Improving Pneumovirus Isolation Using a Centrifugation and AZD1480 Combined Method.

The isolation of respiratory viruses, especially from clinical specimens, often shows poor efficiency with classical cell culture methods. The lack of suitable methods to generate virus particles inhibits the development of diagnostic assays, treatments, and vaccines. We compared three inoculation methods, classical cell culture, the addition of a JAK2 inhibitor AZD1480, and centrifugation-enhanced inoculation (CEI), to replicate human respiratory syncytial virus (HRSV) and human metapneumovirus (HMPV). In addition, a combined method using AZD1480 treatment and CEI was used on throat swabs to verify that this method could increase virus isolation efficiency from human clinical specimens. Both CEI and AZD1480 treatment increased HRSV and HMPV genome replication. Also, the combined method using CEI and AZD1480 treatment enhanced virus proliferation synergistically. The combined method is particularly suited for the isolation of interferon-sensitive or slowly growing viruses from human clinical specimens.

Pneumonia severity index in viral community acquired pneumonia in adults.

Pneumonia severity index (PSI) is an important scoring system that can assess the severity of community acquired pneumonia and determine admission status. However, there is a lack of research on whether this scoring system can be applied to viral community acquired pneumonia. The purpose of this study was to evaluate the usefulness of PSI in viral community acquired pneumonia. This retrospective cohort study included 1,434 adult patients (aged ≥18 years) who were admitted to the emergency department of a university hospital during 2013-2015 because of community-acquired pneumonia. Viral infections were diagnosed by multiplex PCR. Patients diagnosed with non-viral community-acquired pneumonia were included in the control group (N = 1,173). The main outcome was 30-day all-cause mortality. multivariate Cox regression analyses were performed to calculate the risk of death. Respiratory viruses were detected in 261 (18.2%) patients with community-acquired pneumonia. Two types of respiratory viruses were detected in 7 cases. Of the 254 cases detected with only one virus, 62 were influenza A, 18 were influenza B, 65 were rhinovirus, 35 were respiratory syncytial virus, 25 were metapneumovirus, 20 were parainfluenza, 17 were coronavirus, 7 were bocavirus, and 5 were adenovirus. Mortality was not significantly different between patients with respiratory virus and those without respiratory virus; the 30-day all-cause mortality rates were 20.3% and 22.4%, respectively (P = 0.45). Mortality rate increased with an increasing PSI score with or without respiratory viral infection. Pulmonary severity index was significantly associated with mortality adjusted for respiratory virus detection (hazard ratio = 1.024, 95% confidence interval = 1.020-1.028). Pneumonia severity index score is an important factor for assessing the prognosis of patients with community-acquired pneumonia, regardless of respiratory virus detection.

First demonstration of the circulation of a pneumovirus in French pigs by detection of anti-swine orthopneumovirus nucleoprotein antibodies.

The presence of pneumoviruses in pigs is poorly documented. In this study, we used the published sequence of the nucleoprotein (N) of the recently identified Swine Orthopneumovirus (SOV) to express and purify SOV N as a recombinant protein in Escherichia coli. This protein was purified as nanorings and used to set up an enzyme-linked immunosorbent assay, which was used to analyse the presence of anti-pneumovirus N antibodies in swine sera. Sera collected from different pig farms in the West of France and from specific pathogen free piglets before colostrum uptake showed indirectly that a pneumovirus is circulating in pig populations with some variations between animals. Piglets before colostrum uptake were sero-negative for anti-pneumovirus antibodies while most of the other pigs showed positivity. Interestingly, in two farms presenting respiratory clinical signs and negative or under control for some common respiratory pathogens, pigs were detected positive for anti-pneumovirus antibodies. Globally, anti-pneumovirus N antibody concentrations were variable between and within farms. Further studies will aim to isolate the circulating virus and determine its potential pathogenicity. SOV could potentially become a new member of the porcine respiratory complex, important on its own or in association with other viral and bacterial micro-organisms.

European surveillance of emerging pathogens associated with canine infectious respiratory disease.

Canine infectious respiratory disease (CIRD) is a major cause of morbidity in dogs worldwide, and is associated with a number of new and emerging pathogens. In a large multi-centre European study the prevalences of four key emerging CIRD pathogens; canine respiratory coronavirus (CRCoV), canine pneumovirus (CnPnV), influenza A, and Mycoplasma cynos (M. cynos); were estimated, and risk factors for exposure, infection and clinical disease were investigated. CIRD affected 66% (381/572) of the dogs studied, including both pet and kennelled dogs. Disease occurrence and severity were significantly reduced in dogs vaccinated against classic CIRD agents, canine distemper virus (CDV), canine adenovirus 2 (CAV-2) and canine parainfluenza virus (CPIV), but substantial proportions (65.7%; 201/306) of vaccinated dogs remained affected. CRCoV and CnPnV were highly prevalent across the different dog populations, with overall seropositivity and detection rates of 47% and 7.7% for CRCoV, and 41.7% and 23.4% for CnPnV, respectively, and their presence was associated with increased occurrence and severity of clinical disease. Antibodies to CRCoV had a protective effect against CRCoV infection and more severe clinical signs of CIRD but antibodies to CnPnV did not. Involvement of M. cynos and influenza A in CIRD was less apparent. Despite 45% of dogs being seropositive for M. cynos, only 0.9% were PCR positive for M. cynos. Only 2.7% of dogs were seropositive for Influenza A, and none were positive by PCR.

Full-genome analysis of a canine pneumovirus causing acute respiratory disease in dogs, Italy.

An outbreak of canine infectious respiratory disease (CIRD) associated to canine pneumovirus (CnPnV) infection is reported. The outbreak occurred in a shelter of the Apulia region and involved 37 out of 350 dogs that displayed cough and/or nasal discharge with no evidence of fever. The full-genomic characterisation showed that the causative agent (strain Bari/100-12) was closely related to CnPnVs that have been recently isolated in the USA, as well as to murine pneumovirus, which is responsible for respiratory disease in mice. The present study represents a useful contribution to the knowledge of the pathogenic potential of CnPnV and its association with CIRD in dogs. Further studies will elucidate the pathogenicity and epidemiology of this novel pneumovirus, thus addressing the eventual need for specific vaccines.

[Etiological structure of acute respiratory viral infections morbidity in Novosibirsk and Novosibirsk region in epidemic season 2011-2012].

INTRODUCTION: ARI occupying the first place in the structure of total human morbidity. The aim of the study was to investigate the species diversity of the viruses causing AR among residents of the Novosibirsk region during epidemic season (October to April). MATERIALS AND METHODS: 164 nasopharyngeal swabs were collected and analyzed. Viral RNA/DNA, cDNA synthesis and PCR were carried out employing "RIBO-prep" "eReverta-L", "AmpliSens Influenza virus A/B-FL" and "AmpliSens ARI-screen-FL" kits (CRI of Epidemiology). RESULTS: Etiological agent of the disease was found in 69(43%) samples. Monoinfection was found in 58 (35%). In 14 (9%) samples were detected serogroup I coronaviruses, in 13 (8%) rhinoviruses, in 7 (4%) respiratory syncytial virus, in 6 (4%) parainfluenza virus type 1, in 5 (3%) parainfluenza virus type 3. Adenoviruses and bocavirus were identified in 3 (2%) samples. Parainfluenza virus type 2 and 4, metapneumovirus, serogroup Il coronaviruses (HKU1 and OC43) were presented in 2 (1%) samples. In 11 (7%) samples was found mixed infection. CONCLUSION: The majority of common colds were caused by serogroup I coronaviruses (NL63 and 229E), rhinoviruses and mixed infections. The peak of species variability of viruses caused acute respiratory infections was determined in age group of children 2-4 years old. In older age groups the species variability of analyzed viruses was decreased, rhinovirus infection becomes prevalent.

Detection of canine pneumovirus in dogs with canine infectious respiratory disease.

Canine pneumovirus (CnPnV) was recently identified during a retrospective survey of kenneled dogs in the United States. In this study, archived samples from pet and kenneled dogs in the United Kingdom were screened for CnPnV to explore the relationship between exposure to CnPnV and the development of canine infectious respiratory disease (CIRD). Within the pet dog population, CnPnV-seropositive dogs were detected throughout the United Kingdom and Republic of Ireland, with an overall estimated seroprevalence of 50% (n = 314/625 dogs). In the kennel population, there was a significant increase in seroprevalence, from 26% (n = 56/215 dogs) on the day of entry to 93.5% (n = 201/215 dogs) after 21 days (P <0001). Dogs that were seronegative on entry but seroconverted while in the kennel were 4 times more likely to develop severe respiratory disease than those that did not seroconvert (P < 0.001), and dogs with preexisting antibodies to CnPnV on the day of entry were significantly less likely to develop respiratory disease than immunologically naive dogs (P < 0.001). CnPnV was detected in the tracheal tissues of 29/205 kenneled dogs. Detection was most frequent in dogs with mild to moderate respiratory signs and histopathological changes and in dogs housed for 8 to 14 days, which coincided with a significant increase in the risk of developing respiratory disease compared to the risk of those housed 1 to 7 days (P < 0.001). These findings demonstrate that CnPnV is present in the United Kingdom dog population; there is a strong association between exposure to CnPnV and CIRD in the kennel studied and a potential benefit in vaccinating against CnPnV as part of a wider disease prevention strategy.

Is respiratory viral infection really an important trigger of asthma exacerbations in children?

We performed a prospective cohort study from September 2003 to December 2004 to delineate attributing the effect of different respiratory viral infections including newly discovered ones to asthma exacerbations in children in Hong Kong. One hundred and fourteen children aged 6-14 years with chronic stable asthma and on regular inhaled steroid were monitored for respiratory symptoms over a full calendar year from recruitment. They would attend the study clinic if peak expiratory flow rate decreased to below 80% of their baselines, if they met a predefined symptom score, or if parents subjectively felt them developing a cold. Virological diagnosis using virus culture, antigen detection, and polymerase chain reaction methods on nasal swab specimens would be attempted for all these visits irrespective of triggers. Physician diagnosed outcome of each episode was documented. Three hundred and five episodes of respiratory illnesses were captured in the cohort. Nasal specimens were available in 166 episodes, 92 of which were diagnosed as asthma exacerbations, and 74 non-asthma related episodes. Respiratory viruses were detected in 61 of 166 episodes (36.7%). There was no significant difference in virus detection rate between asthma exacerbations (32 out of 97 episodes, 34.8%) and non-asthma respiratory illnesses (29 out of 79 episodes, 39.2%). Although newly discovered respiratory viruses were identified in these episodes, rhinovirus was the commonest organism associated with both asthma exacerbations and non-asthma related episodes. Plausible explanations for much lower virus detection rate than previously reported include improved personal hygiene and precautionary measures taken during respiratory tract infections in the immediate post-severe acute respiratory syndrome period together with a significant contribution of other adverse factors like environmental air pollution. We conclude that not all viral infections in children with asthma lead to an asthma exacerbation and the attributing effect of different triggers of asthma exacerbations in children vary across different time periods and across different localities.

Genomic analysis of a pneumovirus isolated from dogs with acute respiratory disease.

A previously unrecognized virus belonging to the subfamily Pneumovirinae and most closely related to murine pneumovirus (MPV) was identified in domestic dogs in 2 related animal shelters. Additional diagnostic testing yielded 3 new viral isolates and identified 6 additional PCR positive dogs from other USA locations indicating that its distribution is not geographically limited. Nucleotide sequences encompassing 9 of the 10 genes were compared to the only 2 available MPV strains, 15 and J3666. Several features distinguished the canine pneumovirus (CnPnV) from the murine strains. Two regions of diversity were identified in the amino-proximal region of P and the overlapping P2 ORF was only 54 amino acids (aa) compared to 137aa in MPV. The G protein had an amino-terminal cytoplasmic tail 18aa longer than in the MPV strains. The CnPnV SH protein showed the highest divergence with only 90.2% aa identity when compared to MPV strain 15. Like strain 15, the CnPnV SH ORF coded for a protein of 92aa while J3666 has a 114aa variant. Comparison of CnPnV isolates at culture passages 4 and 17 revealed 7nt differences within the 8598nt sequenced. Of note was a substitution at nt 364 in G resulting in a termination codon that would produce a truncated G protein of 122aa. Analysis of early passage and ex vivo samples showed the termination codon in G to be predominant after 6 days in culture indicating rapid selection of the mutation in A72 cells.

Pneumovirus in dogs with acute respiratory disease.

To determine which respiratory viruses circulate among confined dogs, we analyzed nasal and pharyngeal swab specimens from shelter dogs with acute respiratory disease. An unknown virus was isolated. Monoclonal antibody testing indicated that it was probably a pneumovirus. PCR and sequence analysis indicated that it was closely related to murine pneumovirus.

Minimum intravenous infectious dose of ovine progressive pneumonia virus (OPPV).

The minimum intravenous infectious dose for ovine progressive pneumonia virus (OPPV) WLC1 was determined using twenty-four 6month-old lambs. Twelve groups of two 6month-old lambs were inoculated intravenously (i.v.) with tissue culture fluid containing ovine progressive pneumonia virus (OPPV) WLC1 titers ranging from 10(7.6) TCID(50)/lamb down to 10(-3.4) TCID(50)/lamb and were monitored for seroconversion using the OPPV agar gel immunodiffusion assay (AGID). Fifteen of the 16 lambs given equal or greater than 10(0.6) TCID(50) seroconverted, and virus could be isolated from peripheral blood leukocytes in 13 out of the 15 of these lambs. None of the eight lambs receiving less than 10(0.6) TCID(50) seroconverted during the 12months. The results of this study indicated that 10(0.6) or 4 TCID(50)/lamb given i.v. was capable of establishing infection.

Respiratory viruses in pediatric necropsies: an immunohistochemical study.

Infections of the respiratory system are responsible for the majority of hospitalizations and deaths in pediatric patients in developing countries. We selected 177 necropsies of pediatric patients who died as a result of serious respiratory infections. The histopathological findings and epidemiological data were reviewed, and lung tissue samples were separated for immunohistochemistry testing. Conventional immunohistochemistry techniques were used to detect viral antigens in formalin-fixed, paraffin-embedded (FF-PE) lung tissue samples using a pool of monoclonal antibodies against respiratory viruses (respiratory syncytial virus, influenza A and B, adenovirus, and parainfluenza 1, 2, and 3 viruses) as primary antibodies. The histopathological findings were classified into bronchopneumonia (BCP) and interstitial pneumonitis (IP) patterns. The immunohistochemistry results were compared with histopathological patterns and epidemiological data. Positive results for viruses were found in 34% and 62.5% of the BCP and IP cases, respectively. Males and infants below 1 year of age were more frequent in the group that had positive results for viruses. Acute enteritis was the main cause of hospitalization and sepsis the most frequent cause of death in this group. A clear seasonal distribution was observed, with the majority of cases occurring in the 2nd and 3rd trimesters (autumn and winter) of each year in the period studied. Immunohistochemistry is an affordable and easy-to-perform method for viral-antigen detection in FF-PE tissue samples. Although BCP is a classic histopathological pattern found in bacterial infections, it is possible that children with serious respiratory infections had concomitant viral and bacterial infections, regardless of their previous immunologic state.

Comparison of the Seeplex reverse transcription PCR assay with the R-mix viral culture and immunofluorescence techniques for detection of eight respiratory viruses.

This study evaluated the clinical usefulness of a newly introduced multiplex reverse transcription PCR assay (Seeplex RV; Seegene, Inc., Seoul, Korea) in patients with respiratory symptoms. Fifty clinical respiratory specimens (45 from children, 5 from adults) were tested for 8 viruses (influenza virus type A and B, parainfluenza virus type 1, 2, 3, respiratory syncytial virus type A and B, and adenovirus) by Seeplex RV (S-RV) and R-mix viral culture with immunofluorescence (VC-IF). Forty (80%) of the 50 samples showed concordant results between S-RV and VC-IF; 24 of these showed the same positive and 16 showed the same negative results. Among the 10 discrepant samples, 9 were S-RV-positive and VC-IF-negative. Six were obtained in patients with lower respiratory tract infection. Only 1 sample was VC-IF-positive and S-RV-negative. This patient had pneumonia. In 3 cases, more than 1 virus was identified by S-RV. The total running time of S-RV was 6 hr, which shortens the detection time for the viral presence by 2 workdays compared to VC-IF. In conclusion, S-RV is reliable, rapid, relatively easy to perform, and able to detect more than 1 virus simultaneously. Therefore, implementation of the S-RV assay in clinical laboratories will aid rapid diagnosis and treatment of major viral infections of the respiratory tract.

Presence of avian pneumovirus subtypes A and B in Japan.

Four avian pneumovirus (APV) isolates from chickens clinically diagnosed with swollen head syndrome were genetically characterized as to the subtypes of the virus in Japan. The results of reverse transcriptase-polymerase chain reactions based on subtype-specific primers and direct sequence analysis of G genes indicated subtypes A and B but not C or D of APV were present in Japan. Several routes or sources are conceivable for APV to invade into Japan.

Detection of avian pneumovirus in wild Canada (Branta canadensis) and blue-winged teal (Anas discors) geese.

Choanal cleft swab samples from 770 wild Canada geese (Branta canadensis) and 358 blue-winged teal (Anas discors), captured for relocation or banding, were examined for the presence of avian pneumovirus (APV) RNA by reverse transcription (RT)-polymerase chain reaction (PCR) and for virus isolation. The swab samples were pooled into groups of 5 or 10. Sixty eight of 102 (66.7%) pooled goose samples were RT-PCR positive for APV RNA. Thirteen of 52 (25.0%) pooled blue-winged teal samples were RT-PCR positive for APV RNA. APV RNA-positive samples were inoculated onto chick embryo fibroblasts (CEF) and QT-35 cells. Infectious APV was isolated from five Canada goose pooled samples in CEF and from one Canada goose pool in QT-35 cells but not from blue-winged teal.

Detection and differentiation of avian pneumoviruses (metapneumoviruses).

The available detection methods for avian pneumoviruses (turkey rhinotracheitis virus; genus Metapneumovirus) in turkeys, domestic fowl and other species are reviewed. The advantages and disadvantages of virus isolation techniques, virus or genome (polymerase chain reaction) detection and serology are discussed. Some of the problems likely to be encountered are considered, including the detection of yet to be discovered subtypes, as are the factors that are likely to influence the outcome of the work.

Experimental and field evaluation of a live vaccine against avian pneumovirus.

The attenuation of an avian pneumovirus (APV) isolate (APV/MN/turkey/1-a/97) by 63 serial passages in cell culture (seven in chicken embryo fibroblasts and 56 in Vero cells) and its evaluation as a live attenuated vaccine in turkey poults is described. The birds were vaccinated with two different doses of attenuated virus (10(4.5) median tissue culture infectious dose (TCID(50))/ml and 10(2.5) TCID(50) /ml) at 2 weeks of age, and were challenged 2 weeks later with virulent APV. No clinical signs were seen in vaccinated, challenged birds, whereas severe clinical signs were observed in the mock-vaccinated, challenged group. Vaccinated birds developed anti-APV antibodies, which increased in titre following challenge with virulent virus. On challenge, none of the vaccinates was found to shed viral nucleic acid as detected by reverse transcriptase-polymerase chain reaction, but non-vaccinated, challenged birds did. The vaccine virus was also evaluated under field conditions in two farms. At one farm, the 'seeder bird approach' was used and two birds per 1,000 birds were vaccinated by the oculo-nasal route. In the second farm, the virus was given to all birds simultaneously in the drinking water. The birds vaccinated by the drinking water route seroconverted earlier and continued to shed virus for longer as compared with birds inoculated by the seeder bird approach. The overall results of this study indicate that the 63rd passage of APV was sufficiently attenuated and offered protection against challenge with virulent virus.

A single subtype of avian pneumovirus circulates among Minnesota turkey flocks.

The recent emergence of avian pneumovirus (APV) infection among US turkey flocks has resulted in a major economic threat to the turkey industry. In order to elucidate the molecular epidemiology of APV, comparative sequence analysis of the fusion (F) protein gene of APV was performed for 3 cell culture-adapted isolates and 10 APV positive clinical samples recovered from US turkey flocks. Relatively modest levels of nucleotide and amino acid sequence divergence were identified, suggesting the prevalence of a single lineage of APV among US turkey flocks. Additionally, numerous polymorphisms were identified that were only represented in the clinical samples but not in the in vitro propagated isolates of APV. Phylogenetic analyses confirm that the subtype of APV circulating in the upper Midwestern United States is evolutionarily related to, but distinct from, European APV subgroups A and B. Overall, the results of the present investigation suggest that there has been only a single recent introduction of APV into US turkey populations in the upper Midwestern United States.