Comparative genomics of human rubulavirus 2.

Although human rubulavirus 2 (HPIV2) is an important respiratory pathogen, little is known about its molecular epidemiology. We performed a comparative analysis of the full-length genomes of fourteen HPIV2 isolates belonging to different genotypes. Additionally, evolutionary analyses (phylogenetic reconstruction, sequence identity, detection of recombination and adaptive evolution) were conducted. Our study presents a systematic comparative genetic analysis that complements prior analyses and utilizes full-length HPIV2 genomes to provide a basis for future work on the clinical significance, molecular variation and conservation, and evolution of HPIV2.

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.

Full-genome sequencing and phylogenetic analysis of four neurovirulent Mexican isolates of porcine rubulavirus.

We report the complete genome sequences of four neurovirulent isolates of porcine rubulavirus (PorPV) from 2015 and one historical PorPV isolate from 1984 obtained by next-generation sequencing. A phylogenetic tree constructed using the individual sequences of the complete HN genes of the 2015 isolates and other historical sequences deposited in the GenBank database revealed that several recent neurovirulent isolates of PorPV (2008-2015) cluster together in a separate clade. Phylogenetic analysis of the complete genome sequences revealed that the neurovirulent strains of PorPV that circulated in Mexico during 2015 are genetically different from the PorPV strains that circulated during the 1980s.

Acute neurologic disease in Porcine rubulavirus experimentally infected piglets.

The objective of this study was to evaluate the clinical disease, humoral response and viral distribution of recent Porcine rubulavirus (PorPV) isolates in experimentally infected pigs. Four, 6-piglet (5-days old) groups were employed (G1-84, G2-93, G3-147, and G4-T). Three viral strains were used for the experimental infection: the reference strain LPMV-1984 (Michoacán 1984) and two other strains isolated in 2013, one in Queretaro (Qro/93/2013) and the other in Michoacán (Mich/147/2013). Each strain was genetically characterized by amplification and sequencing of the gene encoding hemagglutinin-neuroamidase (HN). The inoculation was performed through the oronasal and ocular routes, at a dose of 1×106TCID50/ml. Subsequently, the signs were evaluated daily and necropsies were performed on 3 different days post infection (dpi). We recorded all micro- and macroscopic lesions. Organs from the nervous, lymphatic, and respiratory system were analyzed by quantifying the viral RNA load and the presence of the infectious virus. The presence of the viral antigen in organs was evidenced through immunohistochemistry. Seroconversion was evaluated through the use of a hemagglutination inhibition test. In the characterization of gene HN, only three substitutions were identified in strain Mich/147/2013, two in strain LPMV/1984 (fourth passage) and one in strain Qro/93/2013, with respect to reference strain LPMV-84, these changes had not been identified as virulence factors in previously reported strains. Neurological alterations associated with the infection were found in all three experimental groups starting from 3dpi. Groups G1-84 and G3-147 presented the most exacerbated nervous signs. Group G2-93 only presented milder signs including slight motor incoordination, and an increased rectal temperature starting from day 5 post infection (PI). The main histopathological findings were the presence of a mononuclear inflammatory infiltrate (lymphocytic/monocytic) surrounding the ventricles in the brain and focal interstitial pneumonitis with distention of the alveolar sacs in the lungs. PorPV and RNA distribution were identified in the organs of the nervous, lymphatic, and respiratory systems of the piglets analyzed at different times (days 5, 10, and 15 PI). The viral antigen was detected in the brain and lungs in most of the assessed groups. Seroconversion was evident in groups G1-84 and G2-93. Groups G1-84 and G3-147 were the most clinically affected by the experimental infection. Both strains were isolated in the state of Michoacán. The virulence of the new isolates maintains similar characteristics to those reported more than 30 years ago.

Isolation of Tioman virus from Pteropus giganteus bat in North-East region of India.

Bat-borne viral diseases are a major public health concern among newly emerging infectious diseases which includes severe acute respiratory syndrome, Nipah, Marburg and Ebola virus disease. During the survey for Nipah virus among bats at North-East region of India; Tioman virus (TioV), a new member of the Paramyxoviridae family was isolated from tissues of Pteropus giganteus bats for the first time in India. This isolate was identified and confirmed by RT-PCR, sequence analysis and electron microscopy. A range of vertebrate cell lines were shown to be susceptible to Tioman virus. Negative electron microscopy study revealed the "herringbone" morphology of the nucleocapsid filaments and enveloped particles with distinct envelope projections a characteristic of the Paramyxoviridae family. Sequence analysis of Nucleocapsid gene of TioV demonstrated sequence identity of 99.87% and 99.99% nucleotide and amino acid respectively with of TioV strain isolated in Malaysia, 2001. This report demonstrates the first isolation of Tioman virus from a region where Nipah virus activity has been noticed in the past and recent years. Bat-borne viruses have become serious concern world-wide. A Survey of bats for novel viruses in this region would help in recognizing emerging viruses and combating diseases caused by them.

Molecular characterisation of Porcine rubulavirus (PorPV) isolates from different outbreaks in Mexico.

Since the report of the initial outbreak of Porcine rubulavirus (PorPV) infection in pigs, only one full-length genome from 1984 (PorPV-LPMV/1984) has been characterised. To investigate the overall genetic variation, full-length gene nucleotide sequences of current PorPV isolates were obtained from different clinical cases of infected swine. Genome organisation and sequence analysis of the encoded proteins (NP, P, F, M, HN and L) revealed high sequence conservation of the NP protein and the expression of the P and V proteins in all PorPV isolates. The V protein of one isolate displayed a mutation that has been implicated to antagonise the antiviral immune responses of the host. The M protein indicated a variation in a short region that could affect the electrostatic charge and the interaction with the membrane. One PorPV isolate recovered from the lungs showed a mutation at the cleavage site (HRKKR) of the F protein that could represent an important factor to determine the tissue tropism and pathogenicity of this virus. The HN protein showed high sequence identity through the years (up to 2013). Additionally, a number of sequence motifs of very high amino acid conservation among the PorPV isolates important for polymerase activity of the L protein have been identified. In summary, genetic comparisons and phylogenetic analyses indicated that three different genetic variants of PorPV are currently spreading within the swine population, and a new generation of circulating virus with different characteristics has begun to emerge.

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.

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.

Efficacy of quantitative RT-PCR for detection of the nucleoprotein gene from different porcine rubulavirus strains.

Blue-eye disease is an emergent viral swine infection caused by porcine rubulavirus (PoRV). We have developed a qRT-PCR method to detect and quantify expression of the nucleoprotein gene for different PoRV strains. The limit of detection for this assay was 10(2) copies of synthetic RNA. Viral RNA from PoRV was detectable at a TCID50 of 0.01. Significant differences were observed between viral RNA quantification and virus titration results for nine PoRV strains. For nasal and oral swab samples that were collected from experimentally infected pigs, the qRT-PCR assay was more sensitive (87.1-83.9 %) for the detection of positive samples than methods involving isolation of virus. The implementation of highly sensitive assays that yield results quickly will be of great assistance in the eradication of PoRV from Mexico. We also believe that the newly developed qRT-PCR assay will help reduce the spread of this viral infection to other countries.

Novel, potentially zoonotic paramyxoviruses from the African straw-colored fruit bat Eidolon helvum.

Bats carry a variety of paramyxoviruses that impact human and domestic animal health when spillover occurs. Recent studies have shown a great diversity of paramyxoviruses in an urban-roosting population of straw-colored fruit bats in Ghana. Here, we investigate this further through virus isolation and describe two novel rubulaviruses: Achimota virus 1 (AchPV1) and Achimota virus 2 (AchPV2). The viruses form a phylogenetic cluster with each other and other bat-derived rubulaviruses, such as Tuhoko viruses, Menangle virus, and Tioman virus. We developed AchPV1- and AchPV2-specific serological assays and found evidence of infection with both viruses in Eidolon helvum across sub-Saharan Africa and on islands in the Gulf of Guinea. Longitudinal sampling of E. helvum indicates virus persistence within fruit bat populations and suggests spread of AchPVs via horizontal transmission. We also detected possible serological evidence of human infection with AchPV2 in Ghana and Tanzania. It is likely that clinically significant zoonotic spillover of chiropteran paramyxoviruses could be missed throughout much of Africa where health surveillance and diagnostics are poor and comorbidities, such as infection with HIV or Plasmodium sp., are common.

Evidence of bat origin for Menangle virus, a zoonotic paramyxovirus first isolated from diseased pigs.

Menangle virus (MenPV) is a zoonotic paramyxovirus capable of causing disease in pigs and humans. It was first isolated in 1997 from stillborn piglets at a commercial piggery in New South Wales, Australia, where an outbreak of reproductive disease occurred. Neutralizing antibodies to MenPV were detected in various pteropid bat species in Australia and fruit bats were suspected to be the source of the virus responsible for the outbreak in pigs. However, previous attempts to isolate MenPV from various fruit bat species proved fruitless. Here, we report the isolation of MenPV from urine samples of the black flying fox, Pteropus alecto, using a combination of improved procedures and newly established bat cell lines. The nucleotide sequence of the bat isolate is 94 % identical to the pig isolate. This finding provides strong evidence supporting the hypothesis that the MenPV outbreak in pigs originated from viruses in bats roosting near the piggery.

Prevalence of henipavirus and rubulavirus antibodies in pteropid bats, Papua New Guinea.

To determine seroprevalence of viruses in bats in Papua New Guinea, we sampled 66 bats at 3 locations. We found a seroprevalence of 55% for henipavirus (Hendra or Nipah virus) and 56% for rubulavirus (Tioman or Menangle virus). Notably, 36% of bats surveyed contained antibodies to both types of viruses, indicating concurrent or consecutive infection.

Molecular characterization of the hemagglutinin-neuraminidase gene of porcine rubulavirus isolates associated with neurological disorders in fattening and adult pigs.

"Blue eye disease" is a viral infection of swine endemic in Mexico, which produces fatal encephalitis accompanied by respiratory signs and corneal opacity in suckling piglets. An atypical blue eye disease outbreak presented high rates of neurological signs in fattening and adult pigs from 2000 to 2003. In order to identify the basis of increased neurovirulence, the hemagglutinin-neuraminidase (HN) gene of several porcine rubulavirus isolates were sequenced and compared with that of La Piedad Michoacan virus and other isolates that did not produce neurological disorders in weaned pigs. Nine amino acid mutations distinguished the high neurovirulent PAC6-PAC9 viruses, whereas five mutations characterized the low neurovirulent PAC2 and PAC3 viruses. HN protein three-dimensional models showed that the main conformation and functional domains were preserved, although substitutions A223T and A291D occurred in PAC2 and PAC3 viruses, as well as A511K and E514K presented in PAC6-PAC9 viruses considerably modified the properties of the HN protein surface. The increased positive charge of the HN protein of PAC6-PAC9 viruses seems to be associated with their increased neurovirulence.

Full-length genome sequence and genetic relationship of two paramyxoviruses isolated from bat and pigs in the Americas.

Mapuera virus (MPRV) was isolated from a fruit bat in Brazil in 1979, but its host range and disease-causing potential are unknown. Porcine rubulavirus (PoRV) was identified as the aetiological agent of disease outbreaks in pigs in Mexico during early 1980s, but the origin of PoRV remains elusive. In this study, the completed genome sequence of MPRV was determined, and the complete genome sequence of PoRV was assembled from previously published protein-coding genes and the non-coding genome regions determined from this study. Comparison of sequence and genome organization indicated that PoRV is more closely related to MPRV than to any other members of the genus Rubulavirus. In the P gene coding region of both viruses, there is an ORF located at the 5' end of the P gene overlapping with the P protein coding region, similar to the C protein ORF present in most viruses of the subfamily Paramyxovirinae, but absent in other known rubulaviruses. Based on these findings, we hypothesise that PoRV may also originate from bats, and spillover events from bats to pigs, either directly or via an intermediate host, were responsible for the sporadic disease outbreaks observed in Mexico.

Completion of the full-length genome sequence of Menangle virus: characterisation of the polymerase gene and genomic 5' trailer region.

Menangle virus (MenV), isolated in 1997 from stillborn piglets during an outbreak of reproductive disease at a large commercial piggery, is the only new paramyxovirus to be identified in Australia since Hendra virus in 1994. Following partial characterisation of the MenV genome, we previously showed that MenV is a novel member of the genus Rubulavirus. Here we report the characterisation of the large (L) polymerase gene and the adjacent 5' trailer region of MenV, which completes the full-length genome sequence of this novel paramyxovirus (15,516 nucleotides), and thereby confirm its taxonomic position within the family Paramyxoviridae.

Full length genome sequence of Tioman virus, a novel paramyxovirus in the genus Rubulavirus isolated from fruit bats in Malaysia.

A novel paramyxovirus in the genus Rubulavirus, named Tioman virus (TiV), was isolated in 1999 from a number of pooled urine samples of Island Flying Foxes (Pteropus hypomelanus) during the search for the reservoir host of Nipah virus. TiV is antigenically related to Menangle virus (MenV) that was isolated in Australia in 1997 during disease outbreak in pigs. Sequence analysis of the full length genome indicated that TiV is a novel member of the genus Rubulavirus within the subfamily Paramyxovirinae, family Paramyxoviridae. However, there are several features of TiV which make it unique among known paramyxoviruses and rubulaviruses in particular: (1) TiV, like MenV, uses the nucleotide G as a transcriptional initiation site, rather than the A residue used by all other known paramyxoviruses; (2) TiV uses C as the +1 residue for all intergenic regions, a feature not seen for rubulaviruses but common for all other members within the subfamily Paramyxovirinae; (3) Although the attachment protein of TiV has structural features that are conserved in other rubulaviruses, it manifests no overall sequence homology with members of the genus, lacks the sialic acid-binding motif N-R-K-S-C-S and has only two out of the six highly conserved residues known to be important for the catalytic activity of neuraminidase.

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.

Analysis of the large (L) protein gene of the porcine rubulavirus LPMV: identification of possible functional domains.

The complete nucleotide sequence of the porcine rubulavirus LPMV (La Piedad Michoacan virus) large (L) protein gene was determined and analysed. The L mRNA was found to span 6,786 nucleotides, containing one single large open reading frame (ORF), putatively encoding a polypeptide of 2,251 amino acids. By aligning the amino acid sequence of the LPMV L-protein with L-protein of a number of viruses belonging to the order mononegavirale, a high degree of similarity between the LPMV L-protein and other rubula virus L-proteins was demonstrated, extending through almost the whole protein. Additionally we could identify several regions as being highly conserved among all studied viruses of the order mononegavirale. The significance of these regions are discussed.

Analysis of the fusion protein gene of the porcine rubulavirus LPMV: comparative analysis of paramyxovirus F proteins.

Complementary DNA clones representing the fusion (F) protein gene of the porcine rubulavirus LPMV were isolated and sequenced. The F gene was found to be 1,845 nucleotides long containing one long open reading frame capable of encoding a protein of 541 amino acids. The cleavage motif for F0 into F1 and F2 is His-Arg-Lys-Lys-Arg. A sequence comparison and a phylogenetic analysis was performed in order to identify possible functional domains of paramyxovirus fusion proteins and also to classify the porcine rubulavirus. The F gene of LPMV is most closely related to the human mumps virus and simian virus type 5 F genes, and is therefore classified into the rubulavirus genus. A coding region for a small hydrophobic protein was however not found between the F and hemagglutinin-neuraminidase (HN) genes as previously found in both SV5 and mumps.