Evolution and Diversity of Bat and Rodent Paramyxoviruses from North America.

Paramyxoviruses are a diverse group of negative-sense, single-stranded RNA viruses of which several species cause significant mortality and morbidity. In recent years the collection of paramyxovirus sequences detected in wild mammals has substantially grown; however, little is known about paramyxovirus diversity in North American mammals. To better understand natural paramyxovirus diversity, host range, and host specificity, we sought to comprehensively characterize paramyxoviruses across a range of diverse cooccurring wild small mammals in southern Arizona. We used highly degenerate primers to screen fecal and urine samples and obtained a total of 55 paramyxovirus sequences from 12 rodent species and 6 bat species. We also performed Illumina transcriptome sequencing (RNA-seq) and de novo assembly on 14 of the positive samples to recover a total of 5 near-full-length viral genomes. We show there are at least two clades of rodent-borne paramyxoviruses in Arizona, while bat-associated paramyxoviruses formed a putative single clade. Using structural homology modeling of the viral attachment protein, we infer that three of the five novel viruses likely bind sialic acid in a manner similar to other respiroviruses, while the other two viruses from heteromyid rodents likely bind a novel host receptor. We find no evidence for cross-species transmission, even among closely related sympatric host species. Taken together, these data suggest paramyxoviruses are a common viral infection in some bat and rodent species present in North America and illuminate the evolution of these viruses. IMPORTANCE There are a number of viral lineages that are potential zoonotic threats to humans. One of these, paramyxoviruses have jumped into humans multiple times from wild and domestic animals. We conducted one of the largest viral surveys of wild mammals in the United States to better understand paramyxovirus diversity and evolution.

Pathogenicity and transmissibility of a novel respirovirus isolated from a Malayan pangolin.

The identification of SARS-CoV-2-like viruses in Malayan pangolins (Manis javanica) has focused attention on these endangered animals and the viruses they carry. We successfully isolated a novel respirovirus from the lungs of a dead Malayan pangolin. Similar to murine respirovirus, the full-length genome of this novel virus was 15 384 nucleotides comprising six genes in the order 3'-(leader)-NP-P-M-F-HN-l-(trailer)-5'. Phylogenetic analysis revealed that this virus belongs to the genus Respirovirus and is most closely related to murine respirovirus. Notably, animal infection experiments indicated that the pangolin virus is highly pathogenic and transmissible in mice, with inoculated mice having variable clinical symptoms and a fatality rate of 70.37 %. The virus was found to replicate in most tissues with the exception of muscle and heart. Contact transmission of the virus was 100 % efficient, although the mice in the contact group displayed milder symptoms, with the virus mainly being detected in the trachea and lungs. The isolation of a novel respirovirus from the Malayan pangolin provides new insight into the evolution and distribution of this important group of viruses and again demonstrates the potential infectious disease threats faced by endangered pangolins.

Clinical characteristics of the lower respiratory tract infection caused by a single infection or coinfection of the human parainfluenza virus in children.

BACKGROUND: Human parainfluenza virus (HPIV), usually combined with other pathogens, causes lower respiratory tract infection (LRTI) in children. However, clinical characteristics of HPIV coinfection with other pathogens were unclear. This study aimed to investigate the viral and atypical bacterial etiology of LRTI in children and compare the clinical characteristics of HPIV single infection with those of coinfection. METHODS: This study included 1335 patients, aged between 1 to 71 months, diagnosed with LRTI in Yuying Children's Hospital, Zhejiang, China, from December 2013 to June 2015. Nasopharyngeal secretions were collected, and respiratory pathogens were detected using Multiplex polymerase chain reaction. The clinical data of patients were collected and analyzed. RESULTS: At least 1 pathogen was detected in 1181/1335 (88.5%) patients. The pathogens identified most frequently were respiratory syncytial virus, human rhinovirus, HPIV, adenovirus, and human metapneumovirus. The coinfection rate was 24.8%. HPIV coinfection with other viruses was more associated with running nose, shortness of breath, and oxygen support compared with HPIV single infection. Moreover, HPIV coinfection with atypical bacteria was more related to running nose, moist rales, and longer hospital duration compared with HPIV single infection, and also to longer hospital duration compared with coinfection with other viruses. CONCLUSIONS: This study demonstrated that viral infections were highly associated with LRTI and the rate of coinfection was high. HPIV single infection was milder than coinfection with other viruses. Moreover, HPIV coinfection with atypical bacteria was more serious than HPIV single infection and coinfection with other viruses.

New clinical and seasonal evidence of infections by Human Parainfluenzavirus.

Human Parainfluenzaviruses (PIVs) account for a significant proportion of viral acute respiratory infections (ARIs) in children, and are also associated with morbidity and mortality in adults, including nosocomial infections. This work aims to describe PIV genotypes and their clinical and epidemiological distribution. Between December 2016 and December 2017, 6121 samples were collected, and submitted to viral culture and genomic quantification, specifically Parainfluenza 1-4 (PIV1-4), Influenza A and B, Respiratory Syncytial Virus (RSV) A and B, Adenovirus, Metapneumovirus, Coronavirus, Rhinovirus, and Enterovirus. Normalized viral load, as (log10) copies/103 cells, was calculated as virus Ct, determined by multiple qRT-PCR, as a function of the Ct of ß-globin. PIV was confirmed in 268 cases (4.37%), and linked to both upper and lower respiratory tract disease, being more frequent in children than in adults (5.23 and 2.43%, respectively). PIV1 and PIV3 were most common (31 and 32.5%, of total PIV positive samples, respectively), with distribution being similar in children and adults, as was viral load. PIV type was correlated with seasonality: PIV3 being more frequent in winter and spring, PIV1 in summer, and PIV 4 in fall. No correlation between vial load and clinical severity was found. Novel findings were that PIV viral load was higher in fall than in other seasons, and PIV4, classically linked to mild respiratory symptoms, was circulating, in children and adults, at all levels of symptoms throughout the year.

Seasonality and clinical impact of human parainfluenza viruses.

BACKGROUND: Widespread availability of rapid diagnostic testing for respiratory viruses allows more in-depth studies of human parainfluenza viruses (HPIV). OBJECTIVES: This study aimed to assess seasonality of HPIV types 1-4, clinical outcomes by HPIV type, and risk factors for illness severity. PATIENTS/METHODS: This retrospective study was performed from January 2013 to December 2015 in children and adults with HPIV, detected by multiplex reverse transcription polymerase chain reaction, participating in a community surveillance study of acute respiratory infections (ARIs) in New York City and patients admitted to a tertiary care center in the same neighborhood. Seasonality trends by HPIV type were compared between the community and hospital groups. The associations between HPIV type, demographics, clinical characteristics, and illness severity were assessed. RESULTS: HPIV was detected in 69 (4%) of 1753 community surveillance participants (median age 9.2 years) and 680 hospitalized patients (median age 6.8 years). Seasonality for HPIV types 1-3 agreed with previously described patterns; HPIV-4 occurred annually in late summer and fall. In the community cohort, 22 (32%) participants sought medical care, 9 (13%) reported antibiotic use, and 20 (29%) reported ≥1 day of missed work or school. Among hospitalized patients, 24% had ≥4 chronic conditions. Multivariable ordinal logistic regression demonstrated that increased severity of illness was significantly associated with HPIV-4 and chronic cardiovascular and respiratory conditions in children and with age ≥65 years and chronic respiratory conditions in adults. CONCLUSIONS: HPIV-4 presented late summer and early fall annually and was associated with increased severity of illness in hospitalized children.

A Novel Squirrel Respirovirus with Putative Zoonotic Potential.

In a globalized world, the threat of emerging pathogens plays an increasing role, especially if their zoonotic potential is unknown. In this study, a novel respirovirus, family Paramyxoviridae, was isolated from a Sri Lankan Giant squirrel (Ratufa macroura), which originated in Sri Lanka and deceased with severe pneumonia in a German zoo. The full-genome characterization of this novel virus, tentatively named Giant squirrel respirovirus (GSqRV), revealed similarities to murine (71%), as well as human respiroviruses (68%) with unique features, for example, a different genome length and a putative additional accessory protein. Congruently, phylogenetic analyses showed a solitary position of GSqRV between known murine and human respiroviruses, implicating a putative zoonotic potential. A tailored real-time reverse transcription-polymerase chain reaction (RT-qPCR) for specific detection of GSqRV confirmed a very high viral load in the lung, and, to a lesser extent, in the brain of the deceased animal. A pilot study on indigenous and exotic squirrels did not reveal additional cases in Germany. Therefore, further research is essential to assess the geographic distribution, host range, and zoonotic potential of this novel viral pathogen.

Detailed genetic analyses of the HN gene in human respirovirus 3 detected in children with acute respiratory illness in the Iwate Prefecture, Japan.

We performed detailed genetic analyses of the partial hemagglutinin-neuraminidase (HN) gene in 34 human respirovirus 3 (HRV3) strains from children with acute respiratory illness during 2013-2015 in Iwate Prefecture, Japan. In addition, we performed analyses of the evolutionary timescale of the gene using the Bayesian Markov chain Monte Carlo (MCMC) method. Furthermore, we analyzed pairwise distances and performed selective pressure analyses followed by linear B-cell epitope mapping and N-glycosylation and phylodynamic analyses. A phylogenetic tree showed that the strains diversified at around 1939, and the rate of molecular evolution was 7.6 × 10-4 substitutions/site/year. Although the pairwise distances were relatively short (0.03 ±â€¯0.018 [mean ±â€¯standard deviation, SD]), two positive selection sites (Cys544Trp and Leu555Ser) and no amino acid substitutions were found in the active/catalytic sites. Six epitopes were estimated in this study, and three mouse monoclonal antibody binding sites (amino acid positions 278, 281, and 461) overlapped with two epitopes belonging to subcluster C3 strains. Bayesian skyline plot analyses indicated that subcluster C3 strains have been increasing from 2004, whereas subcluster C1 strains have declined from 2004. Based on these results, Iwate strains were divided into two subclusters and each subcluster evolved independently. Moreover, our results suggested that some predicted linear epitopes (epitopes 3 and 5) are candidates for an HRV3 vaccine motif. To better understand the details of the molecular evolution of HRV, further studies are needed.

Viral etiology, seasonality and severity of hospitalized patients with severe acute respiratory infections in the Eastern Mediterranean Region, 2007-2014.

INTRODUCTION: Little is known about the role of viral respiratory pathogens in the etiology, seasonality or severity of severe acute respiratory infections (SARI) in the Eastern Mediterranean Region. METHODS: Sentinel surveillance for SARI was conducted from December 2007 through February 2014 at 20 hospitals in Egypt, Jordan, Oman, Qatar and Yemen. Nasopharyngeal and oropharyngeal swabs were collected from hospitalized patients meeting SARI case definitions and were analyzed for infection with influenza, respiratory syncytial virus (RSV), adenovirus (AdV), human metapneumovirus (hMPV) and human parainfluenza virus types 1-3 (hPIV1-3). We analyzed surveillance data to calculate positivity rates for viral respiratory pathogens, describe the seasonality of those pathogens and determine which pathogens were responsible for more severe outcomes requiring ventilation and/or intensive care and/or resulting in death. RESULTS: At least one viral respiratory pathogen was detected in 8,753/28,508 (30.7%) samples tested for at least one pathogen and 3,497/9,315 (37.5%) of samples tested for all pathogens-influenza in 3,345/28,438 (11.8%), RSV in 3,942/24,503 (16.1%), AdV in 923/9,402 (9.8%), hMPV in 617/9,384 (6.6%), hPIV1 in 159/9,402 (1.7%), hPIV2 in 85/9,402 (0.9%) and hPIV3 in 365/9,402 (3.9%). Multiple pathogens were identified in 501/9,316 (5.4%) participants tested for all pathogens. Monthly variation, indicating seasonal differences in levels of infection, was observed for all pathogens. Participants with hMPV infections and participants less than five years of age were significantly less likely than participants not infected with hMPV and those older than five years of age, respectively, to experience a severe outcome, while participants with a pre-existing chronic disease were at increased risk of a severe outcome, compared to those with no reported pre-existing chronic disease. CONCLUSIONS: Viral respiratory pathogens are common among SARI patients in the Eastern Mediterranean Region. Ongoing surveillance is important to monitor changes in the etiology, seasonality and severity of pathogens of interest.

Analysis on the complete genome of a novel caprine parainfluenza virus 3.

Parainfluenza virus type 3 (PIV3) is one of the most important viral respiratory pathogens for humans and for many animals. One unique caprine PIV3 (CPIV3) strain named JS2013 was isolated in Chinese goat flocks with respiratory diseases in 2013. Now, the complete genome sequence of the strain JS2013 had been determined. A total of 15 overlapping DNA clones, covering the entire genome of the virus, were obtained by primer walking RT-PCR. The sequences of the 3' and 5' termini of the viral genome were amplified by 3' and 5' RACE. The viral genome was 15,618 nucleotides (nt) in length, which was consisted of six genes in the order 5'-leader-N-P/C/V-M-F-HN-L-tailer-3'. The junction sequences between two genes were highly conserved gene start and stop signal sequences, and trinucleotide intergenic regions (IGR) similar to those of other reported PIV3 strains. Phylogenetic analysis based on the complete genomes of JS2013 with other strains of genus Respirovirus demonstrated that the JS2013 obviously differed from HPIV1, Sendai virus, HPIV3 and other reported BPIV3 genotypes. Further analysis of HN genes of JS2013 along with two more CPIV3 strains isolated later indicated that CPIV3 strains formed a separate cluster. The results presented here suggested that CPIV3 is a new member of the genus Respirovirus.

Detection of 12 respiratory viruses by duplex real time PCR assays in respiratory samples.

Different viruses can be responsible for similar clinical manifestations of respiratory infections. Thus, the etiological diagnosis of respiratory viral diseases requires the detection of a large number of viruses. In this study, 6 duplex real-time PCR assays, using EvaGreen intercalating dye, were developed to detect 12 major viruses responsible for respiratory diseases: influenza A and B viruses, enteroviruses (including enterovirus spp, and rhinovirus spp), respiratory syncytial virus, human metapneumovirus, coronaviruses group I (of which CoV 229E and CoV NL63 are part) and II (including CoV OC43 and CoV HKU1), parainfluenza viruses type 1, 2, 3 and 4, human adenoviruses and human bocaviruses. The 2 target viruses of each duplex reaction were distinguishable by the melting temperatures of their amplicons. The 6 duplex real time PCR assays were applied for diagnostic purpose on 202 respiratory samples from 157 patients. One hundred fifty-seven samples were throat swabs and 45 were bronchoalveolar lavages. The results of the duplex PCR assays were confirmed by comparison with a commercial, validated, assay; in addition, the positive results were confirmed by sequencing. The analytical sensitivity of the duplex PCR assays varied from 10(3) copies/ml to 10(4) copies/ml. For parainfluenza virus 2 only it was 10(5) copies/ml. Seventy clinical samples (35%) from 55 patients (30 children and 25 adults) were positive for 1 or more viruses. In adult patients, influenza A virus was the most frequently detected respiratory virus followed by rhinoviruses. In contrast, respiratory syncytial virus was the most common virus in children, followed by enteroviruses, influenza A virus and coronavirus NL63. The small number of samples/patients does not allow us to draw any epidemiological conclusion. Altogether, the results of this study indicate that the 6 duplex PCR assays described in this study are sensitive, specific and cost-effective. Thus, this assay could be particularly useful to identify the main respiratory viruses directly from clinical samples, after nucleic acid extraction, and, also, to screen a large number of patients for epidemiological studies.

Novel Atlantic bottlenose dolphin parainfluenza virus TtPIV-1 clusters with bovine PIV-3 genotype B strains.

Parainfluenza virus 3 (PIV-3) is a common viral infection not only in humans, but also in many other species. Serological evidence suggests that nearly 100 % of children in the United States have been infected with PIV-3 by 5 years of age. Similarly, in cattle, PIV-3 is commonly associated with bovine respiratory disease complex. A novel dolphin PIV-3 (TtPIV-1) was described by Nollens et al. in 2008 from a dolphin that was diagnosed with an unknown respiratory illness. At that time, TtPIV-1 was found to be most similar to, but distinct from, bovine PIV-3 (BPIV-3). In the present study, similar viral growth kinetics and pro-inflammatory cytokine (IL-1ß, IL-6, and CXCL8) production were seen between BPIV-3 and TtPIV-1 in BEAS-2B, MDBK, and Vero cell lines. Initial nomenclature of TtPIV-1 was based on partial sequence of the fusion and RNA polymerase genes. Based on the similarities we saw with the in vitro work, it was important to examine the TtPIV-1 genome in more detail. Full genome sequencing and subsequent phylogenetic analysis revealed that all six viral genes of TtPIV-1 clustered within the recently described BPIV-3 genotype B strains, and it is proposed that TtPIV-1 be re-classified with BPIV-3 genotype B strains.

Genetic analysis of human parainfluenza viruses circulating in Korea, 2006.

Human parainfluenza viruses (HPIV) are important causes of respiratory tract infections in young children. To characterize the molecular epidemiology of an HPIV outbreak occurring in Korea during 2006, genetic analysis of 269 cell culture isolates from HPIV-infected children, was conducted using nested reverse transcription-PCR (RT-PCR). HPIV-1 was detected in 70.3% of tested samples (189/269). The detection rate of HPIV-2 and HPIV-3 was 1.5% (4/269) and 9.3% (25/269), respectively. Mixed HPIV-1, -2 and -3 infections were detected in 19.0% (51/269): HPIV-1 and HPIV-2 in 15, HPIV-1 and HPIV-3 in 26, HPIV-2 and HPIV-3 in 6, and HPIV-1, -2 and -3 in 4. Of these positive samples for three different types HIPV-1, -2, and -3, two each representative strains were selected, the full length of hemagglutinin-neuraminidase (HN) gene for HPIV was amplified by RT-PCR, and sequenced. Multiple alignment analysis, based on reference sequence of HPIV-1, -2, and -3 strains available in GenBank, showed that the identity of nucleotide and deduced amino acid sequences was 92.4-97.6% and 92.7-97.9%, respectively, for HPIV-1, 88.5-99.8% and 88.6-100% for HPIV-2, and 96.3-99.5% and 95.0-99.3% for HPIV-3, respectively. Phylogenetic analysis showed that HPIV-1, -2, and -3 strains identified in this study were closely related among the strains in the same type with no significant genetic variability. These results show that HPIV of multiple imported sources was circulating in Korea.

Molecular detection of respiratory viruses.

Over the past several years a wide variety of molecular assays for the detection of respiratory viruses has reached the market. The tests described herein range from kits containing primers and probes detecting specific groups of viruses, to self-contained systems requiring specialized instruments that extract nucleic acids and perform the polymerase chain reaction with little operator input. Some of the tests target just the viruses involved in large yearly epidemics such as influenza, or specific groups of viruses such as the adenoviruses or parainfluenza viruses; others can detect most of the known respiratory viruses and some bacterial agents.

Reassessing conflicting evolutionary histories of the Paramyxoviridae and the origins of respiroviruses with Bayesian multigene phylogenies.

The evolution of paramyxoviruses is still poorly understood since past phylogenetic studies have revealed conflicting evolutionary signals among genes, and used varying methods and datasets. Using Bayesian phylogenetic analysis of full length single and concatenated sequences for the 6 genes shared among paramyxovirus genera, we reassess the ambiguous evolutionary relationships within the family, and examine causes of varying phylogenetic signals among different genes. Relative to a pneumovirus outgroup, the concatenated gene phylogeny, splits the Paramyxovirinae into two lineages, one comprising the avulaviruses and rubulaviruses, and a second containing the respiroviruses basal to the henipaviruses, and morbilliviruses. Phylogenies for the matrix (M), RNA dependent RNA polymerase (L) and the fusion (F) glycoprotein genes, are concordant with the topology from the concatenated dataset. In phylogenies derived from the nucleocapsid (N) and phosphoprotein (P) genes, the respiroviruses form the most basal genus of the Paramyxovirinae subfamily, with the avulaviruses and rubulaviruses in one lineage, and the henipaviruses, and morbilliviruses in a second. The phylogeny of the hemagglutinin (H) gene places the respiroviruses basal to the avula-rubulavirus group, but the relationship of this lineage with henipa and morbillviruses is not resolved. Different genes may be under varying evolutionary pressures giving rise to these conflicting signals. Given the level of conservation in the M and L genes, we suggest that together with F gene, these or concatenated datasets for all six genes are likely to reveal the most reliable phylogenies at a family level, and should be used for future phylogenetic studies in this group. Split decomposition analysis suggests that recombination within genera, may have a contributed to the emergence of dolphin morbillivirus, and several species within respiroviruses. A partial L gene alignment, resolves the relationship of 25 unclassified paramxyoviruses into 4 clades (Chiopteran-, Salmon-, Rodentian- and Ophidian paramyxoviruses) which group with rubula-, respiro-, morbilliviruses, and within the paramxyovirinae respectively.

Characterization of a parainfluenza virus isolated from a bottlenose dolphin (Tursiops truncatus).

A novel member of the parainfluenza virus family was identified in a bottlenose dolphin with respiratory disease. The case animal was a 19-year old male Atlantic bottlenose dolphin (Tursiops truncatus) that presented with signs of respiratory illness, including raspy, foul-odored breaths and cream-colored exudate from the blowhole. Focally extensive pyogranulomatous bronchointerstitial pneumonia with moderate numbers of intralesional yeast organisms was identified on histopathological examination. Other significant microscopic findings included multifocal erosive and ulcerative tracheitis and laryngitis consisting of active laryngeal lymphatic tissue and dilated glands with eosinophilic fluid. The cause of death was attributed to respiratory disease of unknown etiology. In addition to the postmortem isolation of Candida glabrata and mixed bacteria from lung tissue, a virus was isolated from two antemortem affected lung aspirates collected over a 2-month period and two postmortem samples (mediastinal lymph node and left lung tissue homogenate). The morphology of the virions on negative staining and transmission electron microscopy was consistent with that of paramyxoviruses. Two genomic fragments, comprising 532 and 419 nucleotides from the open reading frames that code for the viral polymerase and fusion protein, respectively, were amplified by polymerase chain reaction using degenerate primers. Phylogenetic analyses of the two viral RNA segments showed that the isolate comprised a novel virus strain, tentatively named T. truncatus parainfluenza virus type 1 (TtPIV-1). The virus is monophyletic with, but genetically distinct from, the various bovine parainfluenza virus type 3 strains.

Phylogenetic position of a paramyxovirus from Atlantic salmon Salmo salar.

A paramyxovirus has been isolated from Atlantic salmon Salmo salar suffering from epitheliocystis. This virus does not cause any mortality when used to challenge disease-free salmon, but has been associated with 2 cases of mortality in salmon farms in Norway. Atlantic salmon paramyxovirus (ASPV) has been suggested as a name for the virus. The ASP virus is a slow-growing virus in cell cultures (rainbow trout gill cells: RTgill-W1). Little is known about its importance and its phylogenetic position is uncertain. Hence, the need for a fast and sensitive diagnostic method for studying the prevalence of this virus in salmon farms and for more basic knowledge about its identity were the motivation for this study. A partial nucleotide sequence (816 bp) from the large protein (L protein) gene of the ASP virus has been sequenced from 2 different isolates. The putative amino acid sequence has been compared with the L protein of other paramyxoviruses. This sequence gives strong support to a relationship between the ASP virus and members of the subfamily Paramyxovirinae, genus Respirovirus.

Isolation and partial characterization of a novel paramyxovirus from the gills of diseased seawater-reared Atlantic salmon (Salmo salar L).

A formerly undescribed virus has been isolated from the gills of farmed Atlantic salmon post-smolts in Norway suffering from gill disease. Cytopathic effects appeared in RTgill-W1 cells 9 weeks post-inoculation with gill tissue material. Virus production continued for an extended period thereafter. Light and electron microscopic examination revealed inclusions and replication in the cytoplasm. The viral nucleocapsid consisted of approximately 17 nm thick filaments in a herringbone pattern. Certain areas of the plasma membrane were thickened by the alignment of nucleocapsids on the internal surface and projections of 10 nm long viral glycoprotein spikes on the external surface. Virus assembly and release was achieved by budding through the modified plasma membrane. Negatively stained virions were spherical and partly pleomorphic with a diameter of 150-300 nm as seen by electron microscopy. The virus was sensitive to chloroform, heat and low and high pH, and replication was not inhibited by Br-dU or IdU indicating an RNA genome. Both haemagglutination and receptor-destroying enzyme activity were associated with the virions and the formation of syncytia in infected cultures indicated fusion activity. The receptor-destroying enzyme was identified as neuraminidase. The virus contained five major structural polypeptides with estimated molecular masses of 70, 62, 60, 48 and 37 kDa. Its buoyant density was 1.18-1.19 g ml(-1) in CsCl gradients. From the observed properties we conclude that this new virus belongs to the Paramyxoviridae and suggest the name Atlantic salmon paramyxovirus (ASPV). Furthermore, replication occurred at 6-21 degrees C, suggesting a host range confined to cold-blooded animals.

Biochemical and biophysical analysis of heptad repeat regions from the fusion protein of Menangle virus, a newly emergent paramyxovirus.

Menangle virus is a novel paramyxovirus isolated in Australia in 1997, but its classification position has not yet been finally settled. Here by using a computational program, LearnCoil-VMF, we determined the heptad repeat (HR) regions (HR1 and HR2) of Menangle virus F protein. Subsequently the HR1 and HR2 peptides were expressed as a single chain (named 2-Helix) connected by a six amino-acid linker as a GST fusion protein with an E. coli in vitro expression system. The GST-removed purified 2-Helix protein could form a stable trimer in vitro judging by gel-filtration and chemical cross-linking. CD spectra showed that the 2-Helix protein had a high percentage of alpha-helix and was very thermo-stable. Crystals of the 2-Helix protein preparations have been obtained in many conditions with hanging-drop diffusion method. These results indicated that Menangle virus has the common features of the fusion protein for other paramyxoviruses and should adopt a similar fusion mechanism to other members. As the HR regions of Menangle virus F protein could form stable six-helix bundle coiled coil structure, they should be used as drug target for the design of fusion inhibitors, as successfully used for other parmyxoviruses. This is especially relevant to such a newly emergent virus with zoonotic potentials.

Tioman virus, a novel paramyxovirus isolated from fruit bats in Malaysia.

A search for the natural host of Nipah virus has led to the isolation of a previously unknown member of the family Paramyxoviridae. Tioman virus (TiV) was isolated from the urine of fruit bats (Pteropus hypomelanus) found on the island of the same name off the eastern coast of peninsular Malaysia. An electron microscopic study of TiV-infected cells revealed spherical and pleomorphic-enveloped viral particles (100--500 nm in size) with a single fringe of embedded peplomers. Virus morphogenesis occurred at the plasma membrane of infected cells and morphological features of negative-stained ribonucleoprotein complexes were compatible with that of viruses in the family Paramyxoviridae. Serological studies revealed no cross-reactivity with antibodies against a number of known Paramyxoviridae members except for the newly described Menangle virus (MenV), isolated in Australia in 1997. Failure of PCR amplification using MenV-specific primers suggested that this new virus is related to but different from MenV. For molecular characterization of the virus, a cDNA subtraction strategy was employed to isolate virus-specific cDNA from virus-infected cells. Complete gene sequences for the nucleocapsid protein (N) and phosphoprotein (P/V) have been determined and recombinant N and V proteins produced in baculovirus. The recombinant N and V proteins reacted with porcine anti-MenV sera in Western blot, confirming the serological cross-reactivity observed during initial virus characterization. The lack of a C protein-coding region in the P/V gene, the creation of P mRNA by insertion of 2-G residues, and the results of phylogenetic analyses all indicated that TiV is a novel member of the genus Rubulavirus.