- Email: [email protected]
Parainfluenza Viruses of Humans
Parainfluenza Viruses of Humans
coronaviruses. Some animal paramyxovirus-based vectors can be useful for the development of vaccines against emerging human infections such as H5N1 avian influenza, severe acute respiratory syndrome (SARS), and those caused by Ebola, Marburg, Nipah, and Hendra viruses. Since animal paramyxoviruses can be chosen that are serologically unrelated to common human pathogens, the general human population is susceptible to immunization with animal paramyxovirus-vectored vaccines. Reverse genetics systems are currently available for many but not all animal paramyxoviruses. Therefore, there is a great need to develop reverse genetics systems for the remaining animal paramyxoviruses. Furthermore, it is necessary to develop reverse genetics systems of local paramyxovirus strains for development of effective vaccines against the prevailing virus strains. In addition to vaccine development, it is also important to understand the pathogenesis and determinants of virus virulence and the mechanisms of interspecies transmission of the viruses. Due to the availability of reverse genetics systems for these viruses, we are confident that the next decade will bring a significant improvement in our understanding of their biology and we will witness development of better and safer vaccines against animal diseases.
47
See also: Measles Virus; Mumps Virus; Parainfluenza Viruses of Humans; Viral Pathogenesis; Human Respiratory Syncytial Virus; Rinderpest and Distemper Viruses.
Further Reading Conzelmann KK (2004) Reverse genetics of mononegavirales. Current Topics in Microbiology and Immunology 283: 1–41. Easton AJ, Domachowske JB, and Rosenberg HF (2004) Animal pneumoviruses: Molecular genetics and pathogenesis. Clinical Microbiology Reviews 17: 390–412. Kurath G, Batts WN, Ahme W, and Winton JR (2004) Complete genome sequence of fer-de-lance virus reveals a novel gene in reptilian paramyxoviruses. Journal of Virology 78: 2045–2056. Lamb RA, Collins PL, Kolakofsky D, et al. (2005) Paramyxoviridae. In: Fauquet CM, Mayo MA, Maniloff J, Desselberger U, and Ball LA (eds.) Virus Taxonomy: Eighth Report of the International Committee on Taxonomy of Viruses, pp. 655–668. San Diego, CA: Elsevier Academic Press. Lamb RA and Parks GD (2006) Paramyxoviridae: The viruses and their replication. In: Knipe DM, Howley PM, Griffin DE, et al. (eds.) Fields Virology, 5th edn., pp. 1449–1496. Philadelphia: Lippincott Williams and Wilkins. Li Z, Yu M, Zhang H, et al. (2006) Beilong virus, a novel paramyxovirus with the largest genome of non-segmented negative-stranded RNA viruses. Virology 346: 219–228. Wang LF, Harcourt BH, Yu M, et al. (2001) Molecular biology of Hendra and Nipah viruses. Microbes and Infection 3: 279–287.
Parainfluenza Viruses of Humans E Adderson and A Portner, St. Jude Children’s Research Hospital, Memphis, TN, USA ã 2008 Elsevier Ltd. All rights reserved.
Introduction
Taxonomy and Classification
The human parainfluenza viruses (hPIVs) are an important cause of respiratory disease in infants and children. Four types were discovered between 1956 and 1960. hPIV-1, hPIV-2, and hPIV-3 were first isolated from infants and children with lower respiratory tract (LRT) disease and subsequently shown to be a major cause of croup (type 1) and pneumonia and bronchiolitis (type 3). hPIV-4 was initially isolated from young adults and has been associated with mild upper respiratory tract disease of children and adults. Other viruses antigenically and structurally related to the human paramyxoviruses have been isolated from animals. Sendai virus, a natural pathogen of mice and not of humans, was the first PIV isolated and is antigenically related to human PIV-1. Simian virus (SV5) now PIV-5, recovered from primary monkey kidney cells, causes croup in dogs and is related to human type 2, and bovine shipping fever virus is a subtype of type 3.
The PIVs belong to two genera, human parainfluenza virus types 1 and 3 to Respirovirus and human parainfluenza virus types 2, 4a, and 4b to Rubulavirus, of the subfamily Paramyxovirinae in the family Paramyxoviridae. Some other species found in the Rubulavirus are mumps virus, which causes disease in humans and in the genus Respirovirus Sendai virus in mice and Bovine parainfluenza type 3. The family Paramyxoviridae belongs to the order Mononegavirales, the distinctive feature of which is a negative-stranded RNA genome and a similar strategy of replication, suggesting that all negativestranded viruses may have evolved from an archetypal virus.
Virion Structure, Genome Organization, and Protein Composition The hPIVs are roughly spherical, lipoprotein enveloped particles 150–250 nm in diameter with an internal helical
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nucleocapsid containing the negative-sense single-stranded RNA genome. Projecting from the surface of the virion are the hemagglutinin–neuraminidase (HN) and fusion (F) glycoproteins. These glycoproteins are anchored to the plasma membrane of the infected cell or the virion envelope by a hydrophobic transmembrane region. In paramyxoviruses the transmembrane domain of HN is located near the N-terminus of the molecule and F near the C-terminus. Extending into the cytoplasm of the infected cell or inside the virion membrane is a short hydrophilic tail region, which plays a role in viral assembly through its interaction with the matrix (M) protein which lines the inner surface of the plasma membrane and itself interacts with the nucleocapsid. Inside the lipid bilayer of the viral particle is the RNA nucleocapsid which houses the nonsegmented negative-stranded RNA genome. The genomes of hPIV-1, hPIV-2, and hPIV-3 are approximately 15 000 nt and all genes except L have been sequenced for hPIV-4a and -4b and are certain to fall in this range once the sequencing is complete. The RNA genome serves as a template for transcription of mRNAs specifying six virion structural proteins linked in tandem in the order of 30 -nucleoprotein (NP)–polymerase-associated protein (P/V)–matrix protein (M)–fusion protein (F)–hemagglutinin-neuraminidase (HN)–large protein (L)-50 . The P gene alone is unique in its capacity to express, in addition to the P protein, various other proteins by utilizing internal initiation codons in the same or different reading frames or by RNA editing of the P gene mRNA through insertion of nontemplated G residues. Besides P, these other proteins have been designated V, C, and D, depending on the PIV. The helical nucleocapsid is 18 nm in diameter, contains approximately
2000 molecules of NP bound to the RNA genome, about 200 P and 20 L molecules. The P protein, which forms a polymerase complex with L, is essential for the enzymatic processes of viral RNA transcription and replication including the 30 addition of poly(A), modification of the 50 end, and nucleotide polymerization of viral transcripts. The approximate molecular weights in daltons of the PIV proteins as exemplified by PIV-3 are: NP, 58 000; P, 68 000; M, 40 000; F, 63 000; HN, 72 000; and L, 256 000. A cartoon and an electron micrograph of a naturally occurring PIV are shown in Figure 1.
Attachment Protein The process of infection is initiated by the action of the HN glycoprotein, which binds the virion to sialic acid-containing glycoprotein or glycolipid receptors on the host cell surface. The same process is responsible for the hemagglutination of avian and mammalian erythrocytes. HN also causes the enzymatic (neuraminidase) cleavage of sialic acid residues from the carbohydrate moiety of glycoproteins and glycolipids, which functionally serves to prevent the self-aggregation of virus during release and likely aids in the spread of virus from infected cells. Besides attachment, HN provides an unknown function that is either essential or enhances the fusion activity of the F protein. It is proposed that HN directly interacts with F, possibly altering its conformation, thereby stimulating the fusion activity of F. The morphology of HN based on studies of PIVs such as Sendai virus is envisioned as an N-terminal stalk region
Membrane proteins • HN: receptor-binding, neuraminidase, fusion promotion • F: membrane fusion • M: assembly
Nucleocapsid • NP: encapsidate viral RNA • P: forms polymerase complex with L • L: RNA synthesis, capping, polyadenylation
Figure 1 Parainfluenza virus structure.
Parainfluenza Viruses of Humans
49
of approximately 130 amino acids anchoring a large glycosylated hydrophilic globular head region to the viral envelope. A small uncharged hydrophobic peptide located near the N-terminus spans the viral envelope, and a small hydrophilic domain is internal to the membrane. The globular head contains the active site for virus attachment, neuraminidase activity, and antigenic determinants that induce neutralizing antibodies for hPIVs, as well as the other members of the Respirovirus and Rubulavirus such as Sendai virus. HN exits on the surface of the virion as disulfide-linked homodimers or tetramers. Solution of the three-dimensional structure of the HN protein of NDV and more recently of hPIV-3 and SV5 has resolved many of the previous issues concerning the structure and functions of HN. A significant advance was the determination that the HN sialic acid-binding site and the neuraminidase active site were the same and for NDV that conformational change of the site switches HN activity from sialic acid binding (attachment) to sialic acid hydrolysis (neuraminidase activity). However, similar conformational changes may not occur for hPIV-3 and SV5 HN.
Fl, forms the new hydrophobic N-terminus, which causes membrane fusion. The smaller product, F2, is the original approximately 100 N-terminal residues of FO. Recent solutions of the crystal structure of the F in its prefusion and postfusion conformations reveals dramatic alterations in the F protein structure during membrane fusion and offers insight into the mechanism of F activation.
Fusion Protein
Transcription, Translation, and Replication
Following virus attachment, F, the other surface glycoprotein, mediates the fusion of the virion and host cell-surface membranes, which allows the nucleocapsid to be deposited in the cell cytoplasm where gene expression begins. F expressed on the surface of infected cells also mediates fusion, allowing the extension of infection to uninfected cells. All F proteins are synthesized as inactive precursors (FO) which are post-translationally cleaved by a host cell trypsin-like protease to form the biologically active molecule. For the paramyxoviruses in general, the cleavage site is located about 100 amino acids from the N-terminus of FO. The cleavage site is characterized by a short span of basic amino acids on the amino side of the site and a longer stretch of about 30 hydrophobic residues on the carboxyl side. The number and location of basic amino acids on the amino side of the cleavage site varies with individual PIVs. hPIV-1 and Sendai virus have one basic amino acid immediately adjacent to the cleavage site (Arg), whereas PIV-2 and PIV-3 have two (Arg–Lys) and PIV-5 has five (all Arg). The motif of paired basic amino acids at the cleavage site increases the efficiency of cleavage, host and tissue tropism, and pathogenicity, as the dibasic motif is recognized by a ubiquitous protease, whereas the enzymes that cleave at a monobasic site are found in a limited number of tissues. Cleavage of FO results in two disulfide-linked fragments; the larger one,
M Protein The M protein lines the inner surface of the viral envelope and is thought to play a role in virus maturation by interacting with the envelope glycoproteins and the nucleocapsid. During infection M associates with the inner leaflet of the plasma membrane where it orchestrates the release (budding) of progeny virus by interacting with specific sites on the cytoplasmic tail of the viral glycoproteins and the nucleocapsid and in transport of viral components to the budding site.
The negative-strand strategy of viral replication involves the synthesis of unique mRNA species of each paramyxovirus gene during infection. After introduction of the infecting nucleocapsid into the host cell, transcription is the first step in gene expression. Transcriptional regulation of mRNA abundance is determined by the gene order; the closer the gene is to the 30 end of the genome, the more efficient is the transcription. Thus, the abundance of paramyxovirus proteins is determined mainly by the polarity of the genome. Once protein synthesis is underway, replication of the genome begins, which provides additional templates for transcription and replication. Development of ‘reverse genetics’ systems for SV and hPIV-3 as well as other members of the paramyxovirus family, in which the RNA genomes of these viruses are expressed from a DNA template, has provided tools and facilitated our understanding of PIV protein structures and function, virus assembly, regulation of RNA transcription and replication, and viral pathogenesis.
Geographic and Seasonal Distribution All of the hPIV types 1–4 have a wide geographic distribution. PIV-1–PIV-3 have been identified in most areas where facilities are available for the study of childhood
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respiratory tract diseases. PIV-4 has been isolated in fewer areas but this is likely to be due to the difficulty of isolation and it is probably widely distributed.
Host Range and Viral Propagation The four hPIV types were originally isolated from humans: they cause disease in humans and humans are the primary host. Other laboratory animals can serve as experimental hosts; hamsters, guinea pigs, and ferrets can be infected with hPIV-1, hPIV-2, and hPIV-3 but these infections are usually asymptomatic. hPIV-3 can also infect cotton rats, rhesus, and patas monkeys and chimpanzees, but these animals are not good model systems for studying disease caused by PIVs. Embryonated hen’s eggs can support the growth of some strains of hPIV-1, hPIV-2, and hPIV-3, but are much less sensitive than monkey kidney cells for primary isolation. Sendai virus, a murine subtype of PIV-1, is an exception in that it grows exceedingly well in eggs. All four hPIV types grow well in primary monkey or human kidney cells, which are also used in the isolation of virus from clinical samples. LLC-MK2, a rhesus monkey kidney cell line, offers an efficient system for the isolation of PIVs and an experimental tissue culture system. PIV-2 and PIV-1 require trypsin in the medium to cleave the F glycoprotein for cell growth, but not PIV-3 strains. Viral infection of tissue culture can be detected by hemadsorption with chicken and guinea pig erythrocytes and the cytopathic effects produced by the viruses.
Genetics and Evolution The entire genomic sequences of hPIV-1, hPIV-2, and hPIV-3 have been completed. The genomes of other members of the genera Respirovirus and Rubulavirus (Sendai virus, bPIV-3, and PIV-5) have been sequenced as well. In general, the genome organization is remarkably similar, but differences exist in sequence, intergenic regions, and nonstructural proteins expressed from the P gene. Sequence and immunological analyses suggest that human hPIV-1 and Sendai virus are closely related type 1 PIVs, as is PIV-3 of human and bovine origin. hPIV-1 and -3 are more closely related to each other than to hPIV-2, PIV-5, mumps virus and NDV, which in turn show a closer evolutionary relationship to each other. Evolutionary divergence of hPIV-3 and hPIV-1 is greatest for the P protein (a phenomenon of paramyxovirus P proteins in general), less for the HN and F glycoproteins, and least for M, NP, and L. Similarly,
hPIV-1 and Sendai virus, and human and bovine PIV-3, show the greatest divergence in the P protein and least for NP.
Serologic Relationships and Variability The human paramyxoviruses can be divided into two antigenic groups, comprised of PIV-1/PIV-3 and PIV-2/ PIV-4/mumps virus. Human PIVs share certain antigenic determinants, but structural and antigenic variation occurs between serotypes and, to a lesser degree, within strains belonging to the same type. Antigenic diversity is not generally progressive, but may contribute to PIVs’ propensity to cause recurrent infection. Antibody to F and HN correlates with neutralizing antibody and protective immunity.
Epidemiology and Transmission The hPIVs are an important cause of respiratory tract infections (Table 1). hPIV-3 infections are endemic, whereas infections caused by other PIVs occur in outbreaks. Infections are common before 2 years, especially with hPIV-3, and almost universal by 5 years. hPIVs are highly communicable by respiratory droplets and by contact with surfaces contaminated by respiratory secretions. The incubation period is 2–6 days. Virus is typically shed for 4–21 days.
Pathogenicity hPIVs bind to specific receptors on upper respiratory tract epithelial cells and may subsequently spread to the LRT. Optimal fusion efficiency requires the coordinated action of both F and HN. The virus and host factors contributing to disease pathogenesis are not completely understood. Both viral (F and HN structure) and host features (genetic susceptibility, host cell proteases, immunocompetence) are likely to influence the severity of infection. Table 1 Proportion of viral respiratory tract infections caused by PIV Proportion (%) Rhinitis (common cold) Pharyngitis Laryngotracheitis (croup) Bronchiolitis Pneumonia Otitis media (middle ear)
20 20 50–70 20 10 10
Most common type
PIV-1 PIV-3 PIV-1, PIV-3
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51
Clinical Features
Prevention and Control
hPIVs cause between 15% and 65% of viral respiratory infections, most notably laryngotracheitis (croup) and bronchiolitis (Table 1). Although infections are generally mild, PIVs are responsible for over 10% of pediatric hospitalizations for respiratory infection. Life-threatening LRT infection occurs in patients with cellular immunodeficiencies, particularly those with severe combined immunodeficiency and hematopoietic stem cell and solid organ transplants. Rare cases of parotiditis, myocarditis, aseptic meningitis, and encephalitis have been reported. The gold standard for diagnosis of hPIV infections is culture on primary monkey kidney, human embryonic kidney, or human lung carcinoma cells, which typically requires incubation for 4–7 days. Detection of epithelial cell-associated viral antigens in nasopharyngeal or bronchoalveolar washings is rapid and has a sensitivity and specificity of >80% compared to culture. Polymerase chain reaction (PCR) is rapid and more sensitive, but is not widely available. Detection of specific IgM and IgG in paired acute and convalescent sera has limited utility in the diagnosis of acute infections.
Most hPIV infections require supportive care only. Children with moderate-to-severe croup benefit from inhaled vasoconstrictors or inhaled or systemic corticosteroids, which reduce airway edema. Anecdotal reports describe the successful use of ribavirin in immunocompromised patients. There are currently no licensed vaccines to prevent hPIV. Early formalin-inactivated vaccines were poorly immunogenic and had the potential to enhance pulmonary inflammatory responses to infection with wild-type virus.
Pathology hPIV has direct cytopathic effects, causing ciliary damage, epithelial necrosis, and recruitment of a mononuclear inflammatory response. Recent studies suggest that host inflammatory responses, rather than the direct effects of viral replication, are most accountable for the signs and symptoms of PIV infection. Alterations in HN receptor-binding or -activity influence host inflammatory responses independent of viral replication or the ability to infect epithelial cells. PIV infection may trigger longterm bronchial hyperreactivity in genetically susceptible persons.
Immune Response Viral shedding is more prolonged and disease severity increased in persons with T-cell immunodeficiencies, implying cellular immunity is important in the control of acute hPIV infection. Protection from reinfection is associated with the development of serum and secretory antibody against F and HN. The duration of protection after acute illness is relatively short however, especially in infants, and protection from infection by heterotypic stains is incomplete.
Future Perspectives Novel antiviral therapies being developed for PIV infections include selective inhibitors of HN, fusion, and IMP dehydrogenase, and siRNA against phosphoprotein mRNA. Vaccines currently in early clinical trials include live attenuated human PIV-3, bovine PIV-3, human-bovine chimeras, and bovine PIV or Sendai virus expressing heterologous human PIV F and HN proteins. Besides vaccines, antiviral drugs may be designed that curtail viral replication or prevent viral spread. Knowledge of the threedimensional structure of HN and F, especially those regions involved in viral attachment, penetration, and release, will be important in drug design. See also: Paramyxoviruses of Animals; Human Respiratory Viruses; Viral Membranes.
Further Reading Karron RA and Collins PL (2006) Parainfluenza viruses. In: Knipe DM, Howley PM, Griffin DE, et al. (eds.) Fields Virology, 5th edn., pp. 1497–1526. Philadelphia, PA: Lippincott Williams and Wilkins. Lamb RA and Parks GD (2006) Paramyxoviridae: The viruses and their replication. In: Knipe DM, Howley PM, Griffin DE, et al. (eds.) Fields Virology, 5th edn., pp. 1449–1496. Philadelphia, PA: Lippincott Williams and Wilkins. Lamb RA, Paterson RG, and Jardetzky TS (2006) Paramyxovirus membrane fusion: Lessons from the F and HN atomic structures. Virology 344(1): 30–37. Morrison T and Portner A (1991) Structure, function and intracellular processing of the glycoproteins of the Paramyxoviridae. In: Kingsbury DW (ed.) The Paramyxoviruses, pp. 347–382. New York: Plenum. Subbarao K (2003) Parainfluenza viruses. In: Long SS, Pickering LK, and Prober CG (eds.) Principles and Practice of Pediatric Infectious Diseases, 2nd edn., p. 1131. Philadelphia, PA: Churchill Livingstone. Takimoto T and Portner A (2004) Molecular mechanism of paramyxovirus budding. Virus Research 106: 133–145.
coronaviruses. Some animal paramyxovirus-based vectors can be useful for the development of vaccines against emerging human infections such as H5N1 avian influenza, severe acute respiratory syndrome (SARS), and those caused by Ebola, Marburg, Nipah, and Hendra viruses. Since animal paramyxoviruses can be chosen that are serologically unrelated to common human pathogens, the general human population is susceptible to immunization with animal paramyxovirus-vectored vaccines. Reverse genetics systems are currently available for many but not all animal paramyxoviruses. Therefore, there is a great need to develop reverse genetics systems for the remaining animal paramyxoviruses. Furthermore, it is necessary to develop reverse genetics systems of local paramyxovirus strains for development of effective vaccines against the prevailing virus strains. In addition to vaccine development, it is also important to understand the pathogenesis and determinants of virus virulence and the mechanisms of interspecies transmission of the viruses. Due to the availability of reverse genetics systems for these viruses, we are confident that the next decade will bring a significant improvement in our understanding of their biology and we will witness development of better and safer vaccines against animal diseases.
47
See also: Measles Virus; Mumps Virus; Parainfluenza Viruses of Humans; Viral Pathogenesis; Human Respiratory Syncytial Virus; Rinderpest and Distemper Viruses.
Further Reading Conzelmann KK (2004) Reverse genetics of mononegavirales. Current Topics in Microbiology and Immunology 283: 1–41. Easton AJ, Domachowske JB, and Rosenberg HF (2004) Animal pneumoviruses: Molecular genetics and pathogenesis. Clinical Microbiology Reviews 17: 390–412. Kurath G, Batts WN, Ahme W, and Winton JR (2004) Complete genome sequence of fer-de-lance virus reveals a novel gene in reptilian paramyxoviruses. Journal of Virology 78: 2045–2056. Lamb RA, Collins PL, Kolakofsky D, et al. (2005) Paramyxoviridae. In: Fauquet CM, Mayo MA, Maniloff J, Desselberger U, and Ball LA (eds.) Virus Taxonomy: Eighth Report of the International Committee on Taxonomy of Viruses, pp. 655–668. San Diego, CA: Elsevier Academic Press. Lamb RA and Parks GD (2006) Paramyxoviridae: The viruses and their replication. In: Knipe DM, Howley PM, Griffin DE, et al. (eds.) Fields Virology, 5th edn., pp. 1449–1496. Philadelphia: Lippincott Williams and Wilkins. Li Z, Yu M, Zhang H, et al. (2006) Beilong virus, a novel paramyxovirus with the largest genome of non-segmented negative-stranded RNA viruses. Virology 346: 219–228. Wang LF, Harcourt BH, Yu M, et al. (2001) Molecular biology of Hendra and Nipah viruses. Microbes and Infection 3: 279–287.
Parainfluenza Viruses of Humans E Adderson and A Portner, St. Jude Children’s Research Hospital, Memphis, TN, USA ã 2008 Elsevier Ltd. All rights reserved.
Introduction
Taxonomy and Classification
The human parainfluenza viruses (hPIVs) are an important cause of respiratory disease in infants and children. Four types were discovered between 1956 and 1960. hPIV-1, hPIV-2, and hPIV-3 were first isolated from infants and children with lower respiratory tract (LRT) disease and subsequently shown to be a major cause of croup (type 1) and pneumonia and bronchiolitis (type 3). hPIV-4 was initially isolated from young adults and has been associated with mild upper respiratory tract disease of children and adults. Other viruses antigenically and structurally related to the human paramyxoviruses have been isolated from animals. Sendai virus, a natural pathogen of mice and not of humans, was the first PIV isolated and is antigenically related to human PIV-1. Simian virus (SV5) now PIV-5, recovered from primary monkey kidney cells, causes croup in dogs and is related to human type 2, and bovine shipping fever virus is a subtype of type 3.
The PIVs belong to two genera, human parainfluenza virus types 1 and 3 to Respirovirus and human parainfluenza virus types 2, 4a, and 4b to Rubulavirus, of the subfamily Paramyxovirinae in the family Paramyxoviridae. Some other species found in the Rubulavirus are mumps virus, which causes disease in humans and in the genus Respirovirus Sendai virus in mice and Bovine parainfluenza type 3. The family Paramyxoviridae belongs to the order Mononegavirales, the distinctive feature of which is a negative-stranded RNA genome and a similar strategy of replication, suggesting that all negativestranded viruses may have evolved from an archetypal virus.
Virion Structure, Genome Organization, and Protein Composition The hPIVs are roughly spherical, lipoprotein enveloped particles 150–250 nm in diameter with an internal helical
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nucleocapsid containing the negative-sense single-stranded RNA genome. Projecting from the surface of the virion are the hemagglutinin–neuraminidase (HN) and fusion (F) glycoproteins. These glycoproteins are anchored to the plasma membrane of the infected cell or the virion envelope by a hydrophobic transmembrane region. In paramyxoviruses the transmembrane domain of HN is located near the N-terminus of the molecule and F near the C-terminus. Extending into the cytoplasm of the infected cell or inside the virion membrane is a short hydrophilic tail region, which plays a role in viral assembly through its interaction with the matrix (M) protein which lines the inner surface of the plasma membrane and itself interacts with the nucleocapsid. Inside the lipid bilayer of the viral particle is the RNA nucleocapsid which houses the nonsegmented negative-stranded RNA genome. The genomes of hPIV-1, hPIV-2, and hPIV-3 are approximately 15 000 nt and all genes except L have been sequenced for hPIV-4a and -4b and are certain to fall in this range once the sequencing is complete. The RNA genome serves as a template for transcription of mRNAs specifying six virion structural proteins linked in tandem in the order of 30 -nucleoprotein (NP)–polymerase-associated protein (P/V)–matrix protein (M)–fusion protein (F)–hemagglutinin-neuraminidase (HN)–large protein (L)-50 . The P gene alone is unique in its capacity to express, in addition to the P protein, various other proteins by utilizing internal initiation codons in the same or different reading frames or by RNA editing of the P gene mRNA through insertion of nontemplated G residues. Besides P, these other proteins have been designated V, C, and D, depending on the PIV. The helical nucleocapsid is 18 nm in diameter, contains approximately
2000 molecules of NP bound to the RNA genome, about 200 P and 20 L molecules. The P protein, which forms a polymerase complex with L, is essential for the enzymatic processes of viral RNA transcription and replication including the 30 addition of poly(A), modification of the 50 end, and nucleotide polymerization of viral transcripts. The approximate molecular weights in daltons of the PIV proteins as exemplified by PIV-3 are: NP, 58 000; P, 68 000; M, 40 000; F, 63 000; HN, 72 000; and L, 256 000. A cartoon and an electron micrograph of a naturally occurring PIV are shown in Figure 1.
Attachment Protein The process of infection is initiated by the action of the HN glycoprotein, which binds the virion to sialic acid-containing glycoprotein or glycolipid receptors on the host cell surface. The same process is responsible for the hemagglutination of avian and mammalian erythrocytes. HN also causes the enzymatic (neuraminidase) cleavage of sialic acid residues from the carbohydrate moiety of glycoproteins and glycolipids, which functionally serves to prevent the self-aggregation of virus during release and likely aids in the spread of virus from infected cells. Besides attachment, HN provides an unknown function that is either essential or enhances the fusion activity of the F protein. It is proposed that HN directly interacts with F, possibly altering its conformation, thereby stimulating the fusion activity of F. The morphology of HN based on studies of PIVs such as Sendai virus is envisioned as an N-terminal stalk region
Membrane proteins • HN: receptor-binding, neuraminidase, fusion promotion • F: membrane fusion • M: assembly
Nucleocapsid • NP: encapsidate viral RNA • P: forms polymerase complex with L • L: RNA synthesis, capping, polyadenylation
Figure 1 Parainfluenza virus structure.
Parainfluenza Viruses of Humans
49
of approximately 130 amino acids anchoring a large glycosylated hydrophilic globular head region to the viral envelope. A small uncharged hydrophobic peptide located near the N-terminus spans the viral envelope, and a small hydrophilic domain is internal to the membrane. The globular head contains the active site for virus attachment, neuraminidase activity, and antigenic determinants that induce neutralizing antibodies for hPIVs, as well as the other members of the Respirovirus and Rubulavirus such as Sendai virus. HN exits on the surface of the virion as disulfide-linked homodimers or tetramers. Solution of the three-dimensional structure of the HN protein of NDV and more recently of hPIV-3 and SV5 has resolved many of the previous issues concerning the structure and functions of HN. A significant advance was the determination that the HN sialic acid-binding site and the neuraminidase active site were the same and for NDV that conformational change of the site switches HN activity from sialic acid binding (attachment) to sialic acid hydrolysis (neuraminidase activity). However, similar conformational changes may not occur for hPIV-3 and SV5 HN.
Fl, forms the new hydrophobic N-terminus, which causes membrane fusion. The smaller product, F2, is the original approximately 100 N-terminal residues of FO. Recent solutions of the crystal structure of the F in its prefusion and postfusion conformations reveals dramatic alterations in the F protein structure during membrane fusion and offers insight into the mechanism of F activation.
Fusion Protein
Transcription, Translation, and Replication
Following virus attachment, F, the other surface glycoprotein, mediates the fusion of the virion and host cell-surface membranes, which allows the nucleocapsid to be deposited in the cell cytoplasm where gene expression begins. F expressed on the surface of infected cells also mediates fusion, allowing the extension of infection to uninfected cells. All F proteins are synthesized as inactive precursors (FO) which are post-translationally cleaved by a host cell trypsin-like protease to form the biologically active molecule. For the paramyxoviruses in general, the cleavage site is located about 100 amino acids from the N-terminus of FO. The cleavage site is characterized by a short span of basic amino acids on the amino side of the site and a longer stretch of about 30 hydrophobic residues on the carboxyl side. The number and location of basic amino acids on the amino side of the cleavage site varies with individual PIVs. hPIV-1 and Sendai virus have one basic amino acid immediately adjacent to the cleavage site (Arg), whereas PIV-2 and PIV-3 have two (Arg–Lys) and PIV-5 has five (all Arg). The motif of paired basic amino acids at the cleavage site increases the efficiency of cleavage, host and tissue tropism, and pathogenicity, as the dibasic motif is recognized by a ubiquitous protease, whereas the enzymes that cleave at a monobasic site are found in a limited number of tissues. Cleavage of FO results in two disulfide-linked fragments; the larger one,
M Protein The M protein lines the inner surface of the viral envelope and is thought to play a role in virus maturation by interacting with the envelope glycoproteins and the nucleocapsid. During infection M associates with the inner leaflet of the plasma membrane where it orchestrates the release (budding) of progeny virus by interacting with specific sites on the cytoplasmic tail of the viral glycoproteins and the nucleocapsid and in transport of viral components to the budding site.
The negative-strand strategy of viral replication involves the synthesis of unique mRNA species of each paramyxovirus gene during infection. After introduction of the infecting nucleocapsid into the host cell, transcription is the first step in gene expression. Transcriptional regulation of mRNA abundance is determined by the gene order; the closer the gene is to the 30 end of the genome, the more efficient is the transcription. Thus, the abundance of paramyxovirus proteins is determined mainly by the polarity of the genome. Once protein synthesis is underway, replication of the genome begins, which provides additional templates for transcription and replication. Development of ‘reverse genetics’ systems for SV and hPIV-3 as well as other members of the paramyxovirus family, in which the RNA genomes of these viruses are expressed from a DNA template, has provided tools and facilitated our understanding of PIV protein structures and function, virus assembly, regulation of RNA transcription and replication, and viral pathogenesis.
Geographic and Seasonal Distribution All of the hPIV types 1–4 have a wide geographic distribution. PIV-1–PIV-3 have been identified in most areas where facilities are available for the study of childhood
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Parainfluenza Viruses of Humans
respiratory tract diseases. PIV-4 has been isolated in fewer areas but this is likely to be due to the difficulty of isolation and it is probably widely distributed.
Host Range and Viral Propagation The four hPIV types were originally isolated from humans: they cause disease in humans and humans are the primary host. Other laboratory animals can serve as experimental hosts; hamsters, guinea pigs, and ferrets can be infected with hPIV-1, hPIV-2, and hPIV-3 but these infections are usually asymptomatic. hPIV-3 can also infect cotton rats, rhesus, and patas monkeys and chimpanzees, but these animals are not good model systems for studying disease caused by PIVs. Embryonated hen’s eggs can support the growth of some strains of hPIV-1, hPIV-2, and hPIV-3, but are much less sensitive than monkey kidney cells for primary isolation. Sendai virus, a murine subtype of PIV-1, is an exception in that it grows exceedingly well in eggs. All four hPIV types grow well in primary monkey or human kidney cells, which are also used in the isolation of virus from clinical samples. LLC-MK2, a rhesus monkey kidney cell line, offers an efficient system for the isolation of PIVs and an experimental tissue culture system. PIV-2 and PIV-1 require trypsin in the medium to cleave the F glycoprotein for cell growth, but not PIV-3 strains. Viral infection of tissue culture can be detected by hemadsorption with chicken and guinea pig erythrocytes and the cytopathic effects produced by the viruses.
Genetics and Evolution The entire genomic sequences of hPIV-1, hPIV-2, and hPIV-3 have been completed. The genomes of other members of the genera Respirovirus and Rubulavirus (Sendai virus, bPIV-3, and PIV-5) have been sequenced as well. In general, the genome organization is remarkably similar, but differences exist in sequence, intergenic regions, and nonstructural proteins expressed from the P gene. Sequence and immunological analyses suggest that human hPIV-1 and Sendai virus are closely related type 1 PIVs, as is PIV-3 of human and bovine origin. hPIV-1 and -3 are more closely related to each other than to hPIV-2, PIV-5, mumps virus and NDV, which in turn show a closer evolutionary relationship to each other. Evolutionary divergence of hPIV-3 and hPIV-1 is greatest for the P protein (a phenomenon of paramyxovirus P proteins in general), less for the HN and F glycoproteins, and least for M, NP, and L. Similarly,
hPIV-1 and Sendai virus, and human and bovine PIV-3, show the greatest divergence in the P protein and least for NP.
Serologic Relationships and Variability The human paramyxoviruses can be divided into two antigenic groups, comprised of PIV-1/PIV-3 and PIV-2/ PIV-4/mumps virus. Human PIVs share certain antigenic determinants, but structural and antigenic variation occurs between serotypes and, to a lesser degree, within strains belonging to the same type. Antigenic diversity is not generally progressive, but may contribute to PIVs’ propensity to cause recurrent infection. Antibody to F and HN correlates with neutralizing antibody and protective immunity.
Epidemiology and Transmission The hPIVs are an important cause of respiratory tract infections (Table 1). hPIV-3 infections are endemic, whereas infections caused by other PIVs occur in outbreaks. Infections are common before 2 years, especially with hPIV-3, and almost universal by 5 years. hPIVs are highly communicable by respiratory droplets and by contact with surfaces contaminated by respiratory secretions. The incubation period is 2–6 days. Virus is typically shed for 4–21 days.
Pathogenicity hPIVs bind to specific receptors on upper respiratory tract epithelial cells and may subsequently spread to the LRT. Optimal fusion efficiency requires the coordinated action of both F and HN. The virus and host factors contributing to disease pathogenesis are not completely understood. Both viral (F and HN structure) and host features (genetic susceptibility, host cell proteases, immunocompetence) are likely to influence the severity of infection. Table 1 Proportion of viral respiratory tract infections caused by PIV Proportion (%) Rhinitis (common cold) Pharyngitis Laryngotracheitis (croup) Bronchiolitis Pneumonia Otitis media (middle ear)
20 20 50–70 20 10 10
Most common type
PIV-1 PIV-3 PIV-1, PIV-3
Parainfluenza Viruses of Humans
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Clinical Features
Prevention and Control
hPIVs cause between 15% and 65% of viral respiratory infections, most notably laryngotracheitis (croup) and bronchiolitis (Table 1). Although infections are generally mild, PIVs are responsible for over 10% of pediatric hospitalizations for respiratory infection. Life-threatening LRT infection occurs in patients with cellular immunodeficiencies, particularly those with severe combined immunodeficiency and hematopoietic stem cell and solid organ transplants. Rare cases of parotiditis, myocarditis, aseptic meningitis, and encephalitis have been reported. The gold standard for diagnosis of hPIV infections is culture on primary monkey kidney, human embryonic kidney, or human lung carcinoma cells, which typically requires incubation for 4–7 days. Detection of epithelial cell-associated viral antigens in nasopharyngeal or bronchoalveolar washings is rapid and has a sensitivity and specificity of >80% compared to culture. Polymerase chain reaction (PCR) is rapid and more sensitive, but is not widely available. Detection of specific IgM and IgG in paired acute and convalescent sera has limited utility in the diagnosis of acute infections.
Most hPIV infections require supportive care only. Children with moderate-to-severe croup benefit from inhaled vasoconstrictors or inhaled or systemic corticosteroids, which reduce airway edema. Anecdotal reports describe the successful use of ribavirin in immunocompromised patients. There are currently no licensed vaccines to prevent hPIV. Early formalin-inactivated vaccines were poorly immunogenic and had the potential to enhance pulmonary inflammatory responses to infection with wild-type virus.
Pathology hPIV has direct cytopathic effects, causing ciliary damage, epithelial necrosis, and recruitment of a mononuclear inflammatory response. Recent studies suggest that host inflammatory responses, rather than the direct effects of viral replication, are most accountable for the signs and symptoms of PIV infection. Alterations in HN receptor-binding or -activity influence host inflammatory responses independent of viral replication or the ability to infect epithelial cells. PIV infection may trigger longterm bronchial hyperreactivity in genetically susceptible persons.
Immune Response Viral shedding is more prolonged and disease severity increased in persons with T-cell immunodeficiencies, implying cellular immunity is important in the control of acute hPIV infection. Protection from reinfection is associated with the development of serum and secretory antibody against F and HN. The duration of protection after acute illness is relatively short however, especially in infants, and protection from infection by heterotypic stains is incomplete.
Future Perspectives Novel antiviral therapies being developed for PIV infections include selective inhibitors of HN, fusion, and IMP dehydrogenase, and siRNA against phosphoprotein mRNA. Vaccines currently in early clinical trials include live attenuated human PIV-3, bovine PIV-3, human-bovine chimeras, and bovine PIV or Sendai virus expressing heterologous human PIV F and HN proteins. Besides vaccines, antiviral drugs may be designed that curtail viral replication or prevent viral spread. Knowledge of the threedimensional structure of HN and F, especially those regions involved in viral attachment, penetration, and release, will be important in drug design. See also: Paramyxoviruses of Animals; Human Respiratory Viruses; Viral Membranes.
Further Reading Karron RA and Collins PL (2006) Parainfluenza viruses. In: Knipe DM, Howley PM, Griffin DE, et al. (eds.) Fields Virology, 5th edn., pp. 1497–1526. Philadelphia, PA: Lippincott Williams and Wilkins. Lamb RA and Parks GD (2006) Paramyxoviridae: The viruses and their replication. In: Knipe DM, Howley PM, Griffin DE, et al. (eds.) Fields Virology, 5th edn., pp. 1449–1496. Philadelphia, PA: Lippincott Williams and Wilkins. Lamb RA, Paterson RG, and Jardetzky TS (2006) Paramyxovirus membrane fusion: Lessons from the F and HN atomic structures. Virology 344(1): 30–37. Morrison T and Portner A (1991) Structure, function and intracellular processing of the glycoproteins of the Paramyxoviridae. In: Kingsbury DW (ed.) The Paramyxoviruses, pp. 347–382. New York: Plenum. Subbarao K (2003) Parainfluenza viruses. In: Long SS, Pickering LK, and Prober CG (eds.) Principles and Practice of Pediatric Infectious Diseases, 2nd edn., p. 1131. Philadelphia, PA: Churchill Livingstone. Takimoto T and Portner A (2004) Molecular mechanism of paramyxovirus budding. Virus Research 106: 133–145.