Lingshuang Sun

Virological and Epidemiological Evidence of Avian Influenza Virus Infections Among Feral Dogs in Live Poultry Markets, China: A Threat to Human Health?

TO THE EDITOR— Since its first detection in March 2013, the novel H7N9 avian influenza virus (AIV) has quickly spread among poultry and people in China. As of 16 February 2014, a total of 348 laboratory-confirmed human H7N9 infections in China have been confirmed by the World Health Organization [1–3]. The H7N9 virus has spread widely with little sign of infection among poultry [4]. Epidemiologic studies have identified poultry exposure as an important risk factor for human infections with H5N1 and H7N9, especially for those individuals associated with live poultry markets (LPMs) [5–8]. As dogs in China have been shown to be infected with AIVs, we sought to investigate whether dogs living in close proximity to LPMs and H7N9-affected farms might have been infected with the novel H7N9 virus or other influenza viruses. From August 2011 to August 2013, we studied a total of 2357 dogs that lived in close proximity to LPMs and poultry farms in the rural areas of Shanghai, Guangdong, Zhejiang, and Jiangsu provinces in China where novel H7N9 AIV had been previously detected (forMaterials and Methods, see Supplementary Data). Overall, 68.18% (n = 1607) of the 2357 stray dog samples were collected in rural areas, with the remaining samples collected in LPMs (Table 1). Of the 2357 nasal swab samples collected, 93 (3.9%) were positive for influenza A virus by realtime reverse transcription polymerase chain reaction (PCR), and 11 viruses were isolated from these samples (see Supplementary Data). Hemagglutination inhibition (HI) assays and hemagglutinin antigen–specific enzyme-linked immunosorbent assays against H7N9 viral antigens revealed no evidence of H7N9 infection. Results of the HI and microneutralization (MN) assays are reported in Table 1 and in the Supplementary Data. A total of 19 serum samples had HI antibody titers of ≥1:20 against H5 antigen (Table 1), and 3 of these 19 samples were also positive by MN assay. Dogs that were sampled in LPMs had a greater probability of having elevated HI antibodies against avian H9N2, avian H5N1, and canine H3N2 viruses (Table 2), compared with the dogs that were raised in poultry farms. Our study supports this premise in that, although we failed to find evidence of previous H7N9 infections among the dogs, we found the world’s first evidence of previous H5N1 and H9N2 infection among dogs by real-time PCR, HI, and MN assay. These findings were unexpected but biologically plausible. In LPMs and farms in rural China, stray dogs and cats have considerable contact with poultry or poultry products. This can occur indirectly through aerosol and fecal transmission or directly through the consumption of dead bird carcasses or entrails. LPMs are particularly problematic as they offer a mixing of animal species from often diverse geographical areas, frequent venues for contact with the public, and often nonhygienic behavior of workers who handle and process the birds for sale. Both rural farms and LPMs provide opportunities for wild aquatic birds, domestic poultry, stray dogs, and humans to closely interact and potentially share pathogens (Supplementary Figure 1). Additionally, compared

First Description of Hepacivirus and Pegivirus Infection in Domestic Horses in China: A Study in Guangdong Province, Heilongjiang Province and Hong Kong District.

Since 2012, three viruses, known as equine hepacivirus (EqHV), equine pegivirus (EPgV) and Theiler’s disease-associated virus (TDAV), have been discovered in equines. Given that these viruses are the newest members of the Flaviviridae family, genomic information concerning circulating EqHV, EPgV and TDAV strains around the world is limited. To date, no genetic surveillance studies have been performed on these three viruses in the equine population of China. Here, a total of 177 serum samples were collected from equines across China between 2014 and 2015. Using PCR, we detected viral RNA in the serum samples, six of which were EqHV positive and two of which were EPgV positive. Co-infection with the two viruses was not observed among the Chinese equines studied, and TDAV RNA was not detected in the equine serum samples collected for this study. Phylogenetic analysis of partial NS5B open reading frame (ORF), NS3 ORF, and 5’ untranslated region nucleotide sequences from EqHV as well as partial NS3 ORF sequence from EPgV indicated that EqHV and EPgV have evolved into two main clades by themselves, both of which are circulating in China. Based on the partial NS5B and NS3 ORF sequences of EqHV, the sequences of one clade were also split into two subclades. This study enriches our knowledge of the geographic distribution of these three equine viruses.

Genetic characterization of avian-origin H3N2 canine influenza viruses isolated from Guangdong during 2006–2012

Canine influenza virus (CIV) is an emerging pathogen that causes severe and acute respiratory disease in dogs. In 2006, the H3N2 canine influenza virus was first identified in dogs from Guangdong province in China. Up to now, nine CIVs have been isolated from different populations in Guangdong. The nine isolates were grouped together with the canine H3N2 viruses isolated from dogs and felines in Korea, when the eight phylogenetic trees constructed were compared. These findings emphasize the importance of CIV surveillance in this region for understanding the genesis of this virus, and it is important to remain aware of the potential of H3N2 CIV to be transmitted from dogs to the human population.

Evidence for Subclinical Influenza A(H1N1)pdm09 Virus Infection among Dogs in Guangdong Province, China

ABSTRACT During 2012, we identified sampled dogs with elevated levels of antibodies (≥1:40) against A(H1N1)pdm09 virus by using a hemagglutination inhibition (HI) assay (seroprevalence, 24.7%) and a microneutralization (MN) assay (seroprevalence, 10.8%). These high seroprevalences of A(H1N1)pdm09 among dogs without clinical signs of influenza support the premise that dogs may play a role in the human influenza ecology in China.

Serological report of influenza a (H7N9) infections among pigs in Southern China

BackgroundIn 2013, a novel H7N9 avian influenza virus (AIV) was isolated from ill humans in Shanghai and Anhui Province, China. Since then, the virus has spread quickly throughout China. Previous isolation of H7N2 virus from swine suggests that additional H7 subtype AIVs may be transmitted through pigs. However, prior to the recent zoonosis of H7N9, there were very few studies on the seroprevalence of the H7 subtypes in this species. Thus, there is a need to perform serological surveys for novel H7N9 as well as other H7 subtype AIVs in swine. This surveillance may help us understand risk factors for outbreaks of influenza A (H7N9) virus.ResultsOnly 2.0% (26/1310) of the pig sera had antibodies with an HI titer ≥1:20, and none had an MN titer ≥1:80, against the H7 antigen. Thus, no samples were found to be positive against H7N9. However, 13.6% (178/1310) of the pig sera had antibodies with HI titer ≥1:20 and 8.5% (112/1310) by MN titer ≥1:80 against H9 antigen. Thirty-seven percent (484/1310) of the pig sera had antibodies with HI titer ≥1:20 and 18.2% (238/1310) had MN titer ≥1:80 against pandemic 2009.ConclusionsPigs in southern China have been shown to be infected with multiple avian influenza viruses. As the prevalence of novel influenza A viruses (e.g., H7N9 avian influenza virus) may be increasing among poultry in China, similar seroepidemiological studies of pigs should be conducted in the future.

H9N2 avian influenza infection altered expression pattern of Sphiogosine-1-phosphate Receptor 1 in BALB/c mice

BackgroundThe pathological damage inflicted by virulent AIV strains is often caused by inducing a positive feedback loop of cytokines in immune cells that cause excessive inflammation. Previous research has shown that a G protein-coupled receptor, sphingosine-1-phosphate receptor 1 (S1PR1), plays a crucial role in the development of excessive inflammation in influenza virus infection (Cell 146:861–862, 2011; Cell 146:980–991, 2011). BALB/c mice are common laboratory animals used in research of influenza virus; however the effects of influenza infections on expression patterns of S1PR1 in mice are unknown.MethodsWe investigated the expression patterns of S1PR1 in normal BALB/c mice and those infected by two distinct H9N2 AIV strains, one (A/chicken/Guangdong/V/2008,V) highly pathogenic, and the other (A/chicken/Guangdong/Ts/2004,Ts), non-pathogenic in mice, using quantitative PCR and immunohistochemistry (IHC) to detect S1PR1 mRNA and protein, respectively.ResultsS1PR1 mRNA was ubiquitously expressed in all the tissues examined, and significant differences were seen in mRNA expression between infected Ts, V and control mice in detected tissues, heart, liver, spleen, kidney and brain. S1PR1 protein was expressed in the cytoplasm and also demonstrated quantitative changes in expression in the various tissues between mice infected with the two strains of AIV.ConclusionsOur results provided the first look at differences in S1PR1 expression patterns in BALB/c mice infected by non-pathogenic and highly pathogenic H9N2 influenza viruses. This information will not only be helpful in designing experiments to better understand the role of S1PR1 in virus-host interactions but also in developing novel anti-influenza agents to minimize the mortality and morbidity associated with highly virulent strains in avian and human populations.

Serological evidence of avian influenza virus and canine influenza virus infections among stray cats in live poultry markets, China

From January 2010 to January 2012, we collected sera samples from 700 stray cats living in close proximity to poultry farms or poultry markets in 4 provinces in China. A number of cats had evidence of avian and canine influenza virus infection: avian H9N2 [24 by HI ≥1:20 and 16 by microneutralization (MN) assay ≥1:80]; avian H5N1 (9 by HI ≥1:20 and 3 by MN assay ≥1:80) and canine H3N2 (32 by HI ≥1:20 and 18 by MN ≥1:80). Bivariate analyses revealed that cats sampled near live poultry markets and cats with influenza-like-illness were at increased risk of having elevated antibody titers by HI against avian H9N2, avian H5N1, or canine H3N2 viruses. Hence, cats may play a very important role in the ecology of novel influenza viruses and periodic epidemiological surveillance for novel influenza infections among stray cats could serve as an early warning system for human threats.

Sparse serological evidence of H5N1 avian influenza virus infections in domestic cats, northeastern China.

Today the cross-species transmission of avian influenza viruses (AIV) are a great concern. A number of AIV strains are now enzootic among poultry, with H9N2 and highly pathogenic H5N1 AIV strains prevalent in China. H5N1 strains have been recognized to infect zoo and domestic feline species. In this serological study we sought to examine evidence that H5N1 strains have infected domestic cats in northeastern China. In 2013, we conducted a cross-sectional serological study of 916 healthy cats in Heilongjian, Jilin, and Liaonin Provinces. Sera were screened with a hemagglutinin inhibition (HI) assay and seropositive specimens (HI ≥ 1:20) were further evaluated with a microneutralization (MN) assay against a clade 2.3.2 H5N1 AIV, a H9N2 AIV, A (H1N1)pdm09, and a canine H3N2 virus. While ∼2% of cats had elevated HI assays against H5N1, no elevations were confirmed (MN ≥ 1:80). These data serve as baseline for future surveillance for AIV infections among domestic cats. Conducting such surveillance seems important for geographical areas recognized as endemic for AIVs. This is especially true for countries such as China where domestic cats and poultry are often in close contact.

Newcastle disease virus from domestic mink, China, 2014.

Newcastle disease virus (NDV) is a pathogen that most often infects poultry species. In investigating a 2014 outbreak of encephalitis and death among farmed mink (Mustela vison), we found pathological and later experimental evidence that NDV can infect and cause severe encephalitic and pneumonic disease in these animals. Our findings confirm the host range of NDV.

Lack of exposure of H10N8 avian influenza virus among veterinarians in guangdong province, China

We conducted a retrospective seroepidemiological study for H10N8 avian influenza infection among 400 veterinarians sampled from February 2013 to August 2013 in Guangdong Province, China. None of the veterinarians had evidence of previous infection with the emergent H10N8 AIV. Although there is no evidence of H10N8‐infected veterinarian before the first human index case of H10N8 infection in southern China, a more rigorous and long‐term surveillance remained essential for early warning of novel reassortant viruses and interspecies transmission events. J. Med. Virol. 87:2018–2020, 2015.