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Molecular evolution of the Paramyxoviridae and Rhabdoviridae multiple-protein-encoding P gene.

Presented here is an analysis of the molecular evolutionary dynamics of the P gene among 76 representative sequences of the Paramyxoviridae and Rhabdoviridae RNA virus families. In a number of Paramyxoviridae taxa, as well as in vesicular stomatitis viruses of the Rhabdoviridae, the P gene encodes multiple proteins from a single genomic RNA sequence. These products include the phosphoprotein (P), as well as the C and V proteins. The complexity of the P gene makes it an intriguing locus to study from an evolutionary perspective. Amino acid sequence alignments of the proteins encoded at the P and N loci were used in independent phylogenetic reconstructions of the Paramyxoviridae and Rhabdoviridae families. P-gene-coding capacities were mapped onto the Paramyxoviridae phylogeny, and the most parsimonious path of multiple-coding-capacity evolution was determined. Levels of amino acid variation for Paramyxoviridae and Rhabdoviridae P-gene-encoded products were also analyzed. Proteins encoded in overlapping reading frames from the same nucleotides have different levels of amino acid variation. The nucleotide architecture that underlies the amino acid variation was determined in order to evaluate the role of selection in the evolution of the P gene overlapping reading frames. In every case, the evolution of one of the proteins encoded in the overlapping reading frames has been constrained by negative selection while the other has evolved more rapidly. The integrity of the overlapping reading frame that represents a derived state is generally maintained at the expense of the ancestral reading frame encoded by the same nucleotides. The evolution of such multicoding sequences is likely a response by RNA viruses to selective pressure to maximize genomic information content while maintaining small genome size. The ability to evolve such a complex genomic strategy is intimately related to the dynamics of the viral quasispecies, which allow enhanced exploration of the adaptive landscape.

Evolution, Molecular↗

Resistance of paramyxoviridae to type I interferon-induced Bos taurus Mx1 dynamin.

Typical targets of type I interferon (IFN)-induced antiviral Mx proteins known to date have been shown to share a common profile: single-stranded negative-sense RNA viruses. Among them, human MxA is known to interfere with the replication of measles, human, and bovine parainfluenza-3 viruses (BoPi3V), that is, three members of the Paramyxoviridae family. Recently, bovine Mx1 protein (BoMx1) was included in the group of Mx proteins with authenticated antiviral potential, as it dramatically represses the replication of vesicular stomatitis virus (VSV). As replication in bovine cells of Pi3, respiratory syncytial (RS), and Sendai (Se) viruses, all members of the same family, is known to be reduced on IFN-alpha incorporation into the culture medium, it was hypothesized that the BoMx1 pathway possibly was involved, its antiviral spectrum thus probably extending to Paramyxoviridae. In this study, probing of BoMx1-inhibiting effects was carried out by infecting a transgenic Vero cell line that allows tightly regulated conditional expression of BoMx1 after doxycycline treatment with a wide array of Paramyxoviridae. Expressing and nonexpressing cells displayed similar viability, cytopathic effects (CPEs), and amounts of infectious virus yields, whatever the infecting virus or the multiplicity of infection (moi) imposed. It is, therefore, concluded that BoMx1 does not interfere with Paramyxoviridae.

Animals↗

Molecular evolution of viral fusion and matrix protein genes and phylogenetic relationships among the Paramyxoviridae.

Phylogenetic relationships among the Paramyxoviridae, a broad family of viruses whose members cause devastating diseases of wildlife, livestock, and humans, were examined with both fusion (F) and matrix (M) protein-coding sequences. Neighbor-joining trees of F and M protein sequences showed that the Paramyxoviridae was divided into the two traditionally recognized subfamilies, the Paramyxovirinae and the Pneumovirinae. Within the Paramyxovirinae, the results also showed groups corresponding to three currently recognized genera: Respirovirus, Morbillivirus, and Rubulavirus. The relationships among the three genera of the Paramyxovirinae were resolved with M protein sequences and there was significant bootstrap support (100%) showing that members of the genus Respirovirus and the genus Morbillivirus were more closely related to each other than to members of the genus Rubulavirus. Both F and M phylogenies showed that Newcastle disease virus (NDV) was more closely related to the genus Rubulavirus than to the other two genera but were consistent with the proposal (B. S. Seal et al., 2000, Virus Res. 66, 1-11) that NDV be classified as a separate genus within the Paramyxovirinae. Both F and M phylogenies were also consistent with the proposal (L. Wang et al., 2000, J. Virol 74, 9972-9979) that Hendra virus be classified as a new genus closely related and basal to the genus Morbillivirus. Rinderpest was most closely related to measles and a more derived virus than to canine distemper virus, phocine distemper virus, or dolphin morbillivirus.

Databases, Nucleic Acid↗

Electron microscopy and antigenic studies of uncharacterized viruses. I. Evidence suggesting the placement of viruses in families Arenaviridae, Paramyxoviridae, or Poxviridae.

During approximately 35 years, investigators in various laboratories studying arbovirus ecology and epidemiology accumulated many virus isolates, more than 60 of which were not characterized or placed in taxa. By a combination of electron microscopic and antigenic studies we collected information sufficient to provisionally classify 60 isolates. Electron microscopic observations suggest that 20 are members of the virus family Bunyaviridae, 20 Rhabdoviridae, 14 Reoviridae, one Togaviridae, one Paramyxoviridae (Mapuera virus, from a bat), and one Poxviridae (Yoka virus, from mosquitoes). Serologic studies provided evidence sufficient to place some of these viruses in recognized antigenic groups, within families and genera, and to establish new antigenic groups and taxa for others. Three viruses were found to have morphologic and morphogenetic characteristics consistent with those of members of the family Arenaviridae: Quaranfil virus, a human pathogen, Johnston Atoll virus, isolated from birds and ticks, and Araguari virus, isolated from an opossum. This, the first in a series of three papers, described methods used for these investigations and also presents descriptions of viruses provisionally placed in the families Arenaviridae, Paramyxoviridae, or Poxviridae. Descriptions of viruses provisionally placed in families Bunyaviridae and Reoviridae are published in the second and third papers, respectively. Viruses of the family Rhabdoviridae have been described separately.

Animals↗

Comparison of the deduced matrix and fusion protein sequences of equine morbillivirus with cognate genes of the Paramyxoviridae.

The nucleotide sequence of the matrix protein of equine morbillivirus (EMV) was determined to be 1062 nucleotides and coded for a deduced protein of M(r) 40148 having a net charge of + 19 at neutral pH. The matrix protein gene was separated from the P and F genes by intercistronic regions of 546 and 469 nucleotides, respectively. The nucleotide sequence which coded for the F protein was 1641 nucleotides and coded for a deduced protein of 546 amino acids having an M(r) of 60,447 and a charge + 4 at neutral pH. Partial sequence information was also determined for the P/V proteins. M, P and F protein sequence comparisons revealed that a greater homology existed between EMV and known members of the morbillivirus genus than with other members of the Paramyxoviridae and that this homology resided within the central half of the protein for the fusion protein, the C-terminal half of the matrix protein and at certain sites with the P protein. Far greater homology was seen between the morbilliviruses and EMV than for the other paramyxoviridae. It was inferred from phylogenetic analyses that EMV was a distantly related member of the morbillivirus genus. A conserved sequence of 18 nucleotides (assumed to be the transcriptional editing site) was present in the P gene of EMV. Insertion of a single nucleotide residue within this site generated the C-terminus of a V-like, cysteine rich protein. Likewise, a conserved 'CTT' intergenic region presumed to be the transcription termination and polyadenylation signal was present in EMV between the P-M-F genes. The close sequence homology of these sites with that of morbilliviruses also inferred that EMV was a member of the morbillivirus genus.

Amino Acid Sequence↗

Determination of the disulfide bond arrangement of Newcastle disease virus hemagglutinin neuraminidase. Correlation with a beta-sheet propeller structural fold predicted for paramyxoviridae attachment proteins.

Disulfide bonds stabilize the structure and functions of the hemagglutinin neuraminidase attachment glycoprotein (HN) of Newcastle disease virus. Until this study, the disulfide linkages of this HN and structurally similar attachment proteins of other members of the paramyxoviridae family were undefined. To define these linkages, disulfide-linked peptides were produced by peptic digestion of purified HN ectodomains of the Queensland strain of Newcastle disease virus, isolated by reverse phase high performance liquid chromatography, and analyzed by mass spectrometry. Analysis of peptides containing a single disulfide bond revealed Cys(531)-Cys(542) and Cys(172)-Cys(196) linkages and that HN ectodomains dimerize via Cys(123). Another peptide, with a chain containing Cys(186) linked to a chain containing Cys(238), Cys(247), and Cys(251), was cleaved at Met(249) with cyanogen bromide. Subsequent tandem mass spectrometry established Cys(186)-Cys(247) and Cys(238)-Cys(251) linkages. A glycopeptide with a chain containing Cys(344) linked to a chain containing Cys(455), Cys(461), and Cys(465) was treated sequentially with peptide-N-glycosidase F and trypsin. Further treatment of this peptide by one round of manual Edman degradation or tandem mass spectrometry established Cys(344)-Cys(461) and Cys(455)-Cys(465) linkages. These data, establishing the disulfide linkages of all thirteen cysteines of this protein, are consistent with published predictions that the paramyxoviridae HN forms a beta-propeller structural fold.

Amidohydrolases↗

T cell cross-reactivity among viruses of the paramyxoviridae.

Blastogenesis and delayed-type hypersensitivity assays were used to examine mouse T cell responses to five viruses representing the three genera of the Paramyxoviridae. Cross-reactive T cell responses were observed in a lympho-proliferative assay for measles, mumps, respiratory syncytial, canine distemper and parainfluenza type 3 virus. Confirmation of T cell cross-reactivity among measles, mumps and respiratory syncytial virus was obtained with a delayed-type hypersensitivity test. These results show that T cell cross-reactivity is common for Paramyxoviridae viruses, even though these viruses show virtually no inter-genus antibody cross-reactivity. The cross-reactivity among respiratory syncytial, measles and mumps virus at the T cell level may have implications for usage of the attenuated measles/mumps/rubella (MMR) vaccine. Respiratory syncytial virus is contacted by many children before they receive the MMR vaccine and T cells induced by respiratory syncytial virus may influence subsequent development of immunity to measles and/or mumps virus.

Animals↗

The exceptionally large genome of Hendra virus: support for creation of a new genus within the family Paramyxoviridae.

An outbreak of acute respiratory disease in Hendra, a suburb of Brisbane, Australia, in September 1994 resulted in the deaths of 14 racing horses and a horse trainer. The causative agent was a new member of the family Paramyxoviridae. The virus was originally called Equine morbillivirus but was renamed Hendra virus (HeV) when molecular characterization highlighted differences between it and members of the genus Morbillivirus. Less than 5 years later, the closely related Nipah virus (NiV) emerged in Malaysia, spread rapidly through the pig population, and caused the deaths of over 100 people. We report the characterization of the HeV L gene and protein, the genome termini, and gene boundary sequences, thus completing the HeV genome sequence. In the highly conserved region of the L protein, the HeV sequence GDNE differs from the GDNQ found in almost all other nonsegmented negative-strand (NNS) RNA viruses. HeV has an absolutely conserved intergenic trinucleotide sequence, 3'-GAA-5', and highly conserved transcription initiation and termination sequences similar to those of respiroviruses and morbilliviruses. The large genome size (18,234 nucleotides), the unique complementary genome terminal sequences of HeV, and the limited homology with other members of the Paramyxoviridae suggest that HeV, together with NiV, should be classified in a new genus in this family. The large genome of HeV also fills a gap in the spectrum of genome sizes observed with NNS RNA virus genomes. As such, it provides a further piece in the puzzle of NNS RNA virus evolution.

Amino Acid Sequence↗

Accessory genes of the paramyxoviridae, a large family of nonsegmented negative-strand RNA viruses, as a focus of active investigation by reverse genetics.

The Paramyxoviridae, a large family of nonsegmented negative-strand RNA viruses, comprises several genera each containing important human and animal pathogens. They possess in common six basal genes essential for viral replication and, in addition, a subset of accessory genes that are largely unique to each genus. These accessory genes are either encoded in one or more alternative overlapping frames of a basal gene, which are accessed transcriptionally or translationally, or inserted before or between the basal genes as one or more extra genes. However, the question of how the individual accessory genes contribute to actual viral replication and pathogenesis remained unanswered. It was not even established whether they are dispensable or indispensable for the viral life cycle. The plasmid-based reverse genetics of the full-length viral genome has now come into wide use to demonstrate that most, if not all, of these putative accessory genes can be disrupted without destroying viral infectivity, conclusively defining them as indeed dispensable accessory genes. Studies on the phenotypes of the resulting gene knockout viruses have revealed that the individual accessory genes greatly contribute specifically and additively to the overall viral fitness both in vitro and in vivo.

Animals↗

Zoonotic disease in Australia caused by a novel member of the paramyxoviridae.

Twenty-three horses and three humans in Queensland, Australia, were infected with a novel member of the Paramyxoviridae family of viruses in two geographically distinct outbreaks. Two of the humans died-one died of rapid-onset respiratory illness, and the other died of encephalitis. The third infected human developed an influenza-like illness and made a complete recovery. All infected humans had close contact with sick horses. Since the two outbreaks occurred at sites 1,000 km apart and no known contact between the two groups of humans and horses occurred, extensive testing of animals and birds common to the two areas was conducted. Fruit bats (Pteropus species) were found to carry a virus identical to that found in the infected humans and horses. Although there was no contact between the infected humans and the bats, some form of close contact between the horses and bats is the likely mode of infection.

Adult↗

A novel P/V/C gene in a new member of the Paramyxoviridae family, which causes lethal infection in humans, horses, and other animals.

In 1994, a new member of the family Paramyxoviridae isolated from fatal cases of respiratory disease in horses and humans was shown to be distantly related to morbilliviruses and provisionally called equine morbillivirus (K. Murray et al., Science 268:94-97, 1995). To facilitate characterization and classification, the virus was purified, viral proteins were identified, and the P/V/C gene was cloned and sequenced. The coding strategy of the gene is similar to that of Sendai and measles viruses, members of the Paramyxovirus and Morbillivirus genera, respectively, in the subfamily Paramyxovirinae. The P/V/C gene contains four open reading frames, three of which, P, C, and V, have Paramyxovirinae counterparts. The P and C proteins are larger and smaller, respectively, than are cognate proteins in members of the subfamily, and the V protein is made as a result of a single G insertion during transcription. The P/V/C gene has two unique features. (i) A fourth open reading frame is located between those of the C and V proteins and potentially encodes a small basic protein similar to those found in some members of the Rhabdoviridae and Filoviridae families. (ii) There is also a long untranslated 3' sequence, a feature common in Filoviridae members. Sequence comparisons confirm that although the virus is a member of the Paramyxovirinae subfamily, it displays only low levels of homology with paramyxoviruses and morbilliviruses and negligible homologies with rubulaviruses.

Amino Acid Sequence↗

An apparently new virus (family Paramyxoviridae) infectious for pigs, humans, and fruit bats.

We isolated an apparently new virus in the family Paramyxoviridae from stillborn piglets with deformities at a piggery in New South Wales, Australia. In 1997, the pregnancy rate and litter size at the piggery decreased markedly, while the proportion of mummified fetuses increased. We found serologic evidence of infection in pigs at the affected piggery and two associated piggeries, in humans exposed to infected pigs, and in fruit bats. Menangle virus is proposed as a common name for this agent, should further studies confirm that it is a newly recognized virus.

Agricultural Workers' Diseases↗

Probable human infection with a newly described virus in the family Paramyxoviridae. The NSW Expert Group.

After an apparently new virus in the family Paramyxoviridae was isolated from pigs in August 1997, an investigation was carried out to assess its risk for humans. More than 250 persons with potential exposure to infected pigs were tested serologically. Two piggery workers with intense occupational exposure had high convalescent-phase antibody titers to this new virus. In early June 1997, both workers had an influenzalike illness with rash; serologic testing showed no alternative cause. Strong evidence indicates that the two men became ill from this new virus, but the mode of transmission from pigs to humans remains unknown.

Agricultural Workers' Diseases↗

Paramyxoviridae use distinct virus-specific mechanisms to circumvent the interferon response.

STAT1 and STAT2 are cellular transcription factors involved in interferon (IFN) signaling and are thus critical for the IFN-induced antiviral state. We have previously shown that the paramyxovirus Simian Virus 5 (SV5) blocks both type I and type II interferon (IFN) signaling by targeting STAT1 for proteasome-mediated degradation. To determine whether this is a feature common to all Paramyxoviridae, we examined the abilities of SV5, Sendai virus (SeV), human respiratory syncytial virus (RSV), and human parainfluenza viruses types 2 and 3 (hPIV2 and hPIV3, respectively) to block interferon signaling. The results showed that in reporter assays SV5, SeV, and hPIV3 blocked both type I and type II IFN-signaling; hPIV2 blocked type I but not type II IFN-signaling; and RSV failed to block either type I or type II IFN-signaling. In agreement with these results, SV5 and SeV inhibited the formation of the ISGF3 and GAF transcription complexes (essential for type I and type II signaling, respectively). Surprisingly, although hPIV3 inhibited IFN-induction of the ISGF3 complex, GAF complexes were detected in hPIV3-infected cells. hPIV2 also blocked the formation of the ISGF3 complex but not the GAF complex, whereas RSV failed to block the induction of either complex. SV5 was the only virus that caused the degradation of STAT1. Indeed, in SeV- and hPIV3-infected cells STAT1 was phosphorylated on tyrosine 701 (Y701), a characteristic of IFN receptor activation. However, consistent with these viruses blocking IFN signaling downstream of receptor activation, there was a specific reduction in the levels of serine 727 (S727)-phosphorylated forms of STAT1alpha in SeV- and hPIV3-infected cells. In contrast both (Y701)- and (S727)-phosphorylated forms of STAT1 were detected in hPIV2-infected cells but there was a specific loss of STAT2. Both STAT1 (including Y701- and S727-phosphorylated forms) and STAT2 could readily be detected in RSV-infected cells. Despite not being able to block type I or type II IFN signaling, RSV was able to replicate in human cells that produce and respond to IFN, suggesting that RSV must have an alternative method(s) for circumventing the IFN response. These results demonstrate that, although interference with IFN signaling is a common strategy among Paramyxovirinae, distinct virus-specific mechanisms are used to achieve this end.

Antiviral Agents↗

Avian Paramyxoviridae--recent developments.

This paper reviews the recent developments related to members of the Paramyxoviridae family which infect avian species. These developments include: (1) The identification of the virus responsible for the diseases turkey rhinotracheitis and swollen head syndrome in chickens as a member of the Pneumovirus genus, the first of this type to be isolated from birds. (2) The use of monoclonal antibodies prepared against Newcastle disease virus (NDV) and other avian paramyxoviruses which has enabled more exact and rapid diagnosis and greater understanding of the epizootiology of these viruses. (3) The nucleotide sequencing of the genome of NDV and subsequent amino acid sequencing of the major virus proteins have resulted in greater understanding of the pathogenicity of this economically important virus.

Amino Acid Sequence↗

RNA-dependent RNA polymerase gene analysis of worldwide Newcastle disease virus isolates representing different virulence types and their phylogenetic relationship with other members of the paramyxoviridae.

Nucleotide sequence was determined for the RNA-dependent RNA polymerase (L) gene of 16 Newcastle disease virus (NDV) isolates from diverse geographic and chronological origins. The observed consensus amino acid sequence conformed to the six domains previously identified among paramyxovirus L proteins, and the putative 749QGDNQ753 active site was strictly conserved among all isolates. Analysis of predicted amino acid sequences allowed us to identify a sequencing error in the previously reported L genes for NDV. The correct sequences reported herein provided a more accurate alignment with predicted l-amino acid sequences of other paramyxoviruses. Comparison of L gene coding sequences among isolates revealed that synonymous substitutions dominated non-synonymous substitutions, as observed previously with other NDV genes. However, the overall substitution rate was lower than other genes examined making the L gene the most conserved of the NDV genome. Phylogenetic analysis of L genes among NDV isolates was consistent with previous results that suggested the existence of two major lineages. One group contained strains isolated in North America prior to 1970 and included virulent and vaccine strains, while the second group included virulent viruses isolated worldwide. A comparison of the NDV L coding sequences to other Paramyxoviridae illustrated the unique clustering of the avian-specific paramyxoviruses, further justifying the newly created Avulavirus genus.

Animals↗

Nucleotide sequence analysis of the Newcastle disease virus nucleocapsid protein gene and phylogenetic relationships among the Paramyxoviridae.

The nucleocapsid (N) protein genes from 24 Newcastle disease virus (NDV) isolates representing various pathotypes with different geographical and chronological origins were cloned and sequenced. The N-terminal region of the N protein to residue 401 was highly conserved among isolates with several conservative substitutions occurring that correlated with phylogenetic relationships. Variability of the N protein was detected in the C-terminal portion similar to what has been reported for other members of the Paramyxovirinae. Amino acids previously identified as invariant or highly conserved in N proteins of other paramyxoviruses were also present in the NDV protein. Phylogenetic analysis of N gene coding sequences among NDV isolates again demonstrated the existence of two major groups. One clade contained viruses that included vaccine and virulent strains isolated in the USA prior to 1970 while a second clade included vaccine and virulent viruses isolated worldwide. Comparison of N protein amino acid sequences among members of the Paramyxoviridae resulted in NDV and avian paramyxovirus 6 separating as a cluster distinct from the Rubulavirus genus. This provides further support for avian paramyxoviruses being considered for their own genus among the Paramyxovirinae.

Amino Acid Sequence↗

Molecular evolution of the Newcastle disease virus matrix protein gene and phylogenetic relationships among the paramyxoviridae.

Matrix (M) gene sequences for recent field isolates and older reference Newcastle disease viruses (NDV) were examined to determine phylogenetic relationships and population trends among these viruses. Overall, the M gene has a majority of synonymous nucleotide sequence substitutions occurring among NDV isolates. However, several predicted amino acid changes in the M protein of specific NDV isolates have occurred that correlate to phylogenetic relationships. Nucleotide substitutions in these codons have a greater number of nonsynonymous base changes. The NDV isolates arising since the 1970s belong to a population of viruses that expanded worldwide at an exponential rate. These viruses may have their origins in free-living birds, are present worldwide, and continue to circulate causing disease in poultry. A specific NDV lineage composed of virulent isolates obtained in the US prior to 1970 appears to no longer exists among free-living birds or commercial poultry. However, "vaccine-like" viruses are common in the US and continue to circulate among commercial poultry. Based on M protein amino acid sequences, NDV separates as a clade most closely related to morbilliviruses and not with their current designated category, the rubulaviruses among the Paramyxoviridae. Consequently, avian paramyxoviruses should have their own taxonomic subfamily among the Paramyxovirinae.

Animals↗