Detection of Porcine Respirovirus 1 (PRV1) in Poland: Incidence of Co-Infections with Influenza A Virus (IAV) and Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) in Herds with a Respiratory Disease.

Porcine respirovirus 1 (PRV1) is also known as porcine parainfluenza virus 1 (PPIV1). The prevalence and the role of PRV1 infections for pig health is largely unknown. In order to assess the PRV1 prevalence in Poland, nasal swabs and oral fluids collected from pigs from 30 farms were examined with RT real-time PCR. Additionally, IAV and PRRSV infection statuses of PRV1-positive samples were examined. The results showed that the virus is highly prevalent (76.7% farms positive) and different patterns of PRV1 circulation in herds with mild-moderate respiratory disease were observed. Co-infections with IAV and PRRSV were infrequent and detected in 8 (23.5%) and 4 (11.8%) out of 34 PRV1-positive nasal swab pools from diseased pens, respectively. In one pen PRV1, IAV, and PRRSV were detected at the same time. Interestingly, PRV1 mean Ct value in samples with co-infections was significantly lower (29.8 ± 3.1) than in samples with a single PRV1 infection (32.5 ± 3.6) (p < 0.05), which suggested higher virus replication in these populations. On the other hand, the virus detection in pig populations exhibiting respiratory clinical signs, negative for PRRSV and IAV, suggests that PRV1 should be involved in differential diagnosis of respiratory problems.

Comparison of 11 respiratory pathogens among hospitalized children before and during the COVID-19 epidemic in Shenzhen, China.

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.

Epidemiology and factors associated with the severity of viral acute lower respiratory infection in children hospitalized in Manaus, Amazonas, in 2017-2018: An observational study.

ABSTRACT: To investigate the epidemiology and factors associated with the severity of viral acute lower respiratory infection (ALRI) in children hospitalized in Manaus, Amazonas, in 2017 to 2018.Retrospective cohort study of children hospitalized at the Hospital and Emergency Room Delphina Rinaldi Abdel Aziz, in Manaus, from April 01, 2017 to August 31, 2018, with a clinical diagnosis of ALRI and nasopharyngeal aspirates positive for at least 1 respiratory virus.One hundred forty-six children aged 0.2 to 66 months (median 7 months) were included. Patients were divided into 2 groups according to the disease severity classified by an adapted Walsh et al score: moderate disease, score 0-4, n = 66 (45.2%) and severe disease, score 5-7, n = 80 (54.8%). A greater number of viral ALRI cases were observed in the rainiest months. Respiratory syncytial virus was the most prevalent (n = 103, 70.3%), followed by metapneumovirus (n = 24, 16.4%), influenza virus (n = 17, 11.6%), parainfluenza virus (n = 11, 7.5%), and adenovirus (n = 4, 2.7%). Co-detections of 2 to 3 viruses were found in 12 (8.2%) patients. The presence of viral coinfection was an independent risk factor for disease severity (adjusted relative risk [RR] 1.53; 95% CI 1.10-2.14). Twelve patients (8.2%) died, all with severe disease. Risk factors for death were shock (adjusted RR 10.09; 95% CI 2.31-43.90) and need for vasoactive drugs (adjusted RR 10.63; 95% CI 2.44-46.31).There was a higher incidence of viral ALRI in Manaus in the rainy season. Respiratory syncytial virus was the most prevalent virus. The presence of viral coinfection was an independent risk factor for disease severity.

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

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

Bovine respiratory disease in beef calves supported long transport stress: An epidemiological study and strategies for control and prevention.

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.

Co-detection of Bordetella pertussis and other respiratory organisms in children hospitalised with lower respiratory tract infection.

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.

First report of porcine respirovirus 1 in South America.

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.

Pneumonia in children admitted to the national referral hospital in Bhutan: A prospective cohort study.

OBJECTIVES: The study aim was to describe the etiological profile and clinical characteristics of pneumonia among children hospitalized in Thimphu, Bhutan. METHODS: This prospective study enrolled children aged 2-59 months admitted to the Jigme Dorji Wangchuck National Referral Hospital with World Health Organization (WHO)-defined clinical pneumonia. Demographic and clinico-radiological data were collected through questionnaires, physical examination, and chest radiography. Blood samples and nasopharyngeal washing were collected for microbiological analysis including culture and molecular methods. RESULTS: From July 2017 to June 2018, 189 children were enrolled, of which 53.4% were infants. Pneumonia-related admissions were less frequent over the winter. Chest radiographies were obtained in 149 children; endpoints included pneumonia in 39 cases (26.2%), other infiltrates in 31 (20.8%), and were normal in 79 children (53.0%). Non-contaminated bacterial growth was detected in 8/152 (5.3%) blood cultures, with only two cases of Streptococcus pneumoniae. Viral detection in upper respiratory secretions was common, with at least one virus detected in 103/115 (89.6%). The three most-commonly isolated viruses were respiratory syncytial virus (52/115; 45.2%), rhinovirus (42/115; 36.5%), and human parainfluenza virus (19/115; 16.5%). A third of patients with viral infections showed mixed infections. Case fatality rate was 3.2% (6/189). CONCLUSION: Respiratory viral infections predominated among this cohort of WHO-defined clinical pneumonia cases, whereas bacterial aetiologies were uncommon, highlighting the epidemiologic transition that Bhutan seems to have reached.

Viral etiology of pneumonia among severely malnourished under-five children in an urban hospital, Bangladesh.

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.

An investigation into respiratory tract viruses in children with acute lower respiratory tract infection or wheezing.

BACKGROUND: This study aimed to determine the frequencies of respiratory tract viruses in patient (acute lower respiratory tract infection [LRTI] or wheezing) and control (history of asthma without symptoms) groups. METHODS: Using multiplex-polymerase chain reaction (PCR), respiratory tract viruses were investigated in the respiratory tract specimens from patient and control groups followed in the Pediatric Clinic. RESULTS: The viruses detected in the patient and control groups (P=0.013) were as follows, respectively: rhinoviruses A, B, C (25.6% and 36.7%), influenza virus A (21.1% and 0.0%), parainfluenza virus type 1 (7.8% and 1.7%), parainfluenza virus type 4 (5.6% and 0.0%), adenoviruses A, B, C, D, E (4.4% and 1.7%), parainfluenza virus type 3 (4.4% and 1.7%), coronaviruses 229E and NL63 (4.4% and 1.7%), coronavirus OC43 (3.3% and 0.0%), respiratory syncytial virus A (3.3% and 0.0%), parainfluenza virus type 2 (2.2% and 0.0%), influenza virus B (2.2% and 0.0%), and respiratory syncytial virus B (1.1% and 1.7%). No bocavirus, metapneumovirus or enterovirus was found in any specimen. Statistically significant differences in the detection of influenza virus A (P=0.000), the total detection of parainfluenza viruses (P=0.008) and coinfection (P=0.004) were observed between the patient and control groups. CONCLUSIONS: The advantage of our study compared with other studies is the inclusion of not only wheezing patients but also children with asthma without symptom. The higher detection of rhinoviruses both in patient and control groups give rise to thought that these viruses may be responsible for asthma exacerbations and may be related with long duration of virus shedding.

A rapid and label-free platform for virus capture and identification from clinical samples.

Emerging and reemerging viruses are responsible for a number of recent epidemic outbreaks. A crucial step in predicting and controlling outbreaks is the timely and accurate characterization of emerging virus strains. We present a portable microfluidic platform containing carbon nanotube arrays with differential filtration porosity for the rapid enrichment and optical identification of viruses. Different emerging strains (or unknown viruses) can be enriched and identified in real time through a multivirus capture component in conjunction with surface-enhanced Raman spectroscopy. More importantly, after viral capture and detection on a chip, viruses remain viable and get purified in a microdevice that permits subsequent in-depth characterizations by various conventional methods. We validated this platform using different subtypes of avian influenza A viruses and human samples with respiratory infections. This technology successfully enriched rhinovirus, influenza virus, and parainfluenza viruses, and maintained the stoichiometric viral proportions when the samples contained more than one type of virus, thus emulating coinfection. Viral capture and detection took only a few minutes with a 70-fold enrichment enhancement; detection could be achieved with as little as 102 EID50/mL (50% egg infective dose per microliter), with a virus specificity of 90%. After enrichment using the device, we demonstrated by sequencing that the abundance of viral-specific reads significantly increased from 4.1 to 31.8% for parainfluenza and from 0.08 to 0.44% for influenza virus. This enrichment method coupled to Raman virus identification constitutes an innovative system that could be used to quickly track and monitor viral outbreaks in real time.

Comparison of the Panther Fusion and Allplex assays for the detection of respiratory viruses in clinical samples.

Respiratory viral infections are the most frequent clinical syndrome affecting both children and adults, and early detection is fundamental to avoid infection-related risks and reduce the healthcare costs incurred by unnecessary antibiotic treatments. In this study, performance characteristics of two commercial methods, the Panther Fusion® assay (Hologic Inc., San Diego, CA, USA) were compared to Allplex™ respiratory panels (Seegene, Seoul, South Korea) for the detection of influenza A (Flu A), influenza B (Flu B), respiratory syncytial virus (RSV), parainfluenza virus (PIV), human metapneumovirus (hMPV), rhinovirus (RV) and adenovirus (AdV) targets. A total of 865 specimens collected prospectively and retrospectively were included, and discordant results were further examined using another commercial product, R-GENE™ respiratory kits (bioMérieux, Marcy l'Etoile, France). There was high agreement between both methods, with 98.6% concordance and a kappa (k) value of 0.9 (95% CI: 0.89-0.92). A specific analysis of both methods for each viral agent demonstrated comparable sensitivity and specificity, both ranging from 0.83 to 1 with good predictive values for the prospective part of the study. Good agreement between both methods was also found for the κ values obtained (ranging from 97.55% to 98.9%), with the lowest for hMPV (k = 0.83, 95% CI: 0.75-0.91) and RV (k = 0.73, 95% CI: 0.65-0.81). Amplification efficiency, measured according to the value of the cycle threshold (Ct) obtained in each of the amplifications in both tests, was significantly better with Panther Fusion for Flu A, Flu B, hMPV and RV. Regarding discordant results, R-GENE showed higher agreement with Panther Fusion-positive specimens (negative for Allplex; n = 28/71, 34.9%) than with Allplex-positive samples (negative for Panther Fusion; n = 7/49, 14.3%). In summary, Panther Fusion proved to be a more efficient fully-automated methodology, requiring shorter hands-on and turnaround times than Allplex.

Detection and molecular characterization of respiratory viruses that cause acute respiratory infection in the adult population.

INTRODUCTION: Acute respiratory infections are one of the main causes of morbidity and mortality in older adults and patients with chronic diseases. Among the responsible etiological agents are human respiratory viruses, such as: respiratory syncytial virus, parainfluenza virus and metapneumovirus. OBJECTIVE: To carry out a differential diagnostic study of respiratory viruses circulating and co-circulating in an adult population. METHODS: A pilot study was conducted in patients older than 18 years, who presented signs and symptoms suggestive of acute respiratory infection and whose clinical picture did not exceed 15 days of evolution; end-point polymerase chain reaction assays were performed with the use of specific oligonucleotides for molecular diagnosis. RESULTS: 72 specimens of patients with an age of 51.33 ± 19.33 years, with a predominance of females (4.5:1); original inhabitants of Mexico City; only 22 were positive for respiratory viruses, being mostly metapneumovirus infections. CONCLUSIONS: The knowledge of the circulating viral strains in the population will allow to determine changes that can declare an epidemiological alert leading to the best decision making for the benefit of the patients.

Assessing exhibition swine as potential disseminators of infectious disease through the detection of five respiratory pathogens at agricultural exhibitions.

Widespread geographic movement and extensive comingling of exhibition swine facilitates the spread and transmission of infectious pathogens. Nasal samples were collected from 2862 pigs at 102 exhibitions and tested for five pathogens. At least one pathogen was molecularly detected in pigs at 63 (61.8%) exhibitions. Influenza A virus was most prevalent and was detected in 498 (17.4%) samples. Influenza D virus was detected in two (0.07%) samples. More than one pathogen was detected in 165 (5.8%) samples. Influenza A virus remains a top threat to animal and human health, but other pathogens may be disseminated through the exhibition swine population.

Comparative global epidemiology of influenza, respiratory syncytial and parainfluenza viruses, 2010-2015.

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.

Detection, isolation, and in vitro characterization of porcine parainfluenza virus type 1 isolated from respiratory diagnostic specimens in swine.

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.

Metagenomic analysis of viral nucleic acid extraction methods in respiratory clinical samples.

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.

A lateral flow dipstick combined with reverse transcription recombinase polymerase amplification for rapid and visual detection of the bovine respirovirus 3.

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.

Seasonality and clinical impact of human parainfluenza viruses.

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