Novel Paju Apodemus paramyxovirus 1 and 2, harbored by Apodemus agrarius in the Republic of Korea.

Paramyxoviruses harbored by multiple natural reservoirs pose a potential threat to public health. Jeilongvirus has been proposed as a novel paramyxovirus genus found in rodents, bats, and cats. Paramyxovirus RNA was detected in 108/824 (13.1%) Apodemus agrarius captured at 14 trapping sites in the Republic of Korea. We first present two genetically distinct novel paramyxoviruses, Paju Apodemus paramyxovirus 1 (PAPV-1) and 2 (PAPV-2). The disparity between PAPV-1 (19,716 nucleotides) and -2 (17,475 nucleotides) revealed the presence of the SH gene and length of the G gene in the genome organization. The phylogeny of PAPV-1 and -2 belonged to distinct genetic lineages of Jeilongvirus, respectively, even though these viruses were originated from A. agrarius. PAPV-1 infected human epithelial and endothelial cells, facilitating the induction of type I/III interferons, interferon-stimulated genes, and pro-inflammatory cytokines. Therefore, this study provides insights into the molecular epidemiology, genetic diversity, and virus-host interactions of novel rodent-borne paramyxoviruses.

Genome-wide transposon mutagenesis of paramyxoviruses reveals constraints on genomic plasticity.

The antigenic and genomic stability of paramyxoviruses remains a mystery. Here, we evaluate the genetic plasticity of Sendai virus (SeV) and mumps virus (MuV), sialic acid-using paramyxoviruses that infect mammals from two Paramyxoviridae subfamilies (Orthoparamyxovirinae and Rubulavirinae). We performed saturating whole-genome transposon insertional mutagenesis, and identified important commonalities: disordered regions in the N and P genes near the 3' genomic end were more tolerant to insertional disruptions; but the envelope glycoproteins were not, highlighting structural constraints that contribute to the restricted antigenic drift in paramyxoviruses. Nonetheless, when we applied our strategy to a fusion-defective Newcastle disease virus (Avulavirinae subfamily), we could select for F-revertants and other insertants in the 5' end of the genome. Our genome-wide interrogation of representative paramyxovirus genomes from all three Paramyxoviridae subfamilies provides a family-wide context in which to explore specific variations within and among paramyxovirus genera and species.

Metatranscriptomic Analysis of Virus Diversity in Urban Wild Birds with Paretic Disease.

Wild birds are major natural reservoirs and potential dispersers of a variety of infectious diseases. As such, it is important to determine the diversity of viruses they carry and use this information to help understand the potential risks of spillover to humans, domestic animals, and other wildlife. We investigated the potential viral causes of paresis in long-standing, but undiagnosed, disease syndromes in wild Australian birds. RNA from diseased birds was extracted and pooled based on tissue type, host species, and clinical manifestation for metagenomic sequencing. Using a bulk and unbiased metatranscriptomic approach, combined with clinical investigation and histopathology, we identified a number of novel viruses from the families Astroviridae, Adenoviridae, Picornaviridae, Polyomaviridae, Paramyxoviridae, Parvoviridae, and Circoviridae in common urban wild birds, including Australian magpies, magpie larks, pied currawongs, Australian ravens, and rainbow lorikeets. In each case, the presence of the virus was confirmed by reverse transcription (RT)-PCR. These data revealed a number of candidate viral pathogens that may contribute to coronary, skeletal muscle, vascular, and neuropathology in birds of the Corvidae and Artamidae families and neuropathology in members of the Psittaculidae The existence of such a diverse virome in urban avian species highlights the importance and challenges in elucidating the etiology and ecology of wildlife pathogens in urban environments. This information will be increasingly important for managing disease risks and conducting surveillance for potential viral threats to wildlife, livestock, and human health.IMPORTANCE Wildlife naturally harbor a diverse array of infectious microorganisms and can be a source of novel diseases in domestic animals and human populations. Using unbiased RNA sequencing, we identified highly diverse viruses in native birds from Australian urban environments presenting with paresis. This research included the clinical investigation and description of poorly understood recurring syndromes of unknown etiology: clenched claw syndrome and black and white bird disease. As well as identifying a range of potentially disease-causing viral pathogens, this study describes methods that can effectively and efficiently characterize emergent disease syndromes in free-ranging wildlife and promotes further surveillance for specific pathogens of potential conservation and zoonotic concern.

Differential Features of Fusion Activation within the Paramyxoviridae.

Paramyxovirus (PMV) entry requires the coordinated action of two envelope glycoproteins, the receptor binding protein (RBP) and fusion protein (F). The sequence of events that occurs during the PMV entry process is tightly regulated. This regulation ensures entry will only initiate when the virion is in the vicinity of a target cell membrane. Here, we review recent structural and mechanistic studies to delineate the entry features that are shared and distinct amongst the Paramyxoviridae. In general, we observe overarching distinctions between the protein-using RBPs and the sialic acid- (SA-) using RBPs, including how their stalk domains differentially trigger F. Moreover, through sequence comparisons, we identify greater structural and functional conservation amongst the PMV fusion proteins, as compared to the RBPs. When examining the relative contributions to sequence conservation of the globular head versus stalk domains of the RBP, we observe that, for the protein-using PMVs, the stalk domains exhibit higher conservation and find the opposite trend is true for SA-using PMVs. A better understanding of conserved and distinct features that govern the entry of protein-using versus SA-using PMVs will inform the rational design of broader spectrum therapeutics that impede this process.

Detection and phylogenetic analysis of a new adenoviral polymerase gene in reptiles in Korea.

Over a period of 7 years (2004-2011), samples from 34 diseased reptiles provided by local governments, zoos, and pet shops were tested for viral infection. Animals were diagnosed based on clinical signs, including loss of appetite, diarrhea, rhinorrhea, and unexpected sudden death. Most of the exotic animals had gastrointestinal problems, such as mucosal redness and ulcers, while the native animals had no clinical symptoms. Viral sequences were found in seven animals. Retroviral genes were amplified from samples from five Burmese pythons (Python molurus bivittatus), an adenovirus was detected in a panther chameleon (Furcifer pardalis), and an adenovirus and a paramyxovirus were detected in a tropical girdled lizard (Cordylus tropidosternum). Phylogenetic analysis of retroviruses and paramyxoviruses showed the highest sequence identity to both a Python molurus endogenous retrovirus and a Python curtus endogenous retrovirus and to a lizard isolate, respectively. Partial sequencing of an adenoviral DNA polymerase gene from the lizard isolate suggested that the corresponding virus was a novel isolate different from the reference strain (accession no. AY576677.1). The virus was not isolated but was detected, using molecular genetic techniques, in a lizard raised in a pet shop. This animal was also coinfected with a paramyxovirus.

Evidence for retrovirus and paramyxovirus infection of multiple bat species in china.

Bats are recognized reservoirs for many emerging zoonotic viruses of public health importance. Identifying and cataloguing the viruses of bats is a logical approach to evaluate the range of potential zoonoses of bat origin. We characterized the fecal pathogen microbiome of both insectivorous and frugivorous bats, incorporating 281 individual bats comprising 20 common species, which were sampled in three locations of Yunnan province, by combining reverse transcription polymerase chain reaction (RT-PCR) assays and next-generation sequencing. Seven individual bats were paramyxovirus-positive by RT-PCR using degenerate primers, and these paramyxoviruses were mainly classified into three genera (Rubulavirus, Henipavirus and Jeilongvirus). Various additional novel pathogens were detected in the paramyxovirus-positive bats using Illumina sequencing. A total of 7066 assembled contigs (≥200 bp) were constructed, and 105 contigs matched eukaryotic viruses (of them 103 belong to 2 vertebrate virus families, 1 insect virus, and 1 mycovirus), 17 were parasites, and 4913 were homologous to prokaryotic microorganisms. Among the 103 vertebrate viral contigs, 79 displayed low identity (<70%) to known viruses including human viruses at the amino acid level, suggesting that these belong to novel and genetically divergent viruses. Overall, the most frequently identified viruses, particularly in bats from the family Hipposideridae, were retroviruses. The present study expands our understanding of the bat virome in species commonly found in Yunnan, China, and provides insight into the overall diversity of viruses that may be capable of directly or indirectly crossing over into humans.

Cross-neutralization of four paramyxoviruses by a human monoclonal antibody.

Broadly neutralizing antibodies reactive against most and even all variants of the same viral species have been described for influenza and HIV-1 (ref. 1). However, whether a neutralizing antibody could have the breadth of range to target different viral species was unknown. Human respiratory syncytial virus (HRSV) and human metapneumovirus (HMPV) are common pathogens that cause severe disease in premature newborns, hospitalized children and immune-compromised patients, and play a role in asthma exacerbations. Although antisera generated against either HRSV or HMPV are not cross-neutralizing, we speculated that, because of the repeated exposure to these viruses, cross-neutralizing antibodies may be selected in some individuals. Here we describe a human monoclonal antibody (MPE8) that potently cross-neutralizes HRSV and HMPV as well as two animal paramyxoviruses: bovine RSV (BRSV) and pneumonia virus of mice (PVM). In its germline configuration, MPE8 is HRSV-specific and its breadth is achieved by somatic mutations in the light chain variable region. MPE8 did not result in the selection of viral escape mutants that evaded antibody targeting and showed potent prophylactic efficacy in animal models of HRSV and HMPV infection, as well as prophylactic and therapeutic efficacy in the more relevant model of lethal PVM infection. The core epitope of MPE8 was mapped on two highly conserved anti-parallel ß-strands on the pre-fusion viral F protein, which are rearranged in the post-fusion F protein conformation. Twenty-six out of the thirty HRSV-specific neutralizing antibodies isolated were also found to be specific for the pre-fusion F protein. Taken together, these results indicate that MPE8 might be used for the prophylaxis and therapy of severe HRSV and HMPV infections and identify the pre-fusion F protein as a candidate HRSV vaccine.

Identification and complete genome analysis of three novel paramyxoviruses, Tuhoko virus 1, 2 and 3, in fruit bats from China.

Among 489 bats of 11 species in China, three novel paramyxoviruses [Tuhokovirus 1, 2 and 3 (ThkPV-1, ThkPV-2 and ThkPV-3)] were discovered in 15 Leschenault's rousettes. Phylogenetically, the three viruses are most closely related to Menangle and Tioman virus. Genome analysis showed that their 3'-leader sequences are unique by possessing GA instead of AG at the 5th and 6th positions. Unlike Menangle and Tioman virus, key amino acids for neuraminidase activity characteristic of rubulavirus attachment proteins are present. The genome of ThkPV-1 represents the largest rubulavirus genome. Unique features between the three viruses include perfect complementary 5'-trailer and 3'-leader sequence and a unique cysteine pair in attachment protein of ThkPV-1, G at +1 position in all predicted mRNA sequences of ThkPV-2, and amino acid substitutions in the conserved N-terminal motif of nucleocapsid of ThkPV-3. Analysis of phosphoprotein gene mRNA products confirmed mRNA editing. Antibodies to the viruses were detected in 48-60% of Leschenault's rousettes.

A new paramyxovirus, Tianjin strain, isolated from common cotton-eared marmoset: genome characterization and structural protein sequence analysis.

The complete RNA genome sequence of Tianjin strain, isolated from marmoset, has been determined. The genome was 15,384 nucleotides (nt) in length, which was identical to that of Sendai virus (SeV), and consisted of six genes in the order 3'-NP-P/C-M-F-HN-L-5'. The gene junctions contained highly conserved transcription start and stop signal sequences and trinucleotide intergenic regions (IGR) similar to those of SeV. There was also an overlapping open reading frame and a conserved AnGn site (AAAAAGGG) in the P mRNA. Phylogenetic analysis based on protein sequences from other members of the family Paramyxoviridae showed that Tianjin strain belonged to the genus Respirovirus and was most closely related to SeV. Pairwise comparisons of amino acid identities and phylogenetic analysis with homologous sequences of known SeVs demonstrated that Tianjin strain represented a new evolutionary lineage of SeV.

The complete genome sequence of J virus reveals a unique genome structure in the family Paramyxoviridae.

J virus (J-V) was isolated from feral mice (Mus musculus) trapped in Queensland, Australia, during the early 1970s. Although studies undertaken at the time revealed that J-V was a new paramyxovirus, it remained unclassified beyond the family level. The complete genome sequence of J-V has now been determined, revealing a genome structure unique within the family Paramyxoviridae. At 18,954 nucleotides (nt), the J-V genome is the largest paramyxovirus genome sequenced to date, containing eight genes in the order 3'-N-P/V/C-M-F-SH-TM-G-L-5'. The two genes located between the fusion (F) and attachment (G) protein genes, which have been named the small hydrophobic (SH) protein gene and the transmembrane (TM) protein gene, encode putative proteins of 69 and 258 amino acids, respectively. The 4,401-nt J-V G gene, much larger than other paramyxovirus attachment protein genes sequenced to date, encodes a putative attachment protein of 709 amino acids and distally contains a second open reading frame (ORF) of 2,115 nt, referred to as ORF-X. Taken together, these novel features represent the most significant divergence to date from the common six-gene genome structure of Paramyxovirinae. Although genome analysis has confirmed that J-V can be classified as a member of the subfamily Paramyxovirinae, it cannot be assigned to any of the five existing genera within this subfamily. Interestingly, a recently isolated paramyxovirus appears to be closely related to J-V, and preliminary phylogenetic analyses based on putative matrix protein sequences indicate that these two viruses will likely represent a new genus within the subfamily Paramyxovirinae.

A novel paramyxovirus?

In public databases, we identified sequences reported as human genes expressed in kidney mesangial cells. The similarity of these genes to paramyxovirus matrix, fusion, and phosphoprotein genes suggests that they are derived from a novel paramyxovirus. These genes are sufficiently unique to suggest the existence of a novel paramyxovirus genus.

The classification of viruses infecting the respiratory tract.

Following the boom in respiratory virology in the 1960s, species of rhinoviruses, coronaviruses, enteroviruses, adenoviruses, parainfluenza viruses and respiratory syncytial virus were added to influenza and measles viruses as causes of respiratory tract infection. In restricted patient groups, such as the immunocompromised, members of the family of herpesviruses including herpes simplex, cytomegalovirus, varicella-zoster virus, Epstein-Barr virus and human herpes virus 6 have also been associated with respiratory disease. This list of pathogens was extended last year with the discovery of a novel virus, the human metapneumovirus. More than 200 antigenically distinct viruses have been documented as causes of sporadic or epidemic respiratory infections in infants, children and adults. However, this varied and diverse group can be divided among six distinct families. Understanding some of the basic biology of these families gives an insight into possible strategies for diagnosis, control and therapy.

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.

Phylogenetic analysis of the L and HN gene of ophidian paramyxoviruses. Brief report.

Two reptilian paramyxoviruses, isolated from a neotropical rattlesnake (neotropical virus, NTV, ATCC VR-1408) and a bush viper (bush viper virus, BVV, ATCC VR- 1409), respectively, were analysed to determine their taxonomic position among other reptilian paramyxoviruses investigated previously by Ahne et al.. A 679 bp long region of the hemagglutinin-neuraminidase (HN) gene and a 627 bp long region of the large (L) gene were reverse transcribed, amplified by polymerase chain reaction (PCR), and sequenced. The deduced amino acid sequences were compared to mammalian paramyxoviruses belonging to the genera Respirovirus and Rubulavirus. The deduced amino acid sequences revealed 58.9 to 62% homology for the partial L protein and 41% to 47.1% homology for the partial HN protein. For phylogenetic analyses, a 518 bp L gene and a 352 bp HN gene fragment were used, both generating similar trees consisting of two distinct main groups, and some intermediate isolates. BVV clustered within group "b" while NTV clustered together with the intermediate ophidian paramyxovirus isolate Crot2-OH90.

The cleavage activation and sites of glycosylation in the fusion protein of Hendra virus.

Hendra virus (HeV) is an unclassified member of the Paramyxoviridae family that causes systemic infections in humans, horses, cats, guinea pigs and flying foxes. The fusion protein (F(0)) of members of the Paramyxoviridae family that cause systemic infections in vivo contains a basic amino acid-rich region at which the protein is activated by cleavage into two subunits (F(1) and F(2)). HeV F(0) lacks such a domain. We have determined the cleavage site in HeV F(0) by sequencing the amino terminus of the F(1) subunit and in view of the potential effect of glycosylation on the cleavage process have ascertained the sites at which F(0) is glycosylated. The results indicate that unlike other members of the family that replicate in cultured cells and cause systemic infections in vivo, cleavage of HeV F(0) occurs at a single lysine (reside 109) in the sequence Asp-Val-Lys- downward arrow-Leu. Although HeV genotypically resembles members of the Respirovirus and Rubulavirus genera in having potential N-linked glycosylation sites in both the F(1) and F(2) subunits, we show that phenotypically HeV may more closely resemble members of the Morbillivirus genus that contain N-linked glycans only in the F(2) subunit.

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.

Characterization of Mapuera virus: structure, proteins and nucleotide sequence of the gene encoding the nucleocapsid protein.

The molecular biology of Mapuera virus was studied at both the protein and nucleic acid levels. Seven virus-encoded proteins were detected in infected Vero cells. The sizes and characteristics of each of the proteins determined from various radiolabelling experiments allowed preliminary identification of the proteins as the large (L; 190 kDa), haemagglutinin neuraminidase (HN; 74 kDa), nucleocapsid (N; 66 kDa), fusion (F0; 63 kDa), phosphoprotein (P; 49 kDa), matrix (M; 43 kDa) and non-structural (V; 35 kDa) proteins. Western blot analysis showed that the HN, N and P proteins were major antigens recognized in the mouse. A cDNA library of total virus-infected cellular mRNA was created and screening of the library resulted in the detection of cDNA sequences representing the N mRNA transcript of Mapuera virus. The N mRNA sequence determined from the clones was 1731 nt in length and contained an ORF that encoded 537 amino acids, the complete 3' untranslated region and part of the 5' non-coding region. The calculated M(r) of the N protein was 59 kDa, which is close to the 66 kDa protein observed by SDS-PAGE.

Antigenic and molecular properties of Murayama virus isolated from cynomolgus monkeys: the virus is closely related to avian paramyxovirus type 2.

A new virus that belonged to a member of paramyxovirus was isolated from cynomolgus monkeys showing respiratory disorders about 20 years ago and was named Murayama virus (MrV). Interestingly, it showed no serological relationship with the mammalian paramyxoviruses tested. On the other hand, it was related to Yucaipa (YuV) and Bangor virus (BaV) belonging to avian paramyxovirus type 2 (PMV2). In analysis using anti-MrV monoclonal antibodies, MrV showed the closest relationship with YuV, and furthermore some conserved epitopes were found among avian paramyxovirus, MrV, YuV, BaV, and Newcastle disease virus (NDV). Subsequently, the nucleotide sequences of the F and HN genes of MrV were determined. In comparison with the deduced amino acid sequence of MrV and other paramyxoviruses, MrV showed the highest homology with NDV and higher similarity to HPIV2 group rather than to HPIV1 group. The present study clearly indicates that MrV belongs to PMV2 adapting to monkeys.

Sequence determination of the P gene of simian virus 41: presence of irregular deletions near the RNA-editing sites of paramyxoviruses.

The complete nucleotide sequence of the P gene of simian virus 41 (SV41) was determined. The gene was found to be 1406 nucleotides long and to contain a relatively small open reading frame encoding a cysteine-rich V protein with a calculated M(r) of 24076. We have demonstrated that RNA-editing events occur in SV41 P gene transcripts and that the ratio of edited mRNAs to faithfully copied mRNA (P-mRNA:V-mRNA) is about 1:5 at either 24 or 40 h post-infection. The mRNA with two G insertions was capable of encoding a P protein of 395 amino acids with a predicted M(r) of 41,992. A kinetic study of P and V proteins by Western blot analysis showed that in virus-infected cells the amounts of both proteins were almost equal although the V-mRNA was considerably more abundant than the P-mRNA. Alignment of the SV41 P and V proteins with those of nine other paramyxoviruses demonstrated that irregular gaps were present around the RNA-editing sites.

Humanized animal viruses with special reference to the primate adaptation of morbillivirus.

This review article discusses the evolution of human viruses with special reference to paramyxoviruses. This family of viruses causes epidemics representing the dissemination of infection from one acutely infected host to the next. Since there is no repository for human paramyxoviruses in animals or in the form of persistent infections in man, the history of epidemics afflicting human civilization is short, presumably not exceeding 4000-5000 years. Evolutionary relationships can be deduced for comparison of nucleotide sequences of genes or even complete genomes. The present paramyxovirus genus will probably in the future be divided into two separate genera. In the genus morbillivirus, two pairs of more closely related virus types can be distinguished: canine and phocid viruses, and rinder-pest and measles viruses, respectively. It is speculated that recombination events may have occurred in the evolution of the morbillivirus archetype.