Bioaerosol Sampling to Detect Avian Influenza Virus in Hanoi's Largest Live Poultry Market.

BACKGROUND: Newly emergent and virulent strains of H7N9 avian influenza virus are rapidly spreading in China and threaten to invade Vietnam. We sought to introduce aerosol sampling for avian influenza viruses in Vietnam. METHODS: During October 2017, National Institute for Occupational Safety and Health 2-stage aerosol samplers were assembled on a tripod and run for 4 hours. Concomitantly, up to 20 oropharyngeal (OP) swab samples were collected from chickens and ducks distanced at 0.2-1.5 m from each sampler. RESULTS: The 3 weeks of sampling yielded 30 aerosol samples that were 90% positive for influenza A, by quantitative reverse-transcription polymerase chain reaction, and 116 OP swab sample pools (5 samples per pool) that were 47% positive. Egg cultures yielded 1 influenza A virus (not H5 or H7) from aerosol and 25 influenza A viruses from OP swab sample pools (5 were H5 positive). The association between positive sample types (over time and position) was strong, with 91.7% of positive OP pooled swab samples confirmed by positive aerosol samples and 81% of influenza A positive aerosol samples confirmed by positive OP swab samples. CONCLUSIONS: We posit that aerosol sampling might be used for early warning screening of poultry markets for novel influenza virus detection, such as H7N9. Markets with positive aerosol samples might be followed up with more focused individual bird or cage swabbing, and back-tracing could be performed later to locate specific farms harboring novel virus. Culling birds in such farms could reduce highly pathogenic avian influenza virus spread among poultry and humans.

Characterization of cross-clade monoclonal antibodies against H5N1 highly pathogenic avian influenza virus and their application to the antigenic analysis of diverse H5 subtype viruses.

H5N1 highly pathogenic avian influenza viruses (HPAIVs) are a threat to both animal and public health and require specific and rapid detection for prompt disease control. We produced three neutralizing anti-hemagglutinin (HA) monoclonal antibodies (mAbs) using two clades (2.2 and 2.5) of the H5N1 HPAIV isolated in Japan. Blocking immunofluorescence tests showed that each mAb recognized different epitopes; 3B5.1 and 3B5.2 mAbs against the clade 2.5 virus showed cross-clade reactivity to all 26 strains from clades 1, 2.2, 2.3.2.1, 2.3.2.1a, b, c and 2.3.4, suggesting that the epitope(s) recognized are conserved. Conversely, the 1G5 mAb against the clade 2.2 virus showed reactivity to only clades 1, 2.3.4 and 2.5 strains. An analysis of escape mutants, and some clades of the H5N1 viruses recognized by 3B5.1 and 3B5.2 mAbs, suggested that the mAbs bind to an epitope, including amino acid residues at position 162 in the HA1 protein (R162 and K162). Unexpectedly, however, when five Eurasian-origin H5 low-pathogenic AIV (LPAIV) strains with R162 were examined (EA-nonGsGD clade) as well as two American-origin strains (Am-nonGsGD clade), the mAb recognized only EA-nonGsGD clade strains. The R162 and K162 residues in the HA1 protein were highly conserved among 36 of the 43 H5N1 clades reported, including clades 2.3.2.1a and 2.3.2.1c that are currently circulating in Asia, Africa and Europe. The amino acid residues (158-PTIKRSYNNTNQE-170) in the HA1 protein are probably an epitope responsible for the cross-clade reactivity of the mAbs, considering the epitopes reported elsewhere. The 3B5.1 and 3B5.2 mAbs may be useful for the specific detection of H5N1 HPAIVs circulating in the field.

Characterization of mAbs to chicken anemia virus and epitope mapping on its viral protein, VP1.

Three (MoCAV/F2, MoCAV/F8 and MoCAV/F11) of four mouse mAbs established against the A2/76 strain of chicken anemia virus (CAV) showed neutralization activity. Immunoprecipitation showed a band at ~50 kDa in A2/76-infected cell lysates by neutralizing mAbs, corresponding to the 50 kDa capsid protein (VP1) of CAV, and the mAbs reacted with recombinant VP1 proteins expressed in Cos7 cells. MoCAV/F2 and MoCAV/F8 neutralized the 14 CAV strains tested, whereas MoCAV/F11 did not neutralize five of the strains, indicating distinct antigenic variation amongst the strains. In blocking immunofluorescence tests with the A2/76-infected cells, binding of MoCAV/F11 was not inhibited by the other mAbs. MoCAV/F2 inhibited the binding of MoCAV/F8 to the antigens and vice versa, suggesting that the two mAbs recognized the same epitope. However, mutations were found in different parts of VP1 of the escape mutants of each mAb: EsCAV/F2 (deletion of T89+A90), EsCAV/F8 (I261T) and EsCAV/F11 (E144G). Thus, the epitopes recognized by MoCAV/F2 and MoCAV/F8 seemed to be topographically close in the VP1 structure, suggesting that VP1 has at least two different neutralizing epitopes. However, MoCAV/F8 did not react with EsCAV/F2 or EsCAV/F8, suggesting that binding of MoCAV/F8 to the epitope requires coexistence of the epitope recognized by MoCAV/F2. In addition, MoCAV/F2, with a titre of 1 : 12 800 to the parent strain, neutralized EsCAV/F2 and EsCAV/F8 with low titres of 32 and 152, respectively. The similarity of the reactivity of MoCAV/F2 and MoCAV/F8 to VP1 may also suggest the existence of a single epitope recognized by these mAbs.

Influence of olive-derived hydroxytyrosol on the toll-like receptor 4-dependent inflammatory response of mouse peritoneal macrophages.

Macrophages play important roles in the host innate immune response and are involved in the onset of diseases caused by inflammation. Toll-like receptor 4 (TLR4)-mediated inflammatory responses of macrophages may be associated with diseases such as diabetes and diseases of the cardiovascular system. Hydroxytyrosol (HT) exerts strong antioxidant and anti-inflammatory effects and may be applied in the treatment of inflammatory diseases. In the present study conducted in vitro, we investigated the effects of the TLR4-dependent anti-inflammatory effect of HT on peritoneal macrophage of BALB/c mice. We show here that the elevated levels of iNOS gene expression and nitric oxide production induced by lipopolysaccharide (LPS) (0.25 µg/ml) were suppressed by HT (12.5 µg/ml). LPS-dependent NF-κB gene expression and phosphorylation of NF-κB were not affected by HT under these conditions. In contrast, the expression of TNF-α was significantly increased in the presence of LPS and HT. These results suggest that HT suppressed nitric oxide production by decreasing iNOS gene expression through a mechanism independent of the NF-κB signaling pathway. These novel findings suggest that the modulation by HT of the expression of genes involved in inflammation may involve multiple mechanisms.

Virucidal effect of acidic electrolyzed water and neutral electrolyzed water on avian influenza viruses.

The virucidal effects of two types of electrolyzed water, acidic electrolyzed water (AEW) and neutral electrolyzed water (NEW), on avian influenza viruses were studied. Virus titers of the highly pathogenic H5N1 virus and the low-pathogenic H9N2 virus irreversibly decreased by >5-log at 1 min after the viruses were mixed with NEW containing ≥43 ppm free available chlorine (FAC), but not with NEW containing <17 ppm FAC. The minimum concentration of FAC for a virucidal effect of NEW was estimated at around 40 ppm. In contrast, the virus titers decreased by >5 log at 1 min after the viruses were mixed with AEW, in which the concentration of the FAC ranged from 72 to 0 ppm. Thus, the virucidal effect of AEW did not depend on the presence of FAC. Reverse transcription polymerase chain reaction amplified fragments of the M and NP genes, but not the complete M gene, from RNA extracted from the AEW-inactivated virus. Moderate morphological changes were found under the electron microscope, although no changes were observed in the electrophoresed proteins of the AEW-inactivated virus. No viral genes were amplified from the RNA extracted from the NEW-inactivated virus, regardless of the length of the targeted genes. No viral particles were detected under the electron microscope and no viral proteins were detected by electrophoresis for the NEW-inactivated virus. Thus, this study demonstrated potent virucidal effects of AEW and NEW and differences in the virucidal mechanism of the two types of electrolyzed water.

H5N1 highly pathogenic avian influenza virus isolated from conjunctiva of a whooper swan with neurological signs.

An H5N1 highly pathogenic avian influenza virus was isolated from conjunctiva of a whooper swan with neurological signs, which was captured during the latest H5N1 HPAI outbreak in Japan. The conjunctival swab contained a larger amount of the virus in comparison with the tracheal swab. This is the first report on H5N1 virus isolation from the conjunctiva of a wild bird, and the result may suggest the conjunctival swab to be a critical sample for H5N1 HPAIV detection in waterfowl. Phylogenetic analysis of the HA gene indicated that the virus falls into H5N1 clade 2.3.2.1.

Surveillance and characterization of avian influenza viruses from migratory water birds in eastern Hokkaido, the northern part of Japan, 2009-2010.

Avian influenza virus (AIV) surveillance was conducted around a small pond in Obihiro, eastern Hokkaido, Japan. Eleven AIVs were isolated from a total of 1,269 fecal samples of migratory wild birds collected during 2009 and 2010. The sample number covered approximately 60 % of the total number of birds observed during sampling periods. The subtypes of the isolates included H3N8 (4 isolates), H5N2 (3), H6N2 (2), H6N1 (1), and H11N2 (1). The H3N8 subtype was most prevalent as in the previous studies performed in Hokkaido. The three H5N2 isolates genetically characterized as low pathogenic AIV were closely related to the strains previously isolated from aquatic wild birds in Japan and also to the Korean strains isolated from aquatic birds in recent years. In Korea, H5N2 subtype virus has often been isolated from poultry and wild birds, as well as reassortant viruses generated from duck H5N2 viruses and chicken H9N2 virus, and avian-swine-like reassortant H5N2 viruses. Considering the previous chicken outbreaks caused by highly pathogenic H5N2 viruses, which affected many countries, it should be an important priority to continue, monitoring the evolution of H5N2 viruses circulating in the region.

Surveillance of avian influenza virus in migratory water birds in eastern Hokkaido, Japan.

The epidemiological information has obtained on avian influenza virus (AIV) in eastern Hokkaido, Japan, where AIV surveillance has not been performed. Cloacal or fecal samples obtained from migratory water birds were screened for AIV both by real-time reverse transcriptase polymerase chain reaction to detect the influenza A virus matrix (M) gene and by egg inoculation. Between 2007 and 2009, a total of 2,488 samples were collected from various avian species in Abashiri, Kushiro, Nemuro and Tokachi districts of eastern Hokkaido. AIVs were isolated from 18 of those samples (0.7%). No AIV was isolated from the 1,449 samples collected in Abashiri, Kushiro and Nemuro districts, although 6 were positive for the M gene by RRT-PCR. In contrast, 52 (5.0%) of the 1,039 samples collected from ducks in Tokachi district were M gene positive; AIVs were isolated from 18 of those samples (1.7%). The isolates included H3N5 (1 isolate), H3N6 (1), H3N8 (9), H4N2 (1), H4N6 (2), H6N5 (1), H6N8 (1), and H11N3 (2) subtypes. H3N5 and H11N3 subtypes have not been frequently isolated, and our study is the first to report H3N5 and the second to report H11N3 in Japan. Phylogenetic analysis revealed that the M genes of all isolates belonged to the Eurasian lineage.

A RT-PCR assay for the detection of coronaviruses from four genera.

BACKGROUND: During the past two decades, three novel coronaviruses (CoVs) have emerged to cause international human epidemics with severe morbidity. CoVs have also emerged to cause severe epidemics in animals. A better understanding of the natural hosts and genetic diversity of CoVs are needed to help mitigate these threats. OBJECTIVE: To design and evaluate a molecular diagnostic tool for detection and identification of all currently recognized and potentially future emergent CoVs from the Orthocoronavirinae subfamily. STUDY DESIGN AND RESULTS: We designed a semi-nested, reverse transcription RT-PCR assay based upon 38 published genome sequences of human and animal CoVs. We evaluated this assay with 14 human and animal CoVs and 11 other non-CoV respiratory viruses. Through sequencing the assay's target amplicon, the assay correctly identified each of the CoVs; no cross-reactivity with 11 common respiratory viruses was observed. The limits of detection ranged from 4 to 4 × 102 copies/reaction, depending on the CoV species tested. To assess the assay's clinical performance, we tested a large panel of previously studied specimens: 192 human respiratory specimens from pneumonia patients, 5 clinical specimens from COVID-19 patients, 81 poultry oral secretion specimens, 109 pig slurry specimens, and 31 aerosol samples from a live bird market. The amplicons of all RT-PCR-positive samples were confirmed by Sanger sequencing. Our assay performed well with all tested specimens across all sample types. CONCLUSIONS: This assay can be used for detection and identification of all previously recognized CoVs, including SARS-CoV-2, and potentially any emergent CoVs in the Orthocoronavirinae subfamily.

Characterization of avian paramyxovirus serotype 14, a novel serotype, isolated from a duck fecal sample in Japan.

A hemagglutinating virus isolate designated 11OG0352, was obtained from a duck fecal sample. Genetic and virological analyses indicated that it might represent a novel serotype of avian paramyxovirus (APMV). Electron micrographs showed that the morphology of the virus particle was similar to that of APMV. The complete genome of this virus comprised 15,444 nucleotides complying with the paramyxovirus "rule of six" and contains six open reading frames (3'-N-P-M-F-HN-L-5'). The phylogenetic analysis of the whole genome revealed that the virus was a member of the genus Avulavirus, but that it was distinct from APMV-1 to APMV-13. Although the F-protein cleavage site was TREGK↓L, which resembles a lentogenic strain of APMV-1, the K residue at position -1 of the cleavage site was first discovered in APMV members. The phosphoprotein gene of isolate 11OG0352 contains a putative RNA editing site, 3'-AUUUUCCC-5' (negative sense) which sequence differs from that of other APMVs. The intracerebral pathogenicity index test did not detect virulence in infected chicks. In hemagglutination inhibition (HI) tests, an antiserum against this virus did not detectably react with other APMVs (serotypes 1-4, 6-9) except for low reciprocal cross-reactivity with APMV-6. We designated this isolate, as APMV-14/duck/Japan/11OG0352/2011 and propose that it is a novel APMV serotype. The HI test may not be widely applicable for the classification of a new serotype because of the limited availability of reference antisera against all serotypes and cross-reactivity data. The nucleotide sequence identities of the whole genome of 11OG0352 and other APMVs ranged from 46.3% to 56.1%. Such comparison may provide a useful tool for classifying new APMV isolates. However, the nucleotide sequence identity between APMV-12 and APMV-13 was higher (64%), which was nearly identical to the lowest nucleotide identity (67%) reported in subgroups within the serotype. Therefore, consensus criteria for using whole genome analysis should be established.

Potential of electrolyzed water for disinfection of foot-and-mouth disease virus.

Acidic electrolyzed water (EW) (pH 2.6-5.8) and alkaline EW (pH 11.2-12.1) were examined as potential disinfectants against foot-and-mouth disease virus (FMDV). Using acidic EW with pH 2.6 and alkaline EW with pH >11.7, the viral titer decreased in vitro by > 4.0 log values, 2 min after the virus was mixed with EW at a 1:10 dilution. The strong virucidal effect of acidic EW (pH 2.6), but not that of alkaline EW (>11.7), seemed to depend on the chlorine level in the solution. Genetic analysis revealed that viral RNA was substantially reduced, especially by alkaline EW.

High antiviral effects of hibiscus tea extract on the H5 subtypes of low and highly pathogenic avian influenza viruses.

Viral neuraminidase inhibitors are widely used as synthetic anti-influenza drugs for the prevention and treatment of influenza. However, drug-resistant influenza A virus variants, including H5N1 highly pathogenic avian influenza viruses (HPAIVs), have been reported. Therefore, the discovery of novel and effective antiviral agents is warranted. We screened the antiviral effects of 11 herbal tea extracts (hibiscus, black tea, tencha, rosehip tea, burdock tea, green tea, jasmine tea, ginger tea, lavender tea, rose tea and oak tea) against the H5N1 HPAIV in vitro. Among the tested extracts, only the hibiscus extract and its fractionated extract (frHibis) highly and rapidly reduced the titers of all H5 HPAIVs and low pathogenic AIVs (LPAIVs) used in the pre-treatment tests of Madin-Darby canine kidney (MDCK) cells that were inoculated with a mixture of the virus and the extract. Immunogold electron microscopy showed that anti-H5 monoclonal antibodies could not bind to the deformed H5 virus particles pretreated with frHibis. In post-treatment tests of MDCK cells cultured in the presence of frHibis after infection with H5N1 HPAIV, the frHibis inhibited viral replication and the expression of viral antigens and genes. Among the plants tested, hibiscus showed the most prominent antiviral effects against both H5 HPAIV and LPAIV.

Pathogenicity of an H5N1 avian influenza virus isolated in Vietnam in 2012 and reliability of conjunctival samples for diagnosis of infection.

The continued spread of highly pathogenic avian influenza virus (HPAIV) subtype H5N1 among poultry in Vietnam poses a potential threat to animals and public health. To evaluate the pathogenicity of a 2012 H5N1 HPAIV isolate and to assess the utility of conjunctival swabs for viral detection and isolation in surveillance, an experimental infection with HPAIV subtype H5N1 was carried out in domestic ducks. Ducks were infected with 10(7.2) TCID50 of A/duck/Vietnam/QB1207/2012 (H5N1), which was isolated from a moribund domestic duck. In the infected ducks, clinical signs of disease, including neurological disorder, were observed. Ducks started to die at 3 days-post-infection (dpi), and the study mortality reached 67%. Viruses were recovered from oropharyngeal and conjunctival swabs until 7 dpi and from cloacal swabs until 4 dpi. In the ducks that died or were sacrificed on 3, 5, or 6 dpi, viruses were recovered from lung, brain, heart, pancreas and intestine, among which the highest virus titers were in the lung, brain or heart. Results of virus titration were confirmed by real-time RT-PCR. Genetic and phylogenetic analysis of the HA gene revealed that the isolate belongs to clade 2.3.2.1 similarly to the H5N1 viruses isolated in Vietnam in 2012. The present study demonstrated that this recent HPAI H5N1 virus of clade 2.3.2.1 could replicate efficiently in the systemic organs, including the brain, and cause severe disease with neurological symptoms in domestic ducks. Therefore, this HPAI H5N1 virus seems to retain the neurotrophic feature and has further developed properties of shedding virus from the oropharynx and conjunctiva in addition to the cloaca, potentially posing a higher risk of virus spread through cross-contact and/or environmental transmission. Continued surveillance and diagnostic programs using conjunctival swabs in the field would further verify the apparent reliability of conjunctival samples for the detection of AIV.

Lung cytokine gene expression is correlated with increased severity of disease in a novel H4N8 influenza virus isolated from shorebirds.

The lung cytokine gene expression profiles of mice infected with 2 strains of H4N8 viruses isolated from shorebirds and reference H4 viruses from ducks are compared. Major differences between the two H4N8 strains of shorebirds, one of which causes a severe respiratory disease in mice, are in the PB1 and NS1 genes. In mice with H4N8 virus induced pneumonia, overall expression of TNF-α, IL-6 and IL-12 genes was markedly higher than in mice infected with other H4 viruses tested, although gene expression of type I interferon was not increased until day 4 post viral infection. In contrast, in mice infected with a comparison H4N8 strain, gene expression of type I interferon peaked on day 1 post viral infection. Overall, the cytokine response corresponds with the severity of disease caused by shorebird H4N8 virus. The results obtained in this study provide valuable information to understand the immunopathology induced by a low pathogenic avian influenza virus, which may be useful in preparation for outbreaks of novel influenza A virus.

H4N8 subtype avian influenza virus isolated from shorebirds contains a unique PB1 gene and causes severe respiratory disease in mice.

H4N8 subtype avian influenza viruses were isolated from shorebirds in eastern Hokkaido. All the isolates shared >99.7% nucleotide homology, and all the viral genes except for PB1 were highly related to those of A/red-necked stint/Australia/1/04. Thus, the isolates were regarded as PB1 reassortants. The most similar PB1 gene was identified in A/mallard/New Zealand/1615-17/04 (H4N6) with nucleotide homology of 90.9%. BALB/c mice intranasally inoculated with the H4N8 isolates developed severe respiratory disease, which eventually led to death in some mice. The virus was isolated from the lungs, and viral antigen was detected in the lungs with pneumonia. Other H4 subtype viruses tested did not cause any symptoms in mice, although these viruses were also isolated from the lungs. The PB2 gene of the H4N8 isolates contains K482R, but not the E627K or D701N substitutions. The PB1-F2 gene of the isolates consists of a 101-amino acid unique sequence, but lacks the N66S mutation.