Eptesicus fuscus Orthorubulavirus, a Close Relative of Human Parainfluenza Virus 4, Discovered in a Bat in South Dakota.

Bats are a reservoir for many zoonotic viruses and host large numbers of genetically diverse species in the families Rhabdoviridae, Coronaviridae, and Paramyxoviridae. Viruses from these families have repeatedly spilled over to humans in recent decades, causing significant clinical disease and deaths. Here, metagenomic sequencing of a big brown bat (Eptesicus fuscus) submitted for rabies testing due to human exposure identified a novel paramyxovirus, Eptesicus fuscus orthorubulavirus (EfORV), in South Dakota, United States. The nearly complete 15,814-nucleotide genome shared 72% identity with that of human parainfluenza virus 4 (HPIV4), a virus that causes significant clinical disease, typically bronchiolitis and pneumonia, in children less than 2 years of age. Phylogenetic analysis confirmed a close evolutionary history between EfORV and HPIV4, reminiscent of other orthorubulaviruses with highly similar bat and mammalian species, including conspecific human and bat mumps virus, mammalian parainfluenza virus 5 and bat Alston virus, and porcine La Piedad Michoacán virus and bat Mapuera virus. These results support the idea that bats are a reservoir for diverse paramyxoviruses with closely shared evolutionary histories, compared with a number of significant human pathogens, and expand the range of bat paramyxoviruses to North America. Given the propensity of paramyxoviruses to overcome species barriers, additional surveillance and characterization of EfORV are warranted. IMPORTANCE Bats are a reservoir of large numbers of viruses. Among bat-borne zoonotic viruses, members of Coronaviridae and Paramyxoviridae have had the largest impact on human health. The repeated spillover of bat viruses to humans, often with devastating results, has led to increased surveillance and virus discovery efforts in hot spots for virus emergence, largely Asia and Africa. Apart from rabies virus, little surveillance of viruses in bats is performed in North America. Here, viral metagenomic sequencing identified a close relative to HPIV4 in a big brown bat found in a motel room in South Dakota. The virus, EfORV, was 72% identical to HPIV4, which causes clinically significant respiratory disease, mainly in children; it represents the first bat paramyxovirus identified in North America. Close genetic relationships between bat and mammalian orthorubulaviruses underscore the importance of bats as a reservoir for zoonotic viruses.

Genetic analysis of human parainfluenza virus type 4 associated with severe acute respiratory infection in children in Luohe City, Henan Province, China, during 2017-2018.

During 2017-2018, nasopharyngeal aspirates (NPAs) from 627 hospitalized patients with severe acute respiratory infection at Luohe Center Hospital were tested by RT-PCR for human parainfluenza virus 4 (HPIV-4). Fourteen (2.2%) of the 627 samples were positive for HPIV-4. The complete HN gene was amplified from nine positive samples and sequenced. Sequence comparisons showed that the HPIV-4 strains circulating in the city of Luohe are closely related to HPIV-4A strains. Our study indicated that there were multiple lineages of HPIV-4 circulating in Henan Province in China during the study period. This will improve our understanding of the epidemiological and clinical characteristics of HPIV-4.

Human parainfluenza virus infection in severe acute respiratory infection cases in Beijing, 2014-2016: A molecular epidemiological study.

BACKGROUND: Severe acute respiratory infection (SARI) threatens human health and even survival, causing a huge number of hospitalized patients every year. However, as one of the most common respiratory viruses circulated worldwide, the epidemiological and phylogenetic characteristics of human parainfluenza virus (HPIV) in these cases were not well known. OBJECTIVES: To reveal the epidemiological features of HPIV infection in SARIs in Beijing area from September 2014 to August 2016. METHODS: A total of 1229 SARI cases in Beijing area were enrolled, investigated, sampled, and tested by multiplex real-time PCR to identify HPIVs and other common respiratory viruses. Eighteen HPIV-3 viruses isolated from all HPIV-positive samples in these SARI cases were sequenced and analyzed. RESULTS: Among all enrolled cases, 0.81%, 0.73%, 4.48%, and 0.57% were positive for HPIV-1 to HPIV-4, respectively. The highest yield rate of HPIV infection occurred in children under 5 years old (9.07%), followed by the patients over 60 years old (6.02%). The phylogenetic information of HPIV-3 showed that all viruses belonged to Cluster C3a. CONCLUSIONS: Besides the young children, the elders older than 60 years also showed a relatively high infection rate of HPIVs, which should be given comparable attentions. Moreover, the HPIV-3 circulating in China undergoes continued evolution, suggesting the potential risk of evolved HPIV infection should not be overlooked.

Discovery of a divergent HPIV4 from respiratory secretions using second and third generation metagenomic sequencing.

Molecular detection of viruses has been aided by high-throughput sequencing, permitting the genomic characterization of emerging strains. In this study, we comprehensively screened 500 respiratory secretions from children with upper and/or lower respiratory tract infections for viral pathogens. The viruses detected are described, including a divergent human parainfluenza virus type 4 from GS FLX pyrosequencing of 92 specimens. Complete full-genome characterization of the virus followed, using Single Molecule, Real-Time (SMRT) sequencing. Subsequent "primer walking" combined with Sanger sequencing validated the RS platform's utility in viral sequencing from complex clinical samples. Comparative genomics reveals the divergent strain clusters with the only completely sequenced HPIV4a subtype. However, it also exhibits various structural features present in one of the HPIV4b reference strains, opening questions regarding their lifecycle and evolutionary relationships among these viruses. Clinical data from patients infected with the strain, as well as viral prevalence estimates using real-time PCR, is also described.

A novel duplex real-time PCR for HPIV-4 detects co-circulation of both viral subtypes among ill children during 2008.

The two subtypes of the human parainfluenzavirus type 4 (HPIV-4) are rarely sought in testing for acute respiratory illness (ARI) and this may be confounding our understanding of its role. This study presents a novel duplex real-time RT-PCR assay targeting the P gene that can detect and differentiate the two subtypes in a single reaction. Subtype-specific synthetic RNA positive controls were prepared and used to determine an analytical sensitivity of 10 copies per reaction with an 8log(10) dynamic range. The assays were validated using 1140 clinical specimens mostly nasopharyngeal aspirates collected from children during 2008. These included specimens previously determined to be positive for all commonly considered respiratory viruses. The novel assay did not cross-reaction with any other virus. Fourteen HPIV-4 positives, ten detected in the absence of any co-detections (four with rhinovirus), were identified in 2008 and their subtype confirmed by conventional RT-PCR and sequencing of P gene fragments. Most detections were in children two years of age or younger. Our assay proved suitably sensitive and specific for inclusion in future studies seeking to better understand the role HPIV-4 and other respiratory viruses in children with ARI.

Completion of the full-length genome sequence of human parainfluenza virus types 4A and 4B: sequence analysis of the large protein genes and gene start, intergenic and end sequences.

We have already reported the nucleotide sequences of the NP, P/V, M, F and HN genes of human parainfluenza virus type 4A (hPIV-4A) and type 4B (hPIV-4B). Here, we have determined the sequences of the L protein genes as well as the gene start, intergenic and end sequences, thereby completing the full-length genome sequence of hPIV-4A and 4B. hPIV-4A and 4B have 17,052 and 17,304 nucleotides, respectively. The end sequence of hPIV-4, especially 4B, was extraordinarily long. In a comparison with members of the genus Rubulavirus, the hPIV-4 L proteins were closely related to those of mumps virus (MUV) and hPIV-2, less closely related to those of Menangle virus and Tioman virus, and more distantly related to those of Mapuera virus and porcine rubulavirus.

Clinical and molecular epidemiology of human parainfluenza virus 4 infections in hong kong: subtype 4B as common as subtype 4A.

In this 1-year study, 35 (1.2%) of 2,912 nasopharyngeal aspirates were positive for human parainfluenza virus 4 (HPIV4) by reverse transcription-PCR. Patients with HPIV4 infection were mainly young children and immunocompromised adults. In contrast to the reported predominance of HPIV4A infection, molecular subtyping revealed that 15 (44%) cases were caused by HPIV4B.

Human parainfluenza virus 4 outbreak and the role of diagnostic tests.

Owing to the difficulties in isolating the virus and the lack of routine surveillance, the clinical significance of human parainfluenza virus 4 (HPIV-4) is less well defined than that of the other human parainfluenza viruses. We describe the first outbreak of HPIV-4 infection in a developmental disabilities unit, involving 38 institutionalized children and three staff members, during a 3-week period in autumn 2004. Most subjects had upper respiratory tract infections (URTI), while lower respiratory tract infections (LRTI) occurred in three children (7%), one complicated by respiratory failure requiring ventilation support. All patients recovered. Nasopharyngeal aspirates tested for HPIV-4 were positive by reverse transcriptase PCR (RT-PCR) in all 41 cases (100%), by direct immunofluorescence in 29 of 39 tested cases (74%), and by cell cultures in 6 of 37 cases (16%), and serum was positive for antibodies against HPIV-4 in all 35 cases (100%) with serum samples available. In addition, RT-PCR detected HPIV-4 in four children (three LRTI and one URTI) out of 115 patients with community-acquired respiratory tract infection. Molecular analysis of the 1,198-bp phosphoprotein sequences showed that HPIV-4 isolates among the cases were genetically similar, whereas the community controls were more genetically distant, supporting nosocomial transmission of a single HPIV-4 genotype during the outbreak. Moreover, the HPIV-4 causing the outbreak is more closely related to HPIV-4A than HPIV-4B. HPIV-4 may be an important cause of more severe respiratory illness in children. The present RT-PCR assay is a sensitive, specific, and rapid method for the diagnosing HPIV-4 infection. To better define the epidemiology and clinical spectrum of disease of HPIV-4 infections, HPIV-4 should be included in the routine panels of respiratory virus detection on respiratory specimens.

Simultaneous detection of fourteen respiratory viruses in clinical specimens by two multiplex reverse transcription nested-PCR assays.

There is a need for rapid, sensitive, and accurate diagnosis of lower respiratory tract infections in children, elderly, and immunocompromised patients, who are susceptible to serious complications. The multiplex RT-nested PCR assay has been used widely for simultaneous detection of non-related viruses involved in infectious diseases because of its high specificity and sensitivity. A new multiplex RT-PCR assay is described in this report. This approach includes nested primer sets targeted to conserve regions of human parainfluenza virus haemagglutinin, human coronavirus spike protein, and human enterovirus and rhinovirus polyprotein genes. It permits rapid, sensitive, and simultaneous detection and typing of the four types of parainfluenza viruses (1, 2, 3, 4AB), human coronavirus 229E and OC43, and the generic detection of enteroviruses and rhinoviruses. The testing of 201 clinical specimens with this multiplex assay along with other one formerly described by our group to simultaneously detect and type the influenza viruses, respiratory syncytial viruses, and a generic detection of all serotypes of adenovirus, covers the detection of most viruses causing respiratory infectious disease in humans. The results obtained were compared with conventional viral culture, immunofluorescence assay, and a third multiplex RT-PCR assay for all human parainfluenza viruses types described previously. In conclusion, both multiplex RT-PCR assays provide a system capable of detecting and identifying simultaneously 14 different respiratory viruses in clinical specimens with high sensitivity and specificity, being useful for routine diagnosis and survey of these viruses within the population.