Clinical specimens, such as swabs (nasopharyngeal, conjuncticval, rectal, or other) and washings, corneal scrapings, stool, urine or biopsy and autopsy materials etc. should be transported in viral transport medium.

Viral Isolation in cell cultures is carried out in HeLa, human embryonic kidney (HEK) and human fetal diploid cells (HDFL). A549 cells lines are used for types 1-39.

Subgroup F (serotypes 40, 41) do not grow well in these cell lines, but do grow in Graham-293 (a modified HEK cell line).

Shell vial culture technique aids in faster detection.

Cytopathic effects include swelling and rounding of cells. Cells may become refractile and clustered into irregular clumps.

Isolation of virus from a pharyngeal specimen is more suggestive of a current clinical infection than from fecal specimen.

Rapid detection of enteric types (serotypes 40, 41) is by ELISA or immunofluorescnece antibody. Immune EM (aggregation with sera) may also be used

Other detection methods in current use include electron microscopy, polymerase chain reaction and nucleic acid probes.

Serology is mainly used for epidemiologic studies

Prevention

• Hand washing
• Contact precautions, respiratory precautions in health care settings
• Adequate chlorination of swimming pools
• Sterilization / disinfection of ophthalmologic equipment and use of single dose vials of ophthalmic medications

Vaccine

There is a live, enteric coated, oral vaccine (against types 4 and 7) which will prevent most illness caused by these two adenovirus types. The vaccine is only approved for military personnel 17 through 50 years of age but has side effects, some of which may be severe. Serious problems have been reported by about one per cent of vaccinees within six months of vaccination and include blood in the urine or stool, pneumonia, inflammation of the stomach or intestines
.

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This page last changed on Monday, June 06, 2016
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