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Human parainfluenza viruses (HPIVs) are a significant cause of acute lower respiratory tract infections (ALRTIs) among young children and elderly individuals worldwide. The four types of HPIVs (HPIV1-4) can cause recurrent infections and pose a significant economic burden on health care systems globally. However, owing to the limited availability of complete genome sequences, the genetic evolution of these viruses and the development of vaccines and antiviral treatments are hampered. To address this issue, this study utilized next-generation sequencing to obtain 156 complete genome sequences of HPIV1-4, which were isolated from hospitalized children with ALRTIs in six regions of China between 2015 and 2021. This study revealed multiple clades, lineages, or sublineages of HPIVs circulating in mainland China, with a novel clade D of HPIV1 identified as geographically restricted to China. Moreover, this study identified the endemic dominant genotype of HPIV3, lineage C3, which has widely spread and continuously circulated in China. Bioinformatic analysis of the genome sequences revealed that the proteins of HPIV3 possessed the most variable sites, with the P protein showing more diversity than the other proteins among all types of HPIVs. The HN proteins of HPIV1-3 are all under negative/purifying selection, and two amino acid substitutions in the HN proteins correspond to known mAb neutralizing sites in the two HPIV3 strains. These findings provide crucial insights into the genetic diversity and evolutionary dynamics of HPIVs circulating among children in China and may facilitate research on the molecular diagnosis, vaccine development, and surveillance of HPIVs.IMPORTANCEPhylogenetic analysis revealed the prevalence of multiple clades, lineages, or sublineages of human parainfluenza viruses (HPIVs) circulating in mainland China. Notably, a unique evolutionary branch of HPIV1 containing only Chinese strains was identified and designated clade D. Furthermore, in 2023, HPIV3 strains from Pakistan and Russia formed a new lineage within clade C, named C6. The first HPIV4b sequence obtained in this study from China belongs to lineage C2. Evolutionary rate assessments revealed that both the HN and whole-genome sequences of HPIV3 presented the lowest evolutionary rates compared with those of the other HPIV types, with rates of 6.98E-04 substitutions/site/year (95% HPD: 5.87E-04 to 8.25E-03) and 5.85E-04 substitutions/site/year (95% HPD: 5.12E-04 to 6.62E-04), respectively. Recombination analysis revealed a potential recombination event in the F gene of an HPIV1 strain in this study. Additionally, all the newly obtained HPIV1-3 strains exhibited negative selection pressure, and two mutations were identified in the HN protein of two HPIV3 strains at monoclonal antibody-binding sites., Competing Interests: The authors declare no conflict of interest.

Human parainfluenza virus (HPIV) causes respiratory infections, which are exacerbated in children and older people. Correct evaluation of viral characteristics is essential for the study of countermeasures. However, adaptation of viruses to cultured cells during isolation or propagation might select laboratory passage-associated mutations that modify the characteristics of the virus. It was previously reported that adaptation of HPIV3, but not other HPIVs, was avoided in human airway epithelia. To examine the influence of laboratory passage on the genomes of HPIV1-HPIV4, we evaluated the occurrence of mutations after passage in primary human bronchial/tracheal epithelial cell air-liquid interface (HBTEC-ALI) culture and conventional cultured cells (Vero cells expressing the transmembrane protease, serine 2, and normal Vero cells). The occurrence of mutations was significantly lower in HBTEC-ALI than in conventional culture. In HBTEC-ALI culture, most of the mutations were silent or remained at low variant frequency, resulting in less impact on the viral consensus sequence. In contrast, passage in conventional culture induced or selected genetic mutations at high frequency with passage-associated unique substitutions. High mutagenesis of hemagglutinin-neuraminidase was commonly observed in all four HPIVs, and mutations even occurred in a single passage. In addition, in HPIV1 and HPIV2, mutations in the large protein were more frequent. These results indicate that passage in HBTEC-ALI culture is more suitable than conventional culture for maintaining the original characteristics of clinical isolates in all four HPIVs, which can help with the understanding of viral pathogenesis., Importance: Adaptation of viruses to cultured cells can increase the risk of misinterpretation in virological characterization of clinical isolates. In human parainfluenza virus (HPIV) 3, it has been reported that the human airway epithelial and lung organoid models are preferable for the study of viral characteristics of clinical strains without mutations. Therefore, we analyzed clinical isolates of all four HPIVs for the occurrence of mutations after five laboratory passages in human bronchial/tracheal epithelial cell air-liquid interface (HBTEC-ALI) or conventional culture. We found a high risk of hemagglutinin-neuraminidase mutagenesis in all four HPIVs in conventional cultured cells. In addition, in HPIV1 and HPIV2, mutations of the large protein were also more frequent in conventional cultured cells than in HBTEC-ALI culture. HBTEC-ALI culture was useful for maintaining the original sequence and characteristics of clinical isolates in all four HPIVs. The present study contributes to the understanding of HPIV pathogenesis and antiviral strategies., Competing Interests: The authors declare no conflict of interest.

Respiratory disease is a significant economic issue in pig farming, with a complex aetiology that includes swine influenza A viruses (swIAV), which are common in European domestic pig populations. The most recent human influenza pandemic in 2009 showed swIAV's zoonotic potential. Monitoring pathogens and disease control are critical from a preventive standpoint, and are based on quick, sensitive, and specific diagnostic assays capable of detecting and distinguishing currently circulating swIAV in clinical samples. For passive surveillance, a set of multiplex quantitative reverse transcription real-time PCRs (mRT-qPCR) and MinION-directed sequencing was updated and deployed. Several lineages and genotypes of swIAV were shown to be dynamically developing, including novel reassortants between human pandemic H1N1 and the avian-derived H1 lineage of swIAV. Despite this, nearly 70% (842/1216) of individual samples from pigs with respiratory symptoms were swIAV-negative, hinting to different aetiologies. The complex and synergistic interactions of swIAV infections with other viral and bacterial infectious agents contribute to the aggravation of pig respiratory diseases. Using a newly developed mRT-qPCR for the combined detection of swIAV and the recently described porcine respirovirus 1 (PRV1) and swine orthopneumovirus (SOV) widespread co-circulation of PRV1 (19.6%, 238/1216 samples) and SOV (14.2%, 173/1216 samples) was evident. Because of the high incidence of PRV1 and SOV infections in pigs with respiratory disease, these viruses may emerge as new allies in the porcine respiratory disease syndrome.

Bovine Parainfluenza Type 3 virus (BPIV-3) is an enveloped, non-segmented single-stranded, negative-sense RNA virus belonging to the Paramyxoviridae family (genus Respirovirus) with a well-known role in Bovine Respiratory Disease (BRD) onset. Being isolated for the first time in 1959, BPIV-3 currently circulates worldwide in cattle herds and is routinely tested in suspected BRD cases. Different commercial vaccines are available to prevent infection and/or to reduce the clinical signs associated with BPIV-3 infection, which are essential to prevent secondary infections. Despite years of molecular surveillance, a very limited number of complete genome sequences were made publicly available, preventing thus the understanding of the genetic diversity of the circulating strains in the field. In addition, no data about the genetic identity between field and vaccine strains is currently available. In this study, we sequenced the full-genome and genetically characterized BPIV-3 strains isolated from animals displaying respiratory illness in France and Sweden, as well as the vaccine strains contained in three different commercialized vaccines. Our results show that the sequences from France and Sweden belong to genotype C. However, a third sequence from Sweden from 2017 clustered within genotype A. The sequencing of vaccine strains revealed that two of the vaccine strains clustered within genotype C, whereas the third vaccine strain belonged to genotype A. Altogether, our findings suggest that both genotypes A and C circulate in Europe and that BPIV-3 field and vaccine strains are genetically divergent. Our sequencing results could be useful to better understand the genetic differences between the circulating field and vaccine BPIV-3 strains. This is crucial for a correct interpretation of diagnostic findings and for the assessment of BPIV-3 prevalence in cattle population., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023. Published by Elsevier B.V.)

Introduction: Intranasal vaccination using live vector vaccines based on non-pathogenic or slightly pathogenic viruses is the one of the most convenient, safe and effective ways to prevent respiratory infections, including COVID-19. Sendai virus is the best suited for this purpose, since it is respiratory virus and is capable of limited replication in human bronchial epithelial cells without causing disease. The aim of the work is to design and study the vaccine properties of recombinant Sendai virus, Moscow strain, expressing secreted receptor-binding domain of SARS-CoV-2 Delta strain S protein (RBDdelta) during a single intranasal immunization., Materials and Methods: Recombinant Sendai virus carrying insertion of RBDdelta transgene between P and M genes was constructed using reverse genetics and synthetic biology methods. Expression of RBDdelta was analyzed by Western blot. Vaccine properties were studied in two models: Syrian hamsters and BALB/c mice. Immunogenicity was evaluated by ELISA and virus-neutralization assays. Protectiveness was assessed by quantitation of SARS-CoV-2 RNA in RT-PCR and histological analysis of the lungs., Results: Based on Sendai virus Moscow strain, a recombinant Sen-RBDdelta(M) was constructed that expressed a secreted RBDdelta immunologically identical to natural SARS-CoV-2 protein. A single intranasal administration of Sen-RBDdelta(M) to hamsters and mice significantly, by 15 and 107 times, respectively, reduced replicative activity of SARS-CoV-2 in lungs of animals, preventing the development of pneumonia. An effective induction of virus-neutralizing antibodies has also been demonstrated in mice., Conclusion: Sen-RBDdelta(M) is a promising vaccine construct against SARS-CoV-2 infection and has a protective properties even after a single intranasal introduction.

Porcine respirovirus 1 (PRV1) is a recently emerging porcine respiratory virus that belongs to the genus Respirovirus of the Paramyxoviridae family. Since its first detection in Hong Kong, China in 2009, PRV1 has been subsequently identified in several American and European countries, suggesting that the emerging virus may have been globally distributed. However, in Asia, the virus has been reported only in China. Here, we report that PRV1 was first detected in pigs from 16 farms located in seven provinces across Korea, with a prevalence of 71.4% based on the tested oral fluid samples, suggesting that the virus is already widespread in Korean pig herds. For further genetic characterization of the Korean PRV1 strains, a complete genome and two F gene sequences were obtained from PRV1-positive samples collected from three different pig farms. Phylogenetic analysis based on the complete genome and F gene sequences showed that all three Korean PRV1 strains were grouped into European lineage 1 and were closely related to strains from Hong Kong (China), Germany and Poland. We could not obtain evidence for the origin of Korean PRV1 because of the limited availability of PRV1 sequences. In conclusion, PRV1 was first identified in Korean pig herds and genetically characterized in the present study. These results contribute to a better understanding of the global geographical distribution and genetic characteristics of PRV1., (© 2022 Wiley-VCH GmbH.)

Porcine respirovirus 1 (PRV1) is widely spread in many countries. In this study, we isolated an emgerging PRV1 strain (KS17-258) from a US swine farm. A full-length genome sequence of the virus was obtained, and the mRNA editing mechanism utilized for the expression of V/W proteins by P gene was confirmed. The virus shares 91.3-98% nucleotide sequence identity with the other PRV1 genomes reported previously. Phylogenetic analysis showed that KS17-258 forms a clade with the other US isolates. Infectious clone of the KS17-258 isolate was constructed, which was further explored as a viral vector to express enhanced green fluorescent protein (EGFP). The expression cassette of EGFP in the recombinant virus remained stable for 10 passages in cell culture. The availability of PRV1 infectious clone provides an important tool for study the basic PRV1 replication mechanisms. It also provides a novel platform for potential development of vectored vaccines against swine diseases., (Copyright © 2022 Elsevier Inc. All rights reserved.)

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.

Background: The effect of SARS-CoV-2 on existing respiratory pathogens in circulation remains uncertain. This study aimed to assess the impact of SARS-CoV-2 on the prevalence of respiratory pathogens among hospitalized children., Methods: This study enrolled hospitalized children with acute respiratory infections in Shenzhen Children's Hospital from September to December 2019 (before the COVID-19 epidemic) and those from September to December 2020 (during the COVID-19 epidemic). Nasopharyngeal swabs were collected, and respiratory pathogens were detected using multiplex PCR. The absolute case number and detection rates of 11 pathogens were collected and analyzed., Results: A total of 5696 children with respiratory tract infection received multiplex PCR examination for respiratory pathogens: 2298 from September to December 2019 and 3398 from September to December 2020. At least one pathogen was detected in 1850 (80.5%) patients in 2019, and in 2380 (70.0%) patients in 2020; the detection rate in 2020 was significantly lower than that in 2019.The Influenza A (InfA) detection rate was 5.6% in 2019, but 0% in 2020. The detection rates of Mycoplasma pneumoniae, Human adenovirus, and Human rhinovirus also decreased from 20% (460), 8.9% (206), and 41.8% (961) in 2019 to 1.0% (37), 2.1% (77), and 25.6% (873) in 2020, respectively. In contrast, the detection rates of Human respiratory syncytial virus, Human parainfluenza virus, and Human metapneumovirus increased from 6.6% (153), 9.9% (229), and 0.5% (12) in 2019 to 25.6% (873), 15.5% (530), and 7.2% (247) in 2020, respectively (p < 0.0001)., Conclusions: Successful containment of seasonal influenza as a result of COVID-19 control measures will ensure we are better equipped to deal with future outbreaks of both influenza and COVID-19.Caused by virus competition, the detection rates of Human respiratory syncytial virus, Human parainfluenza virus, and Human metapneumovirus increased in Shenzhen,that reminds us we need to take further monitoring and preventive measures in the next epidemic season., (© 2021. The Author(s).)

BRD is associated with infectious agents, but management and transport-stress are trigger factors. Metaphylactic administration of antimicrobial reduces colonization of respiratory tract by pathogens, but the development of antibiotic-resistance raises public health concerns leading to propose new control strategies. The study analyzed nasopharyngeal swabs of 231 imported cattle, 10% of 49 trucks, transported from France to southern Italy and, through Real-time PCR identified the prevalence of the involved pathogens speculating on strategies to reduce the impact of BRD. The samples were tested by Real-time PCR, for the detection of bovine coronavirus (BCoV), bovine respiratory syncytial virus (BRSV), bovine parainfluenza virus (BPiV), bovine adenovirus (BAdV), Mannheimia haemolytica, Pasteurella multocida, Histophilus somni, and Mycoplasma bovis. Yates-corrected chi squared, or Fisher's exact test were used to compare both animal-health status and positivity/negativity to pathogens, and the relationship between presence/absence of clinical signs and Real-time PCR-positivity. H. somni and BCoV were the most frequently identified pathogens. In BRD-diagnosed cattle, BAdV was detected in 13.8% (19/138), BRSV in 14.5% (20/138) and BPiV in 4.3% (6/138). Healthy cattle were mostly positive for H. somni (89.2%, 83/93). A statistically significant association was observed between clinical signs and positivity to M. haemolytica (p value = 0.016). Although mass-medication and vaccination are used for BRD control, it still remains a primary health problem. Our results highlight that the nasopharyngeal microbiota could be affected by transport and that strategies to enhance calf immunity for reducing BRD-risk development would be more effective if applied at farm of origin prior to loading., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Live-attenuated pediatric vaccines for intranasal administration are being developed for human respiratory syncytial virus (RSV), an important worldwide pediatric respiratory pathogen that lacks a licensed vaccine or suitable antiviral drug. We evaluated a prime-boost strategy in which primary immunization with RSV was boosted by secondary immunization with RSV or with a chimeric recombinant bovine/human parainfluenza virus type 3 (rB/HPIV3) vector expressing the RSV fusion F protein. The vector-expressed F protein had been engineered (DS-Cav1 mutations) for increased stability in the highly immunogenic prefusion (pre-F) conformation, with or without replacement of its transmembrane and cytoplasmic tail domains with their counterparts from bovine parainfluenza virus type 3 (BPIV3) F protein to direct incorporation into the vector virion for increased immunogenicity. In hamsters that received a primary infection with RSV, a booster infection with RSV ∼6 weeks later was completely restricted for producing infectious virus but induced a significant increase in the serum RSV-plaque-reduction neutralizing antibody titer (RSV-PRNT). Boosting instead with the rB/HPIV3-RSV-pre-F vectors resulted in efficient replication and induced significantly higher RSV-PRNTs than RSV. In African green monkeys that received a primary infection with RSV, a booster infection with RSV ∼2, ∼6, or ∼15 months later was highly restricted, whereas booster infections with the vectors had robust replication. Compared with RSV, boosts with the vectors induced 7- to 15-fold higher titers of RSV-specific serum antibodies with high neutralizing activity, as well as significantly higher titers of RSV-specific mucosal IgA antibodies. These findings support further development of this heterologous prime-boost strategy. IMPORTANCE Immune responses to RSV in infants can be reduced due to immunological immaturity and immunosuppression by RSV-specific maternal antibodies. In infants and young children, two infections with wild-type RSV typically are needed to achieve the titers of RSV-specific serum antibodies and protection against illness that are observed in adults. Therefore, a boost might substantially improve the performance of live pediatric RSV vaccines presently being developed. Hamsters and African green monkeys received a primary intranasal infection with RSV and were given a boost with RSV or a parainfluenza virus (PIV) vector expressing RSV fusion protein engineered for enhanced immunogenicity. The RSV boost was highly restricted but induced a significant increase in serum RSV-neutralizing antibodies. The PIV vectors replicated efficiently and induced significantly higher antibody responses. The use of an attenuated PIV vector expressing RSV antigen to boost a primary immunization with an attenuated RSV warrants further evaluation., (Copyright © 2020 American Society for Microbiology.)

Multiple potential pathogens are frequently co-detected among children with lower respiratory tract infection (LRTI). Evidence indicates that Bordetella pertussis has an important role in the aetiology of LRTI. We aimed to study the association between B. pertussis and other respiratory pathogens in children hospitalised with severe LRTI, and to assess clinical relevance of co-detection. Nasopharyngeal (NP) swabs and induced sputa (IS) were tested with a B. pertussis specific PCR; additionally, IS was tested for other pathogens using a multiplex PCR. We included 454 children, median age 8 months (IQR 4-18), 31 (7%) of whom tested positive for B. pertussis. Children with B. pertussis had more bacterial pathogens detected (3 versus 2; P < 0.001). While B. pertussis showed no association with most pathogens, it was independently associated with Chlamydia pneumoniae, Mycoplasma pneumoniae and parainfluenza viruses with adjusted risk ratios of 4.01 (1.03-15.64), 4.17 (1.42-12.27) and 2.13 (1.03-4.55), respectively. There was a consistent increased risk of severe disease with B. pertussis. Patterns indicated even higher risks when B. pertussis was co-detected with any of the three organisms although not statistically significant. Improving vaccine coverage against B. pertussis would impact not only the incidence of pertussis but also that of severe LRTI generally.

Porcine respirovirus 1 (PRV1) is an emerging virus in pigs that has been previously described in the USA and China. There are no reports of its presence in the rest of the world. The objective of this study was to determine the occurrence of PRV1 in Chile and to determine its phylogeny. Thus, we collected samples (oral fluids, nasal swabs, and lungs) from a swine influenza A virus (IAV) surveillance program, most of which belonged to pigs with respiratory disease. The samples were analyzed by RT-PCR, and the viral sequencing was obtained using RNA whole-genome sequencing approach. Maximum likelihood phylogeny was constructed with the available references. Thirty-one of 164 samples (18.9 %) were RT-PCR positive for PRV1: 62.5 % oral fluids, 19.0 % nasal swabs, and 8.6 % lungs. All 6 farms in this study had at least one positive sample, with 6-40 % of positive results per farm, which suggests that PRV1 is disseminated in Chilean swine farms. Twenty-one of 31 (677%) PRV1-positive samples were also positive for IAV, so the role of PRV1 as secondary pathogen in respiratory disease needs to be further evaluated. Near to complete genome of two PRV1s were obtained from two farms. The phylogenies, in general, showed low bootstrap support, except the concatenated genome and the L gene trees which showed clustering of the Chilean PRV1 with Asian sequences, suggesting a close genetic relationship. This is the first report of PRV1 in the Southern Hemisphere. Further studies are necessary to determine the genetic diversity of this virus in Chile., Competing Interests: Declaration of Competing Interest The authors declare no conflict of interest., (Copyright © 2020 The Authors. Published by Elsevier B.V. All rights reserved.)

Background: In Bangladesh, pneumonia has a higher mortality among malnourished children aged <5 years. Evaluating pneumonia etiology among malnourished children may help improve empiric treatment guidelines., Methods: During April 2015-December 2017, we conducted a case-control study among severe acute malnourished (SAM) children aged <5 years admitted to the Dhaka hospital of International Centre for Diarrhoeal Disease Research, Bangladesh (icddr,b). We enrolled hospital admitted SAM children with clinical or radiological pneumonia as cases (during April 2015 to March 2017) and hospital admitted SAM children without any respiratory symptom in the past 10 days before admission as controls (during February 2016 to December 2017). We tested nasopharyngeal wash from both case and control for respiratory syncytial virus (RSV), human metapneumovirus (HMPV), influenza viruses, human parainfluenza viruses (HPIV), rhinovirus and adenovirus by singleplex real-time reverse transcriptase polymerase chain reaction. To identify the independent association of pneumonia with viral pathogens during February 2016 to March 2017, we used multivariable logistic regression for calculating adjusted odds ratios., Results: We enrolled 360 cases and 334 controls. For case and control the median age was 8 months (IQR: 5-13) and 11 months (IQR: 6-18) (p = 0.001) respectively. Weight/age Z-score was -4.3 (SD ±0.7) for cases and -4.1 (SD ±1.1) for controls (p = 0.01). Among cases 68% had both clinical and radiological pneumonia, 1% had clinical pneumonia and 31% had only radiological pneumonia. Respiratory virus detection was high in cases compared to controls [69.9% (251) vs. 44.8% (148), p = 0.0001]. The most frequently detected viruses among cases were rhinoviruses (79, 22.0%) followed by RSV (32, 8.9%), adenovirus (23, 6.4%), HPIV (22, 6.1%), influenza virus (16, 4.5%), and HMPV (16, 4.5%). Among the controls, rhinoviruses (82, 24.8%) were most commonly detected one followed by adenovirus (26,7.9%), HMPV (5, 1.5%), HPIV (4, 1.2%), RSV (3, 0.9%), and influenza virus (2, 0.6%). RSV (OR 13.1; 95% CI: 1.6, 106.1), influenza virus (OR 8.7; 95% CI: 1.0, 78.9), HPIV (3.8; 95% CI: 1.0, 14.8), and HMPV (2.7; 95% CI: 1.3, 5.5) were independently associated with pneumonia while compared between 178 cases and 174 controls., Conclusion: Viral etiology of pneumonia in SAM children were mainly attributable to RSV, influenza, HPIV and HMPV. Our study findings may help in planning further studies targeting vaccines or drugs against common respiratory viruses responsible for pneumonia among SAM children., Competing Interests: The authors have declared that no competing interests exist.

Objectives: To improve our understanding of the global epidemiology of common respiratory viruses by analysing their contemporaneous incidence at multiple sites., Methods: 2010-2015 incidence data for influenza A (IAV), influenza B (IBV), respiratory syncytial (RSV) and parainfluenza (PIV) virus infections were collected from 18 sites (14 countries), consisting of local (n = 6), regional (n = 9) and national (n = 3) laboratories using molecular diagnostic methods. Each site submitted monthly virus incidence data, together with details of their patient populations tested and diagnostic assays used., Results: For the Northern Hemisphere temperate countries, the IAV, IBV and RSV incidence peaks were 2-6 months out of phase with those in the Southern Hemisphere, with IAV having a sharp out-of-phase difference at 6 months, whereas IBV and RSV showed more variable out-of-phase differences of 2-6 months. The tropical sites Singapore and Kuala Lumpur showed fluctuating incidence of these viruses throughout the year, whereas subtropical sites such as Hong Kong, Brisbane and Sydney showed distinctive biannual peaks for IAV but not for RSV and PIV., Conclusions: There was a notable pattern of synchrony of IAV, IBV and RSV incidence peaks globally, and within countries with multiple sampling sites (Canada, UK, Australia), despite significant distances between these sites., (Copyright © 2019. Published by Elsevier Ltd.)

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., (© 2019 Wiley Periodicals, Inc.)

Background: Caprine parainfluenza virus type 3 (CPIV3) is major pathogen of goat herds causing serious respiratory tract disease and economic losses to the goat industry in China. We analyzed the differential proteomics of CPIV3-infected Madin-Darby bovine kidney (MDBK) cells using quantitative iTRAQ coupled LC-MS/MS. In addition, four DEPs were validated by qRT-PCR and western blot analysis., Results: Quantitative proteomics analysis revealed 163 differentially expressed proteins (DEPs) between CPIV3-infected and mock-infected groups (p-value < 0.05 and fold change > 1.2), among which 91 were down-regulated and 72 were up-regulated. Gene ontology (GO) analysis showed that these DEPs were involved in molecular functions, cellular components and biological processes. Biological functions in which the DEPs were involved in included diseases, genetic information processing, metabolism, environmental information processing, cellular processes, and organismal systems. STRING analysis revealed that four heat shock proteins (HSPs) included HSPA5, HSPA1B, HSP90B1 and HSPA6 may be associated with proliferation of CPIV3 in MDBK cells. qRT-PCR and western blot analysis showed that the selected HSPs were identical to the quantitative proteomics data., Conclusion: To our knowledge, this is the first report of the proteomic changes in MDBK cells after CPIV3 infection.

Porcine parainfluenza virus type 1 (PPIV-1) is a member of the genus Respirovirus in the family Paramyxoviridae. The PPIV-1 was initially detected in 2013 from slaughter pigs in Hong Kong, China although its role in respiratory disease has remained unknown without virus isolates for experimental inoculation in swine. The objective of this study was to determine the relative frequency of PPIV-1 detection in diagnostic samples collected from swine in the United States, describe the cell culture isolation of PPIV-1, and characterize PPIV-1 cell culture isolates in vitro. Among 842 porcine specimens submitted to the Iowa State University Veterinary Diagnostic Laboratory during 2016-2017, 43.3% were PPIV-1 positive by a real-time, reverse transcriptase PCR suggesting PPIV-1 may be common in swine. Two strains of PPIV-1 were successfully isolated in an LLC-MK2 cell line from a PPIV-1 RT-qPCR positive nasal swab (USA/MN25890NS/2016) and lung (USA/IA84915LG/2017). The PPIV-1 cytopathic effect was demonstrated in tissue culture and enveloped viral particles were observed by electron microscopy. The whole genome, F, and HN gene sequences of both isolates share 98.2%, 98.5%, and 98.2% nucleotide homology, respectively, and phylogenetic analysis indicated they are closely related to other PPIV-1 strains detected in swine from the United States. Whole virus PPIV-1-specific monoclonal antibodies were generated for PPIV-1 detection in infected LLC-MK2 cells by indirect immunofluorescence and immunocytochemistry assays. The virus isolates and monoclonal antibodies obtained in the present study can be used to investigate the pathogenesis of PPIV-1 and develop new diagnostic tests., (Copyright © 2018 The Authors. Published by Elsevier B.V. All rights reserved.)

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/10 3 cells, was calculated as virus Ct, determined by multiple qRT-PCR, as a function of the Ct of β-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.

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., (Published 2018. This article is a U.S. Government work and is in the public domain in the USA. Influenza and Other Respiratory Viruses published by John Wiley &Sons Ltd.)

Background: Numerous protocols for viral enrichment and genome amplification have been created. However, the direct identification of viral genomes from clinical specimens using next-generation sequencing (NGS) still has its challenges. As a selected viral nucleic acid extraction method may determine the sensitivity and reliability of NGS, it is still valuable to evaluate the extraction efficiency of different extraction kits using clinical specimens directly., Results: In this study, we performed qRT-PCR and viral metagenomic analysis of the extraction efficiency of four commonly used Qiagen extraction kits: QIAamp Viral RNA Mini Kit (VRMK), QIAamp MinElute Virus Spin Kit (MVSK), RNeasy Mini Kit (RMK), and RNeasy Plus Micro Kit (RPMK), using a mixed respiratory clinical sample without any pre-treatment. This sample contained an adenovirus (ADV), influenza virus A (Flu A), human parainfluenza virus 3 (PIV3), human coronavirus OC43 (OC43), and human metapneumovirus (HMPV). The quantity and quality of the viral extracts were significantly different among these kits. The highest threshold cycle(Ct)values for ADV and OC43 were obtained by using the RPMK. The MVSK had the lowest Ct values for ADV and PIV3. The RMK revealed the lowest detectability for HMPV and PIV3. The most effective rate of NGS data at 67.47% was observed with the RPMK. The other three kits ranged between 12.1-26.79% effectiveness rates for the NGS data. Most importantly, compared to the other three kits the highest proportion of non-host reads was obtained by the RPMK. The MVSK performed best with the lowest Ct value of 20.5 in the extraction of ADV, while the RMK revealed the best extraction efficiency by NGS analysis., Conclusions: The evaluation of viral nucleic acid extraction efficiency is different between NGS and qRT-PCR analysis. The RPMK was most applicable for the metagenomic analysis of viral RNA and enabled more sensitive identification of the RNA virus genome in respiratory clinical samples. In addition, viral RNA extraction kits were also applicable for metagenomic analysis of the DNA virus. Our results highlighted the importance of nucleic acid extraction kit selection, which has a major impact on the yield and number of viral reads by NGS analysis. Therefore, the choice of extraction method for a given viral pathogen needs to be carefully considered.

Bovine respirovirus 3 also known as Bovine parainfluenza virus type 3 (BPIV3) is one of the most important viral respiratory agents of both young and adult cattle. Rapid diagnosis could contribute greatly in containing epidemics and thus avoid economic losses. However, the lack of robust isothermal visual method poses difficulty. In this study, a novel isothermal assay for detecting BPIV3 was established. The method includes a lateral flow dipstick (LFD) assay combined with reverse transcription recombinase polymerase amplification (RT-RPA). First, the analytical sensitivity and specificity of BPIV3 LFD RT-RPA were tested. The LFD RT-RPA assay has a detection limit of up to 100 copies per reaction in 30 min at 38 °C. Then the performance of LFD RT-RPA was evaluated using 95 clinical samples. Compared to qPCR, the LFD RT-RPA assay showed a clinical sensitivity of 94.74%, a clinical specificity of 96.05% and 0.8734 kappa coefficient. These results have demonstrated the efficiency and effectiveness of the method to be developed into a point of care protocol for the diagnosis of BPIV3., (Copyright © 2018. Published by Elsevier Ltd.)

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.

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 ± 0.018 [mean ± 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., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Caprine parainfluenza virus type 3 (CPIV3) is a newly emerging pathogenic respiratory agent infecting both young and adult goats, and it was identified in eastern China in 2013. Cellular microRNAs (miRNAs) have been reported to be important modulators of the intricate virus-host interactions. In order to elucidate the role of miRNAs in madin-darby bovine kidney (MDBK) cells during CPIV3 infection. In this study, we performed high-throughput sequencing technology to analyze small RNA libraries in CPIV3-infected and mock-infected MDBK cells. The results showed that a total of 249 known and 152 novel candidate miRNAs were differentially expressed in MDBK cells after CPIV3 infection, and 22,981 and 22,572 target genes were predicted, respectively. In addition, RT-qPCR assay was used to further confirm the expression patterns of 13 of these differentially expressed miRNAs and their mRNA targets. Functional annotation analysis showed these up- and downregulated target genes were mainly involved in MAPK signaling pathway, Jak-STAT signaling pathway, Toll-like receptor signaling pathway, p53 signaling pathway, focal adhesion, NF-kappa B signaling pathway, and apoptosis, et al. To our knowledge, this is the first report of the comparative expression of miRNAs in MDBK cells after CPIV3 infection. Our finding provides information concerning miRNAs expression profile in response to CPIV3 infection, and offers clues for identifying potential candidates for antiviral therapies against CPIV3.

This study aimed to identify a broad spectrum of respiratory pathogens from hospitalized and not-preselected children with acute respiratory tract infections in the Farhat Hached University-hospital of Sousse, Tunisia. Between September 2013 and December 2014, samples from 372 children aged between 1 month and 5 years were collected, and tested using multiplex real-time RT-PCR by a commercial assay for 21 respiratory pathogens. In addition, samples were screened for the presence of Streptococcus pneumoniae 16S rDNA using real-time PCR. The viral distribution and its association with clinical symptoms were statistically analyzed. Viral pathogens were detected in 342 (91.93%) of the samples of which 28.76% were single positive and 63.17% had multiple infections. The most frequent detected viruses were rhinovirus (55.64%), respiratory syncytial virus A/B (33.06%), adenovirus (25.00%), coronavirus NL63, HKU1, OC43, and 229E (21.50%), and metapneumovirus A/B (16.12%). Children in the youngest age group (1-3 months) exhibited the highest frequencies of infection. Related to their frequency of detection, RSV A/B was the most associated pathogen with patient's demographic situation and clinical manifestations (p<0.05). Parainfluenza virus 1-4 and parechovirus were found to increase the risk of death (p<0.05). Adenovirus was statistically associated to the manifestation of gastroenteritis (p = 0.004). Rhinovirus infection increases the duration of oxygen support (p = 0.042). Coronavirus group was statistically associated with the manifestation of bronchiolitis (p = 0.009) and laryngitis (p = 0.017). Streptococcus pneumoniae DNA was detected in 143 (38.44%) of tested samples. However, only 53 samples had a concentration of C-reactive protein from equal to higher than 20 milligrams per liter, and 6 of them were single positive for Streptocuccus pneumoniae. This study confirms the high incidence of respiratory viruses in children hospitalized for acute respiratory tract infections in the Sousse area, Tunisia.

Respiratory syncytial virus (RSV) is the most important viral agent of severe pediatric respiratory tract disease worldwide, but it lacks a licensed vaccine or suitable antiviral drug. A live attenuated chimeric bovine/human parainfluenza virus type 3 (rB/HPIV3) was developed previously as a vector expressing RSV fusion (F) protein to confer bivalent protection against RSV and HPIV3. In a previous clinical trial in virus-naive children, rB/HPIV3 was well tolerated but the immunogenicity of wild-type RSV F was unsatisfactory. We previously modified RSV F with a designed disulfide bond (DS) to increase stability in the prefusion (pre-F) conformation and to be efficiently packaged in the vector virion. Here, we further stabilized pre-F by adding both disulfide and cavity-filling mutations (DS-Cav1), and we also modified RSV F codon usage to have a lower CpG content and a higher level of expression. This RSV F open reading frame was evaluated in rB/HPIV3 in three forms: (i) pre-F without vector-packaging signal, (ii) pre-F with vector-packaging signal, and (iii) secreted pre-F ectodomain trimer. Despite being efficiently expressed, the secreted pre-F was poorly immunogenic. DS-Cav1 stabilized pre-F, with or without packaging, induced higher titers of pre-F specific antibodies in hamsters, and improved the quality of RSV-neutralizing serum antibodies. Codon-optimized RSV F containing fewer CpG dinucleotides had higher F expression, replicated more efficiently in vivo , and was more immunogenic. The combination of DS-Cav1 pre-F stabilization, optimized codon usage, reduced CpG content, and vector packaging significantly improved vector immunogenicity and protective efficacy against RSV. This provides an improved vectored RSV vaccine candidate suitable for pediatric clinical evaluation. IMPORTANCE RSV and HPIV3 are the first and second leading viral causes of severe pediatric respiratory disease worldwide. Licensed vaccines or suitable antiviral drugs are not available. We are developing a chimeric rB/HPIV3 vector expressing RSV F as a bivalent RSV/HPIV3 vaccine and have been evaluating means to increase RSV F immunogenicity. In this study, we evaluated the effects of improved stabilization of F in the pre-F conformation and of codon optimization resulting in reduced CpG content and greater pre-F expression. Reduced CpG content dampened the interferon response to infection, promoting higher replication and increased F expression. We demonstrate that improved pre-F stabilization and strategic manipulation of codon usage, together with efficient pre-F packaging into vector virions, significantly increased F immunogenicity in the bivalent RSV/HPIV3 vaccine. The improved immunogenicity included induction of increased titers of high-quality complement-independent antibodies with greater pre-F site Ø binding and greater protection against RSV challenge., (Copyright © 2017 American Society for Microbiology.)

Objective: To establish a T7 promoter based reverse genetics system competent for the rescue of bovine parainfluenza virus type 3 (BPIV3)., Methods: We constructed three helper plasmids of px8δT-PT1-bPIV3-NP, px8δT-PT1-bPIV3-P and px8δT-PT1-bPIV3-L and one minigenome plasmid of pSC11-bPIV3-EGFP containing open reading frame (ORF) of the enhanced green fluorescent protein (EGFP) and cis-acting elements including BPIV3 leader region, gene start (GS), gene end (GE) and trailer region. All these plasmids are under the control of T7 promoter and identified by restriction endonuclease analysis. We rescued the pSC11-bPIV3-EGFP by two different methods. Then, we observed the fluorescence expression over time with fluorescence microscopy., Results: We successfully constructed a reverse genetic system based 4 plasmids under the control of T7 promoter and finished the rescue operation to the minigenome of BPIV3., Conclusion: This system can be further applied to investigate the function of BPIV3 genome by deletion and mutation of its genes.

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., (Copyright © 2015. Published by Elsevier B.V.)

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-1β, 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.

Pneumonia is a leading cause of morbidity and mortality among infants and young children. The most common causes of pneumonia in children are respiratory viruses. In Thailand, the epidemiology of the viruses causing community-acquired pneumonia (CAP) among children is poorly defined. In this cross sectional study we used nasopharyngeal samples collected from hospitalized children diagnosed with severe CAP in accordance with WHO criteria between June 2013 and May 2014 to determine the causes of infection. The samples were analyzed for respiratory syncytial virus (RSV), parainfluenza viruses (PIV) types 1,2 and 3, adenovirus, rhinovirus, influenza viruses types A and B and coronavirus by polymerase chain reaction (PCR) and reverse transcriptase-polymerase chain reaction (RT-PCR). Of 102 cases of severe CAP, samples were obtained in 91 cases and 48 (52.7%) were positive for respiratory viruses. The most common viruses were RSV (n = 22; 45.8%), rhinovirus (n = 11; 22.9%) and adenovirus (n = 9; 18.7%). Patients were aged 1 month to 4 years 5 months, with a median age of 1 year 1 month. Thirty-seven (77.1%) were male. Asthma was the most common co-morbidity affecting 5 (10.4%) of the 48 cases with an identified virus. The peak prevalence occurred during October (n = 17). All patients required oxygen therapy and 17 (35.4%) required mechanical ventilation. The median length of hospitalization was 11 days. Preterm infants had a significantly higher rate of RSV infection than other respiratory viruses (8 of 21; 38% vs 3 of 27; 11.1%) (p = 0.02). Viruses were most commonly associated with severe CAP among children aged less than 1 year. The peak prevalence occurred during the rainy season. Our findings suggest that young and preterm infants with CAP should be monitored closely due to their high risk for developing serious complications.

Acute Respiratory Infections (ARIs) are responsible for considerable morbidity and mortality worldwide. Documentation of respiratory specimens can help for an appropriate clinical management with a significant effect on the disease progress in patient, the antimicrobial therapy used and the risk of secondary spread of infection. Here, we compared the performances of four commercial multiplex kits used in French University Hospital diagnostic microbiology laboratories for the detection of ARI pathogens (i.e., the xTAG Respiratory Viral Panel Fast, RespiFinder SMART 22, CLART PneumoVir and Fast Track Diagnostics Respiratory Pathogen 33 kits). We used a standardised nucleic acids extraction protocol and a comprehensive comparative approach that mixed reference to well established real-time PCR detection techniques and analysis of convergent positive results. We tested 166 respiratory clinical samples and identified a global high degree of correlation for at least three of the techniques (xTAG, RespiFinder and FTD33). For these techniques, the highest Youden's index (YI), positive predictive (PPV) and specificity (Sp) values were observed for Core tests (e.g., influenza A [YI:0.86-1.00; PPV:78.95-100.00; Sp:97.32-100.00] & B [YI:0.44-1.00; PPV:100.00; Sp:100.00], hRSV [YI:0.50-0.99; PPV:85.71-100.00; Sp:99.38-100.00], hMPV [YI:0.71-1.00; PPV:83.33-100.00; Sp:99.37-100.00], EV/hRV [YI:0.62-0.82; PPV:93.33-100.00; Sp:94.48-100.00], AdV [YI:1.00; PPV:100.00; Sp:100.00] and hBoV [YI:0.20-0.80; PPV:57.14-100.00; Sp:98.14-100.00]). The present study completed an overview of the multiplex techniques available for the diagnosis of acute respiratory infections.

Aim: Study the circulation of respiratory viruses in children with community-acquired pneumonia (CAP) during the period from October 2012 to May 2013., Materials and Methods: 136 children with CAP aged from 3 months to 16 years with ARI symptoms at the disease debut were studied. RNA/DNA of influenza A, B, parainfluenza (PI); adeno-, rhino-, RS-viruses, corona-, metapneumo- (MPV) and bocaviruses were detected in nasopharynx smears by PCR with hybridization-fluorescent detection in real time. Antibodies against influenza viruses A/H1N1/pdm09 California/07/09, epidemic reference strains of influenza viruses A/H1N1/Brisbane/59/07, A/ H3N2/Victoria/361/201 1, B/Wisconsin/1/10, against PI viruses type 1, 2, 3 were determined in paired sera by HAI., Results: In February-March 2013 the number of children protected by antibodies against influenza decreased, and circulation of influenza viruses A/H3N2 and A/H1N1/ pdm09 was detected. Rhinoviruses and PI viruses were determined throughout the epidemic season, bocavirus and adenoviruses--during the autumn-winter period, RS-virus and MPV--during winter-spring. Coronaviruses were not detected. The peak of virus detection was established in February when the threshold of influenza and ARI morbidity was exceeded. The main pathogens of children of the first 3 years of life are rhinoviruses, RS-virus, PI viruses and bocavirus. RS-virus infection at the debut of CAP in children younger than 3 years in 55.5% of cases is associated with the development of broncho-obstructive syndrome. Bocavirus infection in 50% of cases progresses with laryngo-tracheitis and bronchiolitis., Conclusion: The fraction of viruses in etiologic structure ofARI in children varies depending on immune layer, season and age of children. Etiology of viral infection at the debut of CAP could only be proven using specialized laboratory studies.

The seasonal distribution of the respiratory viruses for the period of 2011-2012 is presented. The ARI etiological structure among children 0-17 years, who were admitted to the hospital for respiratory disease in Belarus, was defined by the PCR-method. It was found that the etiological agents of the infections were not only influenza viruses A and B, parainfluenza types 1-4, adeno- and respiratory syncytial viruses, but also described boca- and metapneumoviruses. The most complete spectrum of the respiratory viruses was detected among children aged 0-4 years.

Inefficiency of RT-PCR can be associated with the suboptimal process of reverse transcription as only 40-80% of RNA is converted to cDNA. We employed a novel method, RT-Bst, to enrich the concentration of cDNA for subsequent multiplex PCR detection of selected RNA viruses. The RT-Bst method amplifies cDNA through reverse transcription of viral RNA using reverse transcriptase and amplification of cDNA using Bst DNA polymerase. Viral RNA was extracted from 25 nasopharyngeal samples for detection of influenza A, B and C; parainfluenza 1-4; human coronaviruses 229E and OC43; respiratory syncytial virus (RSV) and rhinovirus. Both multiplex one-step RT-PCR and RT-Bst PCR were used to compare their performances for detection of virus sequences. These findings were compared with routine laboratory detection. When using RT-Bst PCR, 28% of samples yielded a viral pathogen compared to 20% with RT-PCR and 12% using routine diagnostic tests. RT-Bst PCR was shown to have particular utility in the detection of RSV RNA as this was present in 20% of the samples studied compared to 8% when using RT-PCR. For one patient, RT-Bst PCR was able to detect RSV five days earlier than conventional hospital diagnostic testing. RT-Bst and RT-Bst PCR can be used as alternative approaches to reverse transcription and one-step RT-PCR, respectively, for sequence-independent amplification of RNA virus sequences and a larger scale analysis of this new diagnostic approach is warranted.

A surface plasmon resonance (SPR)-based biosensor was developed for specific detection of nine common respiratory virus, including influenza A and influenza B, H1N1, respiratory syncytial virus (RSV), parainfluenza virus 1-3 (PIV1, 2, 3), adenovirus, and severe acute respiratory syndrome coronavirus (SARS). The SPR biosensor was developed by immobilizing nine respiratory virus-specific oligonucleotides in an SPR chip. To increase the biosensor sensitivity, biotin was used to label the PCR primer and further amplify the signal by introducing streptavidin after hybridization. Throat swab specimens representing nine common respiratory viruses were tested by the innovative SPR-based biosensor to evaluate the sensitivity, specificity and reproducibility of this method. Results suggest that this biosensor has the potential to simultaneously identify common respiratory viruses.

Chemically synthesized short interfering RNA (siRNA) has ushered a new era in the application of RNA interference (RNAi) against viral genes. We have paid particular attention to respiratory viruses that wreak heavy morbidity and mortality worldwide. The clinically significant ones include respiratory syncytial virus (RSV), parainfluenza virus (PIV) (two Paramyxoviruses), and influenza virus (an Orthomyxovirus). As the infection by these viruses is clinically restricted to the respiratory tissues, mainly the lungs, the logical route for the application of the siRNA was also the same, i.e., via the nasal route. Following the initial success of single intranasal siRNA against RSV, we now offer two new strategies: (1) second-generation siRNAs, used against the paramyxoviral RNA polymerase large subunit (L), (2) siRNA cocktail with a novel transfection reagent, used against influenza virus. Based on these results, we propose the following consensus for designing intranasal antiviral siRNAs: (a) modified 19-27 nt-long double-stranded siRNAs are functional in the lung, (b) excessive 2'-OMe and 2'-F modifications in either or both strands of these siRNAs reduce efficacy, (c) limited modifications in the sense strand are beneficial, although their precise efficacy may be position-dependent, (d) cocktail of multiple siRNAs can be highly effective against multiple viral strains and subtypes.

Parainfluenza virus type 3 (PIV3) is one of the most important viral respiratory pathogens for humans and for many animals, but goat infection has been rarely reported. Starting in Aug 2013, goats in the Jiangsu and Anhui provinces of eastern China suffered severe respiratory diseases. In order to identify the causative agent, numerous related pathogens were tested with RT-PCR or PCR. A unique PIV3 strain was detected in most of the clinical nasal swabs or serum samples. The virus was isolated on MDBK cells and characterized by RT-PCR, nucleotide sequence analysis and hemagglutination test. The entire M and F gene coding regions, HN, 5'-UTR-N and L gene fragments were amplified using pairs of degenerate primers. Nucleotide, amino acid sequence alignments and phylogenetic analyses based on these genes indicated that the goat-derived PIV3 strain was distinct from previously reported BPIV3 genotypes and HPIV3 strains. The novel isolate, named JS2013, might be a potentially new member of the respirovirus genus. Goats were experimentally infected with JS2013 culture. The virus-inoculated goats displayed coughing and nasal discharges that were related to respiratory diseases. Viremia and virus shedding were detected during 4-10 days post-inoculation (dpi). Virus-specific HI antibodies became positive from 14 dpi. This is the first report of the detection of PIV3 from Chinese goat herds and genetic and pathogenetic characterization of the novel goat-derived PIV3., (Copyright © 2014. Published by Elsevier B.V.)

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., (© 2014 Wiley Periodicals, Inc.)

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., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Untreated rabies virus (RABV) infection leads to death. Vaccine and postexposure treatment have been effective in preventing RABV infection. However, due to cost, rabies vaccination and treatment have not been widely used in developing countries. There are 55,000 human death caused by rabies annually. An efficacious and cost-effective rabies vaccine is needed. Parainfluenza virus 5 (PIV5) is thought to contribute to kennel cough, and kennel cough vaccines containing live PIV5 have been used in dogs for many years. In this work, a PIV5-vectored rabies vaccine was tested in mice. A recombinant PIV5 encoding RABV glycoprotein (G) (rPIV5-RV-G) was administered to mice via intranasal (i.n.), intramuscular (i.m.), and oral inoculation. The vaccinated mice were challenged with a 50% lethal challenge dose (LD(50)) of RABV challenge virus standard 24 (CVS-24) intracerebrally. A single dose of 10(6) PFU of rPIV5-RV-G was sufficient for 100% protection when administered via the i.n. route. The mice vaccinated with a single dose of 10(8) PFU of rPIV5-RV-G via the i.m. route showed very robust protection (90% to 100%). Intriguingly, the mice vaccinated orally with a single dose of 10(8) PFU of rPIV5-RV-G showed a 50% survival rate, which is comparable to the 60% survival rate among mice inoculated with an attenuated rabies vaccine strain, recombinant LBNSE. This is first report of an orally effective rabies vaccine candidate in animals based on PIV5 as a vector. These results indicate that rPIV5-RV-G is an excellent candidate for a new generation of recombinant rabies vaccine for humans and animals and PIV5 is a potential vector for oral vaccines.

Objective: To establish a convenient and high-throughput respiratory virus detection method to facilitate epidemiological viral monitoring., Methods: We used high-throughput microsphere-based flexible multi-analyte profiling technology (xMAP) coupled with signal amplification molecules to simultaneously detect RNAs of 8 viruses including influenza viruses A and B, parainfluenza viruses type 1, 2 and 3, respiratory syncytial viruses A and B, and metapneumovirus in a 96-well plate format. The sensitivity and specificity of the method for the synthetic viral RNAs were evaluated., Results: There was no cross-reactivity among the 8 respiratory viral target RNAs. The detection limits for the 8 viral in intro-transcribed RNAs ranged from 1204 to 4695 RNA copies., Conclusion: We establish a sensitive, specific, convenient, and high-throughput multiplex detection method suitable for detecting multiple respiratory viral RNAs for epidemiological viral monitoring.

Interferon (IFN) induces an antiviral state in cells that results in alterations of the patterns and levels of parainfluenza virus type 5 (PIV5) transcripts and proteins. This study reports that IFN-stimulated gene 56/IFN-induced protein with tetratricopeptide repeats 1 (ISG56/IFIT1) is primarily responsible for these effects of IFN. It was shown that treating cells with IFN after infection resulted in an increase in virus transcription but an overall decrease in virus protein synthesis. As there was no obvious decrease in the overall levels of cellular protein synthesis in infected cells treated with IFN, these results suggested that ISG56/IFIT1 selectively inhibits the translation of viral mRNAs. This conclusion was supported by in vitro translation studies. Previous work has shown that ISG56/IFIT1 can restrict the replication of viruses lacking a 2'-O-methyltransferase activity, an enzyme that methylates the 2'-hydroxyl group of ribose sugars in the 5'-cap structures of mRNA. However, the data in the current study strongly suggested that PIV5 mRNAs are methylated at the 2'-hydroxyl group and thus that ISG56/IFIT1 selectively inhibits the translation of PIV5 mRNA by some as yet unrecognized mechanism. It was also shown that ISG56/IFIT1 is primarily responsible for the IFN-induced inhibition of PIV5.

Live-attenuated respiratory syncytial virus (RSV) vaccines offer several advantages for immunization of infants and young children: (1) they do not cause vaccine-associated enhanced RSV disease; (2) they broadly stimulate innate, humoral, and cellular immunity, both systemically and locally in the respiratory tract; (3) they are delivered intranasally; and (4) they replicate in the upper respiratory tract of young infants despite the presence of passively acquired maternally derived RSV neutralizing antibody. This chapter describes early efforts to develop vaccines through the classic methods of serial cold-passage and chemical mutagenesis, and recent efforts using reverse genetics to derive attenuated derivatives of wild-type (WT) RSV and to develop parainfluenza vaccine vectors that express RSV surface glycoproteins.

There is no vaccine currently approved for paramyxovirus-induced respiratory diseases in humans, despite their major clinical importance. We review the development and evaluation of new vaccine strategies based on live-attenuated chimeric and recombinant vaccines against human respiratory syncytial virus, human metapneumovirus and human parainfluenza viruses types 1 to 3, which are significant causes of upper and lower tract respiratory diseases. Most promising strategies are based on virus attenuation through (i) mutations in key genes involved in replication; (ii) deletion of accessory genes; or (iii) the use of a corresponding animal viral vector, such as bovine parainfluenza type 3 and Sendai virus, as a background for the expression of a viral glycoprotein. Indeed, the fusion (F), or attachment (HN/H/G) glycoproteins are the most immunogenic antigens in paramyxoviruses. For each strategy, we will review the immunogenicity (increase in neutralising antibody titres) and the protection conferred by the most promising recombinant vectored vaccines and list ongoing clinical trials. We will conclude by discussing the most important challenges regarding the introduction of such vaccines into immunisation programmes., (Copyright © 2012 John Wiley & Sons, Ltd.)

Background: In this study, we evaluated the analytical performance and clinical potential of a one-step multiplex real-time PCR assay for the simultaneous detection of 14 types of respiratory viruses using the AdvanSure RV real-time PCR Kit (LG Life Sciences, Korea)., Methods: Three hundred and twenty clinical specimens were tested with the AdvanSure RV real-time PCR Kit and conventional multiplex reverse transcription (RT)-PCR assay. The assay results were analyzed and the one-step AdvanSure RV real-time PCR Kit was compared with the conventional multiplex RT-PCR assay with respect to the sensitivity and specificity of the detection of respiratory viruses., Results: The limit of detection (LOD) was 1.31 plaque-forming units (PFU)/mL for human rhinoviruses (hRVs), 4.93 PFU/mL for human coronavirus HCoV-229E/NL63, 2.67 PFU/mL for human coronavirus HCoV-OC43, 18.20 PFU/mL for parainfluenza virus 1 (PIV)-1, 24.57 PFU/mL for PIV-2, 1.73 PFU/mL for PIV-3, 1.79 PFU/mL for influenza virus group (Flu) A, 59.51 PFU/mL for FluB, 5.46 PFU/mL for human respiratory syncytial virus (hRSV)-A, 17.23 PFU/mL for hRSV-B, 9.99 PFU/mL for human adenovirus (ADVs). The cross-reactivity test for this assay against 23 types of non-respiratory viruses showed negative results for all viruses tested. The agreement between the one-step AdvanSure multiplex real-time PCR assay and the conventional multiplex RT-PCR assay was 98%., Conclusions: The one-step AdvanSure RV multiplex real-time PCR assay is a simple assay with high potential for specific, rapid and sensitive laboratory diagnosis of respiratory viruses compared to conventional multiplex RT-PCR.

Background: Pneumonia is the leading cause of childhood death in Bangladesh. We conducted a longitudinal study to estimate the incidence of virus-associated pneumonia in children aged <2 years in a low-income urban community in Dhaka, Bangladesh., Methods: We followed a cohort of children for two years. We collected nasal washes when children presented with respiratory symptoms. Study physicians diagnosed children with cough and age-specific tachypnea and positive lung findings as pneumonia case-patients. We tested respiratory samples for respiratory syncytial virus (RSV), rhinoviruses, human metapneumovirus (HMPV), influenza viruses, human parainfluenza viruses (HPIV 1, 2, 3), and adenoviruses using real-time reverse transcription polymerase chain reaction assays., Results: Between April 2009-March 2011, we followed 515 children for 730 child-years. We identified a total of 378 pneumonia episodes, 77% of the episodes were associated with a respiratory viral pathogen. The overall incidence of pneumonia associated with a respiratory virus infection was 40/100 child-years. The annual incidence of pneumonia/100 child-years associated with a specific respiratory virus in children aged < 2 years was 12.5 for RSV, 6 for rhinoviruses, 6 for HMPV, 4 for influenza viruses, 3 for HPIV and 2 for adenoviruses., Conclusion: Young children in Dhaka are at high risk of childhood pneumonia and the majority of these episodes are associated with viral pathogens. Developing effective low-cost strategies for prevention are a high priority.

Viruses are the major cause of pediatric respiratory tract infection and yet many suspected cases of illness remain uncharacterized. This study aimed to determine the distribution of several respiratory viruses in children diagnosed as having influenza-like illness, over the winter period of 2005-2008. Molecular assays including conventional and real time PCR protocols, were employed to screen respiratory specimens, collected by clinicians of the Influenza sentinel system and of outpatient pediatric clinics, for identification of several respiratory viruses. Of 1,272 specimens tested, 814 (64%) were positive for at least one virus and included 387 influenza viruses, 160 rhinoviruses, 155 respiratory syncytial viruses, 95 adenoviruses, 81 bocaviruses, 47 parainfluenza viruses, 44 metapneumoviruses, and 30 coronaviruses. Simultaneous presence of two or three viruses was observed in 173 of the above positive cases, 21% of which included influenza virus and rhinovirus. The majority of positive cases occurred during January and February. Influenza virus predominated in children older than 1 year old, with type B being the dominant type for the first season and subtypes A/H3N2 and A/H1N1 the following two winter seasons, respectively. Respiratory syncytial virus prevailed in children younger than 2 years old, with subtypes A and B alternating from year to year. This is the most comprehensive study of the epidemiology of respiratory viruses in Greece, indicating influenza, rhinovirus and respiratory syncytial virus as major contributors to influenza-like illness in children., (Copyright © 2011 Wiley-Liss, Inc.)

In children under 5 years of age, human parainfluenza viruses (HPIVs) as a group are the second most common etiology of acute respiratory illness leading to hospitalization, surpassed only by respiratory syncytial virus but ahead of influenza viruses. Using reverse genetics systems for HPIV serotypes 1, 2 and 3 (HPIV1, 2 and 3), several live-attenuated HPIVs have been generated and evaluated as intranasal vaccines in adults and in children. Two vaccines against HPIV3 were found to be well tolerated, infectious and immunogenic in Phase I trials in HPIV3-seronegative infants and children and should progress to proof-of-concept trials. Vaccines against HPIV1 and HPIV2 are less advanced and have just entered pediatric trials.

Macrophages regulate immune responses during many viral infections, and can be a major determinant of pathogenesis, virus replication and immune response to infection. Here, we have addressed the question of the outcome of infection of primary human macrophages with parainfluenza virus 5 (PIV5) and a PIV5 mutant (P/V-CPI-) that is unable to counteract interferon (IFN) responses. In cultures of naïve monocyte-derived macrophages (MDMs), WT PIV5 established a highly productive infection, whereas the P/V-CPI- mutant was restricted for replication in MDMs by IFN-beta. Restricted replication in vitro was relieved in MDM that had been activated by prior exposure to heat killed Gram positive bacteria, including Listeria monocytogenes, Streptococcus pyogenes, and Bacillus anthracis. Enhanced replication of the P/V mutant in MDM previously activated by bacterial components correlated with a reduced ability to produce IFN-beta in response to virus infection, whereas IFN signaling was intact. Activated MDM were found to upregulate the synthesis of IRAK-M, which has been previously shown to negatively regulate factors involved in TLR signaling and IFN-beta production. We discuss these results in terms of the implications for mixed bacteria-virus infections and for the use of live RNA virus vectors that have been engineered to be attenuated for IFN sensitivity., (Copyright © 2010 Institut Pasteur. Published by Elsevier SAS. All rights reserved.)