Isolation, identification and pathogenic characteristics of tick-derived parainfluenza virus 5 in northeast China.

The number of parainfluenza virus 5 (PIV5) infection cases has increased worldwide over the past six decades; however, factors underlying this increase remain unclear. PIV5 has been emerging or re-emerging in humans and animal species. To date, no information is yet available regarding PIV5 infection in arthropod ticks. Here, we successfully isolated tick-derived PIV5 from the Ixodes persulcatus species designated as HLJ/Tick/2019 in Heilongjiang, China. Phylogenetic analysis revealed that the tick-derived PIV5 is closely related to subclade 2.2.6, which has become the dominant subtype prevalent in dogs, pigs and wildlife across China. Further experiments to understand the importance of this virus as an infectious vector revealed that a ferret animal model experimentally infected with Tick/HLJ/2019 via the oronasal and ocular inoculation routes developed moderate respiratory distress with pneumonia and neurologic tissue damage from inflammation for the first time. Further surveillance of PIV5 in vectors of viral transmission is necessary to enhance our knowledge of its ecology in reservoirs and facilitate the control of re-emerging diseases.

Comparison of hemagglutination inhibition tests, immunoperoxidase monolayer assays, and serum neutralizing tests in detecting antibodies against blue eye disease in pigs.

Blue eye disease (BED) of pigs was identified in the early 1980s in La Piedad, Michoacan, Mexico. The causal agent is Porcine orthorubulavirus (PRV), which affects pigs of all ages, producing nervous, respiratory, and reproductive disorders. BED is geographically endemic to the center of Mexico, where 75% of the country's swine industry is concentrated. Due to its adverse effects on the swine industry and the risk of dissemination to other countries, it is essential to have reliable diagnostic methods for BED. The objective of this study was to establish the optimal conditions for three serological tests, hemagglutination inhibition (HI), immunoperoxidase monolayer assay (IPMA), and serum neutralization (SN), and to compare their sensitivity, specificity, kappa coefficient, and predictive values. Twelve different HI protocols (9408 tests), one SN protocol and one IPMA protocol (784 tests, each) were evaluated. Forty-nine sera were analyzed, and thirty-seven sera showed true positive results, while twelve showed true negative results. The kappa coefficient was used to assess the variation in each test. The best HI protocol registered a sensitivity and specificity of 89 and 100%, respectively, the IPMA test showed values of 85 and 100%, and the SN test registered a sensitivity of 91% and a specificity of 96%. One of the disadvantages of the HI test is that when chicken red blood cells (RBCs) are used, elution occurs in a short incubation time, which would decrease the specificity. The use of bovine RBCs increases the specificity of the testy and makes it more stable, but it decreases the sensitivity. The results of HI and SN revealed the importance of eliminating the complement system of the serum and removing other inhibitors to avoid test nonspecificity. The IPMA test does not use an active virus; hence, it is considered safe and does not present any risk of disseminating PRV.

Molecular detection and whole genome characterization of Canine Parainfluenza type 5 in Thailand.

Parainfluenza virus type 5 (PIV-5) causes respiratory infection in several animal species and humans. Canine parainfluenza virus type 5 (CPIV-5) causes respiratory disease in domestic dogs worldwide. In this study, we conducted a cross-sectional survey of CPIV-5 in dogs with respiratory symptoms from small animal hospitals in Thailand from November 2015 to December 2018. Our results showed that 32 out of 571 nasal swab samples (5.6%) were positive for CPIV-5 by RT-PCR specific to the NP gene. To characterize the viruses, three representative CPIV-5 were subjected to whole genome sequencing, and an additional ten CPIV-5 were subjected to HN, F, SH and V/P gene sequencing. Pairwise sequence comparison and phylogenetic analysis showed that Thai CPIV-5 was closely related to the CPIV-5 isolated from China and Korea. In conclusion, this study constitutes a whole genome characterization of CPIV-5 from dogs in Thailand. The surveillance of CPIV-5 should be further investigated at a larger scale to determine the dynamics, distribution and potential zoonotic transmission of CPIV-5.

Respiratory infections and coinfections: geographical and population patterns.

INTRODUCTION: Acute respiratory infections are the second cause of mortality in children younger than five years, with 150.7 million episodes per year. Human orthopneumovirus (hOPV) and metapneumovirus (hMPV) are the first and second causes of bronchiolitis; type 2 human orthorubulavirus (hORUV) has been associated with pneumonia in immunocompromised patients. OBJECTIVE: To define hOPV, hMPV and hORUV geographical distribution and circulation patterns. METHOD: An observational, prospective cross-sectional pilot study was carried out. Two-hundred viral strains obtained from pediatric patients were genotyped by endpoint reverse transcription polymerase chain reaction (RT-PCR). RESULTS: One-hundred and eighty-six positive samples were typed: 84 hOPV, 43 hMPV, two hORUV and 57 co-infection specimens. Geographical distribution was plotted. hMPV, hOPV, and hORUV cumulative incidences were 0.215, 0.42, and 0.01, respectively. Cumulative incidence of hMPV-hORUV and hMPV-hOPV coinfection was 0.015 and 0.23; for hOPV-hMPV-hORUV, 0.035; and for hORUV-hOPV, 0.005. The largest number of positive cases of circulating or co-circulating viruses occurred between January and March. CONCLUSIONS: This study successfully identified circulation and geographical distribution patterns of the different viruses, as well as of viral co-infections.

INTRODUCCIÓN: Las infecciones respiratorias agudas constituyen la segunda causa de mortalidad en los niños menores de cinco años, con 150.7 millones de episodios anuales. Entre los principales agentes etiológicos están Orthopneumovirus (hOPV) y metapneumovirus (hMPV) humanos como primera y segunda causa de bronquiolitis, respectivamente; Orthorubulavirus humano tipo 2 (hORUV) se ha asociado a neumonía en pacientes inmunocomprometidos. OBJETIVO: Definir patrones de distribución geográfica y de circulación de hOPV, hMPV y hORUV. MÉTODO: Se llevó a cabo un estudio piloto transversal prospectivo observacional. Se genotipificaron 200 aislamientos virales de pacientes pediátricos mediante transcripción inversa seguida de reacción en cadena de la polimerasa en punto final (RT-PCR). RESULTADOS: Se tipificaron 186 muestras positivas: 84 de hOPV, 43 de hMPV, dos de hORUV y 57 de coinfecciones. Se trazó la distribución geográfica. Las incidencias acumuladas de hMPV, hOPV y hORUV fueron de 0.215, 0.42 y 0.01, respectivamente. Las incidencias acumuladas de la coinfección de hMPV-hORUV y hMPV-hOPV fueron de 0.015 y 0.23; de hOPV-hMPV-hORUV, de 0.035; y de hORUV-hOPV, de 0.005. El mayor número de casos positivos de virus circulantes o cocirculantes se presentó entre enero y marzo. CONCLUSIONES: Fue posible identificar patrones de circulación y distribución geográfica de los diferentes virus, así como de las coinfecciones virales.

Respiratory infections and coinfections: geographical and population patterns / Infecciones y coinfecciones respiratorias: patrón geográfico y de circulación poblacional

Abstract Introduction: Acute respiratory infections are the second cause of mortality in children younger than five years, with 150.7 million episodes per year. Human orthopneumovirus (hOPV) and metapneumovirus (hMPV) are the first and second causes of bronchiolitis; type 2 human orthorubulavirus (hORUV) has been associated with pneumonia in immunocompromised patients. Objective: To define hOPV, hMPV and hORUV geographical distribution and circulation patterns. Method: An observational, prospective cross-sectional pilot study was carried out. Two-hundred viral strains obtained from pediatric patients were genotyped by endpoint reverse transcription polymerase chain reaction (RT-PCR). Results: One-hundred and eighty-six positive samples were typed: 84 hOPV, 43 hMPV, two hORUV and 57 co-infection specimens. Geographical distribution was plotted. hMPV, hOPV, and hORUV cumulative incidences were 0.215, 0.42, and 0.01, respectively. Cumulative incidence of hMPV-hORUV and hMPV-hOPV coinfection was 0.015 and 0.23; for hOPV-hMPV-hORUV, 0.035; and for hORUV-hOPV, 0.005. The largest number of positive cases of circulating or co-circulating viruses occurred between January and March. Conclusions: This study successfully identified circulation and geographical distribution patterns of the different viruses, as well as of viral co-infections.

Resumen Introducción: Las infecciones respiratorias agudas constituyen la segunda causa de mortalidad en los niños menores de cinco años, con 150.7 millones de episodios anuales. Entre los principales agentes etiológicos están Orthopneumovirus (hOPV) y metapneumovirus (hMPV) humanos como primera y segunda causa de bronquiolitis, respectivamente; Orthorubulavirus humano tipo 2 (hORUV) se ha asociado a neumonía en pacientes inmunocomprometidos. Objetivo: Definir patrones de distribución geográfica y de circulación de hOPV, hMPV y hORUV. Método: Se llevó a cabo un estudio piloto transversal prospectivo observacional. Se genotipificaron 200 aislamientos virales de pacientes pediátricos mediante transcripción inversa seguida de reacción en cadena de la polimerasa en punto final (RT-PCR). Resultados: Se tipificaron 186 muestras positivas: 84 de hOPV, 43 de hMPV, dos de hORUV y 57 de coinfecciones. Se trazó la distribución geográfica. Las incidencias acumuladas de hMPV, hOPV y hORUV fueron de 0.215, 0.42 y 0.01, respectivamente. Las incidencias acumuladas de la coinfección de hMPV-hORUV y hMPV-hOPV fueron de 0.015 y 0.23; de hOPV-hMPV-hORUV, de 0.035; y de hORUV-hOPV, de 0.005. El mayor número de casos positivos de virus circulantes o cocirculantes se presentó entre enero y marzo. Conclusiones: Fue posible identificar patrones de circulación y distribución geográfica de los diferentes virus, así como de las coinfecciones virales.

Common and unique mechanisms of filamentous actin formation by viruses of the genus Orthorubulavirus.

We previously found that infection with human parainfluenza virus type 2 (hPIV-2), a member of the genus Orthorubulavirus, family Paramyxoviridae, causes filamentous actin (F-actin) formation to promote viral growth. In the present study, we investigated whether similar regulation of F-actin formation is observed in infections with other rubulaviruses, such as parainfluenza virus type 5 (PIV-5) and simian virus 41 (SV41). Infection with these viruses caused F-actin formation and RhoA activation, which promoted viral growth. These results indicate that RhoA-induced F-actin formation is important for efficient growth of these rubulaviruses. Only SV41 and hPIV-2 V and P proteins bound to Graf1, while the V and P proteins of PIV-5, mumps virus, and hPIV-4 did not bind to Graf1. In contrast, the V proteins of these rubulaviruses bound to both inactive RhoA and profilin 2. These results suggest that there are common and unique mechanisms involved in regulation of F-actin formation by members of the genus Orthorubulavirus.

Parainfluenza Virus 5 Infection in Neurological Disease and Encephalitis of Cattle.

The etiology of viral encephalitis in cattle often remains unresolved, posing a potential risk for animal and human health. In metagenomics studies of cattle with bovine non-suppurative encephalitis, parainfluenza virus 5 (PIV5) was identified in three brain samples. Interestingly, in two of these animals, bovine herpesvirus 6 and bovine astrovirus CH13 were additionally found. We investigated the role of PIV5 in bovine non-suppurative encephalitis and further characterized the three cases. With traditional sequencing methods, we completed the three PIV5 genomes, which were compared to one another. However, in comparison to already described PIV5 strains, unique features were revealed, like an 81 nucleotide longer open reading frame encoding the small hydrophobic (SH) protein. With in situ techniques, we demonstrated PIV5 antigen and RNA in one animal and found a broad cell tropism of PIV5 in the brain. Comparative quantitative analyses revealed a high viral load of PIV5 in the in situ positive animal and therefore, we propose that PIV5 was probably the cause of the disease. With this study, we clearly show that PIV5 is capable of naturally infecting different brain cell types in cattle in vivo and therefore it is a probable cause of encephalitis and neurological disease in cattle.

Human parainfluenza virus circulation, United States, 2011-2019.

BACKGROUND: Human parainfluenza viruses (HPIVs) cause upper and lower respiratory tract illnesses, most frequently among infants and young children, but also in the elderly. While seasonal patterns of HPIV types 1-3 have been described, less is known about national patterns of HPIV-4 circulation. OBJECTIVES: To describe patterns of HPIVs circulation in the United States (US). STUDY DESIGN: We used data from the National Respiratory and Enteric Virus Surveillance System (NREVSS), a voluntary passive laboratory-based surveillance system, to characterize the epidemiology and circulation patterns of HPIVs in the US during 2011-2019. We summarized the number of weekly aggregated HPIV detections nationally and by US census region, and used a subset of data submitted to NREVSS from public health laboratories and several clinical laboratories during 2015-2019 to analyze differences in patient demographics. RESULTS: During July 2011 - June 2019, 2,700,135 HPIV tests were reported; 122,852 (5 %) were positive for any HPIV including 22,446 for HPIV-1 (18 %), 17,474 for HPIV-2 (14 %), 67,649 for HPIV-3 (55 %), and 15,283 for HPIV-4 (13 %). HPIV testing increased substantially each year. The majority of detections occurred in children aged ≤ 2 years (36 %) with fluctuations in the distribution of age by type. CONCLUSIONS: HPIVs were detected year-round during 2011-2019, with type-specific year-to-year variations in circulation patterns. Among HPIV detections where age was known, the majority were aged ≤ 2 years. HPIV-4 exhibited an annual fall-winter seasonality, both nationally and regionally. Continued surveillance is needed to better understand national patterns of HPIV circulation.

Novel Mammalian orthorubulavirus 5 Discovered as Accidental Cell Culture Contaminant.

A distinct Russian Mammalian orthorubulavirus 5 (PIV5) was detected in cell culture exhibiting cytopathic effect and hypothesized to be contaminated by a scientist with respiratory symptoms. The identification of the divergent strain indicated a lack of knowledge on the diversity of PIV5 strains and calls for surveillance of global PIV5 strains.

Profilin2 is required for filamentous actin formation induced by human parainfluenza virus type 2.

We previously reported that human parainfluenza virus type 2 (hPIV-2) promoted RhoA activation and subsequent filamentous actin (F-actin) formation. Actin-binding proteins, such as profilin and cofilin, are involved in the regulation of F-actin formation by RhoA signaling. In the present study, we identified profilin2 as a key molecule that is involved in hPIV-2-induced F-actin formation. Immunoprecipitation assays demonstrated that hPIV-2 V protein binds to profilin2 but not to profilin1. Mutation of Trp residues within C-terminal region of V protein abolished the binding capacity to profilin2. Depletion of profilin2 resulted in the inhibition of hPIV-2-induced F-actin formation and the suppression of hPIV-2 growth. Overexpression of wild type V but not Trp-mutated V protein reduced the quantity of actin co-immunoprecipitated with profilin2. Taken together, these results suggest that hPIV-2 V protein promotes F-actin formation by affecting actin-profilin2 interaction through its binding to profilin2.

Claudin-1 inhibits human parainfluenza virus type 2 dissemination.

Tight junctions enable epithelial cells to form physical barriers that act as an innate immune defense against respiratory infection. However, the involvement of tight junction molecules in paramyxovirus infections, which include various respiratory pathogens, has not been examined in detail. Human parainfluenza virus type 2 (hPIV2) infects airway epithelial cells and causes respiratory illness. In the present study, we found that hPIV2 infection of cultured cells induces expression of claudin-1 (CLDN1), an essential component of tight junctions. This induction seemed to be intrinsically restricted by V, an accessory protein that modulates various host responses, to enable efficient virus propagation. By generating CLDN1 over-expressing and knockout cell lines, we showed that CLDN1 is involved in the restriction of hPIV2 spread via cell-to-cell contact. Taken together, we identified CLDN1 an inhibitory factor for hPIV2 dissemination, and that its V protein acts to counter this.

Complete Genome Sequence of Parainfluenza Virus 5 (PIV5) from a Sunda Pangolin (Manis javanica) in China.

We report here the complete genome sequence of the parainfluenza virus PIV5-GD18 strain, isolated from a wild Sunda pangolin (Manis javanica) in China in 2017. It was 15,246 nucleotides with four nucleotides substitutions, which resulted in four changes of amino acid that were found only in PIV5-GD18, which further broadens the PIV5 infection host spectrum and will aid in our understanding of the complete genome sequence of PIV5 in different hosts.

Angiomotin-Like 1 Links Paramyxovirus M Proteins to NEDD4 Family Ubiquitin Ligases.

To define the links between paramyxovirus budding and cellular ESCRT machinery, we previously identified angiomotin-like 1 (AMOTL1) in a screen for host factors that bind to the matrix (M) protein of parainfluenza virus 5 (PIV5). This protein harbors three L/PPXY sequences, allowing it to interact with WW domain containing proteins including NEDD4 family members. We hypothesize that paramyxoviruses use AMOTL1 as a linker to indirectly recruit the same NEDD4 ubiquitin ligases for budding that other enveloped viruses recruit directly through their PPXY late domains. In support of this hypothesis, we found that AMOTL1 could link together M proteins and NEDD4 family proteins in three-way co-IP experiments. Both PIV5 and mumps virus M proteins could be linked to the NEDD4 family proteins NEDD4-1, NEDD4L, and NEDL1, provided that AMOTL1 was co-expressed as a bridging protein. AMOT and AMOTL2 could not substitute for AMOTL1, as they lacked the ability to bind with paramyxovirus M proteins. Attachment of a PPXY late domain sequence to PIV5 M protein obviated the need for AMOTL1 as a linker between M and NEDD4 proteins. Together, these results suggest a novel host factor recruitment strategy for paramyxoviruses to achieve particle release.

Co-Circulation and Excretion Dynamics of Diverse Rubula- and Related Viruses in Egyptian Rousette Bats from South Africa.

The Egyptian rousette bat (Rousettus aegyptiacus) has previously been implicated as the natural host of a zoonotic rubulavirus; however, its association with rubulaviruses has been studied to a limited extent. Urine, spleen, and other organs collected from the R. aegyptiacus population within South Africa were tested with a hemi-nested RT-PCR assay targeting a partial polymerase gene region of viruses from the Avula- and Rubulavirus genera. Urine was collected over a 14-month period to study the temporal dynamics of viral excretion. Diverse rubulaviruses, including viruses related to human mumps and parainfluenza virus 2, were detected. Active excretion was identified during two peak periods coinciding with the host reproductive cycle. Analysis of additional organs indicated co-infection of individual bats with a number of different putative rubulaviruses, highlighting the limitations of using a single sample type when determining viral presence and diversity. Our findings suggest that R. aegyptiacus can harbor a range of Rubula- and related viruses, some of which are related to known human pathogens. The observed peaks in viral excretion represents potential periods of a higher risk of virus transmission and zoonotic disease spill-over.

Alston Virus, a Novel Paramyxovirus Isolated from Bats Causes Upper Respiratory Tract Infection in Experimentally Challenged Ferrets.

Multiple viruses with zoonotic potential have been isolated from bats globally. Here we describe the isolation and characterization of a novel paramyxovirus, Alston virus (AlsPV), isolated from urine collected from an Australian pteropid bat colony in Alstonville, New South Wales. Characterization of AlsPV by whole-genome sequencing and analyzing antigenic relatedness revealed it is a rubulavirus that is closely related to parainfluenza virus 5 (PIV5). Intranasal exposure of mice to AlsPV resulted in no clinical signs of disease, although viral RNA was detected in the olfactory bulbs of two mice at 21 days post exposure. Oronasal challenge of ferrets resulted in subclinical upper respiratory tract infection, viral shedding in respiratory secretions, and detection of viral antigen in the olfactory bulb of the brain. These results imply that AlsPV may be similar to PIV5 in its ability to infect multiple mammalian host species. This isolation of a novel paramyxovirus with the potential to transmit from bats to other mammalian species reinforces the importance of continued surveillance of bats as a source of emerging viruses.

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.

Genotype replacement of the human parainfluenza virus type 2 in Croatia between 2011 and 2017 - the role of neutralising antibodies.

Previously we reported on the HPIV2 genotype distribution in Croatia 2011-2014. Here we expand this period up to 2017 and confirm that G1a genotype has replaced G3 genotype from the period 2011-2014. Our hypothesis was that the G1a-to-G3 genotype replacement is an antibody-driven event. A cross-neutralisation with anti-HPIV2 sera specific for either G1a or G3 genotype revealed the presence of genotype-specific antigenic determinants. By the profound, in silico analyses three potential B cell epitopic regions were identified in the hemagglutinin neuraminidase (regions 314-361 and 474-490) and fusion protein (region 440-484). The region identified in the fusion protein does not show any unique site between the G1a and G3 isolates, five differentially glycosylated sites in the G1a and G3 genotype isolates were identified in epitopic regions of hemagglutinin neuraminidase. All positively selected codons were found to be located either in the region 314-316 or in the region 474-490 what indicates a strong positive selection in this region and reveals that these regions are susceptible to evolutionary pressure possibly caused by antibodies what gives a strong verification to our hypothesis that neutralising antibodies are a key determinant in the inherently complex adaptive evolution of HPIV2 in the region.

Seroprevalence of Human Parainfluenza Virus Types 1-4 Among Healthy Children Under 5 Years of Age in Korea.

Human parainfluenza viruses (HPIVs) are among the major causes of respiratory infections in children, worldwide, including in Korea. There are four types of HPIVs, each with different epidemiological characteristics. HPIV3 is the most frequently circulating HPIV type, while the epidemiology of HPIV4 remains unclear. The aim of this study was to investigate the age-stratified seropositivity rates of HPIV types 1-4 among children in Korea. These data will be useful to determine vaccine requirements. This study included 245 participants categorized into four age groups: 6-11 months, 1 year, 2 years, and 3-5 years. Hemagglutination inhibition (HAI) assay was used to measure the antibody titers in the serum samples of the subjects. Overall, a significantly higher seropositivity rate (68%) was observed for HPIV3 (p < 0.001), indicating the predominant circulation of this type. In the 3- to 5-year-old group, 97% of the participants displayed seropositivity for HPIV3, suggesting that most Korean children acquire HPIV3 infection by the age of 5 years. The seropositivity rate for HPIV3 increased with age (p < 0.001); a prompt rise was observed between the 6-11 months age group and the 1-year age group. The seropositivity rates of HPIV1, HPIV2, and HPIV4 were found to increase with age (p < 0.001), with a marked increase recorded after the age of 2 years. HPIV1, HPIV2, and HPIV4 tended to infect children later than HPIV3. Older children showed high antibody titer ranges for HPIV3 (p < 0.001), suggesting that children experience multiple HPIV3 infections. An increasing trend of HPIV4 seropositivity rates with age was observed and this was comparable to theHPIV1 and HPIV2 seropositivity rates, indicating that its incidence may have been underestimated. To reduce HPIV infection, the administration of a HPIV3 vaccine to children 1 year of age is likely to be the most effective option.

Human parainfluenza virus type 2 V protein inhibits caspase-1.

The multifunctional V protein of human parainfluenza virus type 2 (hPIV2) plays important roles in controlling viral genome replication, inhibiting the host interferon response and promoting virus growth. We screened a yeast two-hybrid library using V protein as bait to identify host factors that are important for other functions of V. One of several positive clones isolated from HeLa cell-derived cDNA library encodes caspase-1. We found that the C-terminal region of V interacts with the C-terminal region of caspase-1 in mammalian cells. Moreover, the V protein repressed caspase-1 activity and the formation of interleukin-1ß (IL-1ß) in a dose-dependent manner. IL-1ß secretion induced by wild-type hPIV2 infection in human monocytic THP-1 cells was significantly lower than that induced by recombinant hPIV2 lacking V protein or having a mutant V. These data suggest that hPIV2 V protein inhibits caspase-1-mediated maturation of IL-1ß via its interaction with caspase-1.