Highly Pathogenic Avian Influenza A(H5N8) Virus in Swans, China, 2020.

In October 2020, highly pathogenic avian influenza A(H5N8) viruses were detected in 2 dead swans in Inner Mongolia, China. Genetic analysis showed that the H5N8 isolates belong to clade 2.3.4.4b and that the isolates cluster with the H5N8 viruses isolated in Eurasia in the fall of 2020.

New H6 influenza virus reassortment strains isolated from Anser fabalis in Anhui Province, China.

BACKGROUND: H6 subtype avian influenza viruses are globally distributed and, in recent years, have been isolated with increasing frequency from both domestic and wild bird species as well as infected humans. Many reports have examined the viruses in the context of poultry or several wild bird species, but there is less information regarding their presence in migratory birds. METHODS: Hemagglutination and hemagglutination inhibition tests were used to measure HA activity for different HA subtypes. Whole viral genomes were sequenced and analysed using DNAstar and MEGA 6 to understand their genetic evolution. Pathogenicity was evaluated using a mouse infection model. RESULTS: We isolated 13 strains of H6 virus from faecal samples of migratory waterfowl in Anhui Province of China in 2014. Phylogenetic analysis showed gene reassortment between Eurasian and North American lineages. Five of the identified H6 strains had the ability to infect mice without adaptation. CONCLUSION: Our findings suggest that regular surveillance of wild birds, especially migratory birds, is important for providing early warning and control of avian influenza outbreaks.

Construction and characterization of a recombinant reticuloendotheliosis virus expressing enhanced green fluorescent protein.

Reticuloendotheliosis virus (REV) causes an immunosuppressive and oncogenic disease in chickens and other birds. In this study, based on an infectious clone of REV, named HLJR0901, a recombinant virus containing the enhanced green fluorescence protein (EGFP) gene was constructed by inserting the EGFP expression cassette downstream of the 3' terminus of the viral env gene. An EGFP-tagged REV that stably expresses EGFP was rescued. This visible recombinant REV could contribute to the further understanding of the molecular mechanism involved in the replication and pathogenicity of REV.

Experimental infection of dogs with H6N1 avian influenza A virus.

H6N1 avian influenza A viruses, which have spread across North America, Europe and Asia, have been shown to be infectious not only for birds but also for mammals. Because humans lack immunity to H6N1 avian influenza A viruses, the emergence of these viruses in humans would probably cause a pandemic. Replication of H6N1 avian influenza A viruses in dogs may facilitate their adaptation in humans because dogs are often in close contact with humans. However, the susceptibility of dogs to these viruses is unknown. To address this question, we infected beagles intranasally (i.n.) with an H6N1 avian influenza A virus that was isolated from a mallard. Inoculation of this virus into beagles resulted in the virus being detectable in the lung and seroconversion with no clinical signs except for a fever at 1 day post-inoculation (dpi). In addition, the virus was transiently shed from the nose and in the feces of the infected beagles. Our results suggest that dogs can be subclinically infected with H6N1 avian influenza A viruses, which, like H7N9, have low pathogenicity in birds and may serve as an intermediate host to transfer this virus to humans. Certain actions may be taken to prevent the potential transmission of these viruses, including the development of H6N1 avian influenza vaccines for prevention.

Genetic diversity and phylogenetic analysis of glycoprotein gp85 of avian leukosis virus subgroup J wild-bird isolates from Northeast China.

Avian leukosis virus subgroup J (ALV-J), first isolated in 1989, preferentially infects meat-type birds. Chinese layer flocks have experienced outbreaks of this virus since 2008. To analyze the status of ALV-J infection in wild birds in China, 585 wild birds collected from three provinces of Northeast China from 2010 to 2012 were tested, and six ALV-J strains were isolated for the first time. Furthermore, the gp85 genes of the six strains were amplified, cloned, and sequenced. The results indicated that two different ALV-J strains coexisted in Chinese wild birds from 2010 to 2012. These results not only expand the epidemiological data available for ALV-J and provide necessary information for the further understanding of the evolution of ALV-J, but they also highlight the potential role of wild-bird migration in the spread of ALV-J.

First isolation of reticuloendotheliosis virus from mallards in China.

Reticuloendotheliosis virus (REV) causes an oncogenic, immunosuppressive and runting syndrome in many avian hosts worldwide. REV infection has never been reported in mallard ducks, however. To identify REV infection in mallards, we collected 40 mallard duck samples from Jilin Province of China. In this study, the REV strain, DBYR1102, was first isolated from a mallard in China and identified by PCR, indirect immunofluorescence assay and electron microscopy. The gp90 gene and complete LTR of DBYR1102 were amplified and sequenced. Phylogenetic analysis based on gp90 genes of REV indicated that the REV strain DBYR1102 is closely related to strain HLJR0901 from northeastern China, the prairie chicken isolate APC-566, and REV subtype III, represented by chick syncytial virus. This new strain is distantly related to two other subtypes of REV, 170A and SNV. Phylogenetic analysis based on the LTR yielded information similar to that obtained with the gp90 genes. The results of this study not only expand our epidemiological understanding of REV in the wild birds of China but also demonstrate the potential role of wild waterfowl in REV transmission.

Complete nucleotide sequence of avian paramyxovirus type 6 strain JL isolated from mallard ducks in China.

A new strain of avian paramyxovirus type 6 (APMV-6), JL, has been isolated from mallard ducks in China, and its complete genome has been sequenced and analyzed. This work is the first announced complete genome sequence of APMV-6 from mallards.

Characterization of integron-mediated antimicrobial resistance among Escherichia coli strains isolated from a captive population of Amur tigers in China.

The present study was undertaken to identify and characterize integrons and integrated resistance gene cassettes among multidrug resistant Escherichia coli isolates from a captive population of Amur tigers (Panthera tigris altaica) in China. In addition, the prevalence of antimicrobial resistance and class I integrons was assessed in E. coli strains (n = 61) isolated from a captive population of Amur tigers in Heilongjiang Amur Tiger Park, China. Among the isolates, 52.46% (32 of 61) were positive for intI1, but no isolates carried intI2 or intI3. Most isolates were susceptible to amoxicillin/clavulanic acid, aztreonam, and polymyxin B, while they also exhibited high incidence rates of resistance to ampicillin, doxycycline, chloramphenicol, tetracycline, and dihydrofolate reductase. Sequencing analysis revealed three gene cassettes, which encoded resistance to dihydrofolate reductase (dfrA15), dihydrofolate reductase (dfrA12), and adenyltransferase (aadA2). The gene cassette arrays dfrA15 (31%) and dfrA12-aadA2 (19%) were most prevalent among these isolates.

[Identification and production of monoclonal antibody of Siberian tiger's immunoglobulin].

OBJECTIVE: To purify immunoglobulin (Ig) of Siberian Tiger and prepare monoclonal antibody (mAb) against the Ig,which can be used to develop immunological diagnostic kits for diagnosing infectious disease in Siberian Tiger. METHODS: The Ig of Siberian tigers was purified with saturated ammonium sulfate combined with recombinant Protein G. The C57BL/6 mice were immunized with the purified Ig. Spleno-cytes of the mice immunized were collected and fused with the mouse myeloma cell line (Sp2/0-Ag14). The positive hybridoma clones were selected by ELISA and were identified by western blot. The sandwich ELISA was used to detect immunocompetence of the purified Ig and the mAb. RESULTS: We obtained three mouse hybridoma clones that produced mAbs against Ig of Siberian Tiger. The derived McAbs could recognize Ig heavy chain of Siberian Tiger specifically. The biological activity of the Ig and obtained McAbs also could be identified by detecting the antibody induced by panleukopenia virus (FPV-HLJ) vaccine in Siberian Tiger. The antibody also would be useful for assess the vaccine efficacy against the infectious disease on the Siberian Tiger. CONCLUSION: Protein G can be used in Ig purification of Siberian Tiger. The obtained McAbs from the hybridoma ADT11 in this study owned strong ability to bind Ig of Siberian Tiger and have a stable immunocompetence. They can be used to develop diagnostic methods for detecting infectious disease in Siberian Tiger and vaccine research.

Novel Reassortant Avian Influenza A(H9N2) Virus Isolate in Migratory Waterfowl in Hubei Province, China.

In December 2017, an influenza A(H9N2) virus (B51) was isolated from migratory waterfowl in Hubei Province, China. Phylogenetic analysis demonstrated that B51 is a novel reassortant influenza virus containing segments from human H7N4 virus and North American wild bird influenza viruses. This suggest that B51 has undergone multiple reassortment events.

Novel H5N6 avian influenza virus reassortants with European H5N8 isolated in migratory birds, China.

Five novel H5N6 influenza viruses, including four highly pathogenic avian influenza viruses and one low pathogenic avian influenza virus, were isolated from migratory birds in Ningxia, China, in November 2017. To understand the genetic origination of the novel H5N6 virus, and the infectivity and pathogenicity of the four highly pathogenic avian influenza viruses in mammals, phylogeographic analyses and infection studies in mice were performed. The phylogenetic and phylogeographic analyses showed that the H5N6 isolates, which are closely related to the viruses from Korea, Japan and the Netherlands, originated from reassortant virus between H5N8 and HxN6 viruses from western Russia. The animal study revealed that the SBD-87 isolate presented moderate virulence in mice, suggesting a potential public risk to humans and a potential threat to public health.

[Preparation of the vaccine with inactivated Feline Panleukopenia Virus isolated from tiger and the preliminary application].

OBJECTIVE: To prepare a vaccine with inactivated Feline Panleukopenia Virus (FPV) isolated from Siberian tigers and to evaluate its immunological effect. METHODS: FPV-HLJ, an FPV strain previously isolated from Siberian tiger in our lab was used to inoculate cat kidney cell line F81 with dose 1/10(v/v) using the synchronizing inoculation method. Inoculated F81 cell line was cultured at 37 degrees C and collected when cytopathic effect was up to 75%. Viral suspension was inactivated by using formaldehyde for 24 h and inactive vaccine preapred by adding aluminium hydroxide gel as adjuvant to the suspension. The inactive vaccine was applied to 2-month-old kitten tigers of hypodermically after protection effect was proved in 2-month-old nonimmunized cats by using the same vaccination procedure. RESULTS: Antibody level in vaccinated cats revealed an increasing trend that the valence of antibody reached 1:1024-2048 after three times of vaccination. All vaccinated cats survived challenges of virulent FPV virus. The valence of antibody reached 1:1024 in most vaccinated tigers 15 days after the third vaccination. CONCLUSION: The results indicated that the inactivated vaccine can produce immunoprotection for tigers.

Genetic characterization of a hantavirus isolated from Heilongjiang province, China.

Hantavirus is the causative agent of hemorrhagic fever with renal syndrome (HFRS). Heilongjiang Province is experiencing an epidemic of HFRS, the main causative agent is a variant of hantavirus called Seoul virus (SEOV). In this study, the entire genome of one SEOV, the DN2 strain, was sequenced and analyzed. The alignment analysis of the sequences indicated that the DN2 strain shares the highest homology with the SEOV-LYO852 strain. The nucleotide identity is 97.6% for the S segment, 97.7% for the M segment, and 98.0% for the L segment. The corresponding amino acid sequence homologies are 99.1%, 98.9% and 99.8%. The phylogenetic analysis of the segments suggests that the DN2 strain has a high genetic relationship with SEOV strains and no genetic recombination occurs.

L'Hantavirus est l'agent causal de la fièvre hémorragique avec syndrome rénal (FHSR). La province d'Heilongjiang est au prise avec une épidémie de FHSR, l'agent causal principal est un variant de l'Hantavirus dénommé virus Séoul (SEOV). Dans la présente étude, les séquences complètes d'un SEOV, la souche DN2, ont été séquencées et analysées. L'analyse d'appariement des séquences a démontré que la souche DN2 partage la plus forte homologie avec la souche SEOV-LYO852. L'identité de nucléotides est de 97,6 % pour le segment S, 97,7 % pour le segment M, et de 98,0 % pour le segment L. L'homologie des séquences d'acides aminés correspondants est de 99,1 %, 98,9 %, et 99,8 %. L'analyse phylogénétique des segments suggéraient que la souche DN2 avait une parenté génétique la plus élevée avec les souches de SEOV et qu'aucune recombinaison génétique ne s'est produite.(Traduit par Docteur Serge Messier).

Detection of reassortant avian influenza A (H11N9) virus in wild birds in China.

Human infectious avian influenza virus (AIV) H7N9 emerged in China in 2013. The N9 gene of H7N9, which has the ability to cause death in humans, originated from an H11N9 influenza strain circulating in wild birds. To investigate the frequency and distribution of the N9 gene of the H11N9 and H7N9 influenza virus circulating in wild birds between 2006 and 2015, 35,604 samples were collected and tested. No H7N9 but four strains of the H11N9 subtype AIV were isolated, and phylogenetic analyses showed that the four H11N9 viruses were intra-subtype and inter-subtype reassortant viruses. A sequence analysis revealed that all six internal genes of A/wild bird/Anhui/L306/2014 (H11N9) originated from an H9N2 AIV isolated in Korea. The H9N2 strain, which is an inner gene donor reassorted with other subtypes, is a potential threat to poultry and even humans. It is necessary to increase monitoring of the emergence and spread of H11N9 AIV in wild birds.

[Monitoring influenza A virus and Newcastle disease virus in migratory waterfowls in Sanjiang natural reserve of Heilongjiang Province].

OBJECTIVE: In order to monitor the present situation of Avian influenza virus (AIV) and Newcastle disease virus (NDV) in migratory waterfowls effectively, 158 tracheal and cloacal swab samples for wild birds were collected from Sanjiang natural reserve during migratory seasons in October 2005, April 2006 and October 2006. METHODS: Serial passages in specific pathogen free embryonated chicken eggs, haemagglutination activity (HA) text, hemagglutination inhibition (HI) text and RT-PCR detection were used to isolate and identify AIV and NDV. RESULTS: Twenty AIV isolates and 13 NDV isolates were collected in the test. Twenty AIV isolates were all from aquatic birds in October 2006, and among these isolates, 12 AIV subtypes were identified definitely, 11 subtypes were found in mallards-H2N2 (2/20), H2N6 (2/20), H3N4 (1/20), H3N6 (2/20), H3N7 (2/20), H3N8 (2/20), H6N2 (2/20), H11N2 (1/20), H11N3 (1/20), H11N5 (2/20), H11N6 (1/20), and 1 subtype was found in garganey-H5N2 (1/20). Thirteen NDV isolates were collected in all three migratory seasons from 5 different species of waterfowls, including mallard (8/13), bean goose (1/13), white-fronted goose (1/13), common teal (1/13) and mandarin duck (2/13). CONCLUSION: The results indicated that mallard, which possesses huge population size and world wide distribution, could be considered one of the most important natural carrier of AIV and NDV and may have more important ecological significance on viruses transmission than other species of wild birds.

A 627K variant in the PB2 protein of H9 subtype influenza virus in wild birds.

BACKGROUND: Wild birds are gaining increasing attention as gene-mixing reservoirs for influenza viruses. To investigate the molecular properties of the viruses isolated and epidemiological analysis of H9N2 subtype AIV in wild birds, we studied samples obtained over two years (2014-2015) from wetlands in Anhui province, China. METHODS: A total of 4534 samples were collected from migratory waterfowl in Anhui in 2014-2015, and 8 strains of H9 subtype AIV were isolated. RESULTS: Phylogenetic analysis showed different degrees of gene segment reassortment in H9 viruses between the Eurasian lineage and the North American lineage. Most importantly, two viruses harbored the E627K mutation in the polymerase PB2 (PB2) protein. This is the first report of the mutation of this virus from low pathogenicity to high pathogenicity in wild birds. CONCLUSIONS: The continued surveillance of wild birds, especially migratory birds, is important to provide early warning and control of AIV outbreaks. Our results highlight the high genetic diversity of AIV along the Eurasian-Australian migration flyway and the need for more extensive AIV surveillance in eastern China.

[Prokaryotic expression and detective application of the main antigenic region of VP2 protein of Aleutian mink disease parvovirus].

To research safer diagnosis antigen for ADV, the main antigenic region VP2a and VP2b gene of ADV were obtained by restriction digestion of the recombinant plasmids pMD-VP2a and pMD-VP2b. Then the genes were respectively cloned into pMAL-c2 to get two prokaryotic recombinant plasmids pMAL-VPa and pMAL-VPb. The target genes were successfully expressed in the host cell TB1 when induced by IPTG. The Western blot analysis proved the recombinant proteins have good antigenic. The recombinant proteins were purified by KCL dyeing method, and were used as antigen to establish VP2-CIEP for AD diagnoses. The detection result shared 94.3% identity with that of CIEP. The results reported here show that VP2-CIEP is highly sensitive and specific and can benefit the research on the serodiagnosis to AD.

Baylisascaris schroederi Infection in Giant Pandas (Ailuropoda melanoleuca) in Foping National Nature Reserve, China.

The giant panda (Ailuropoda melanoleuca) is the most iconic endangered species in the world, but there is little information about the spatial and temporal distribution of parasites in the wild giant panda population. In total, 193 fecal samples from giant pandas in the Foping National Nature Reserve, People's Republic of China, were analyzed for parasite eggs using a modification of the McMaster technique. The morphology and size of Baylisascaris schroederi eggs were observed under an optical microscope. The prevalence and intensity of B. schroederi infection during the sampling year 2012 were 52.3% (101/193) and 89 eggs/g of feces, respectively, among giant pandas in this population. The prevalence of B. schroederi in the pandas varied during different months of the year, from 7% to 100%, and the prevalences in spring, summer, autumn, and winter were 71, 77, 23, and 18%, respectively. The prevalence was not significantly different between giant pandas that ate two different types of bamboo, but the intensity of infection was higher in the group eating Arundinaria fargesii (P=0.043). Altitude, temperature, and dew point were correlated with the infection intensity (r=-0.224, P<0.001; r=0.328, P<0.001; r=0.328, P=0.028, respectively). There was no correlation between infection intensity and distance to rivers. This study provides a better understanding of B. schroederi prevalence among the wild giant pandas in Foping National Nature, China.

Epidemic of wild-origin H1NX avian influenza viruses in Anhui, China.

BACKGROUND: As the natural hosts of avian influenza viruses (AIVs), aquatic and migratory birds provide a gene pool for genetic transfer among species and across species, forming transient "genome constellations." This work describes the phylogenetic dynamics of H1NX based on the complete molecular characterization of eight genes of viruses that were collected from 2014 to 2015 in Anhui Province, China. METHODS: Hemagglutination and hemagglutination inhibition tests were used to determine the hemagglutination (HA) activity of the HA subtypes. The entire genomes of the viruses were sequenced on an ABI PRISM 3500xl DNA Analyzer. The sequences were genetically analysed to study their genetic evolution using DNASTAR and MEGA 6. The pathogenic effects of the viruses were evaluated using mouse infection models. RESULTS: Seven strains of the H1 subtype avian influenza virus were isolated. Phylogenetic analysis indicated natural recombination of the H1 influenza viruses between the Eurasian lineage and the North American lineage. Some genes had high sequence identity with A/bean goose/Korea/220/2011(H9N2), which is a typical case involving viral reassortment between the Eurasian lineage and the North American lineage. The results of infection experiments in mice showed that the viruses could acquire the ability to multiply in mouse respiratory organs without adaptation. CONCLUSIONS: These findings suggest that continued surveillance of wild birds, particularly migratory birds, is important to provide early warning of possible H1 influenza epidemics and to understand the ecology of the virus.

Amino Acid Substitutions Associated with Avian H5N6 Influenza A Virus Adaptation to Mice.

At least 15 cases of human beings infected with H5N6 have been reported since 2014, of which at least nine were fatal. The highly pathogenic avian H5N6 influenza virus may pose a serious threat to both public health and the poultry industry. However, the molecular features promoting the adaptation of avian H5N6 influenza viruses to mammalian hosts is not well understood. Here, we sequentially passaged an avian H5N6 influenza A virus (A/Northern Shoveler/Ningxia/488-53/2015) 10 times in mice to identify the adaptive amino acid substitutions that confer enhanced virulence to H5N6 in mammals. The 1st and 10th passages of the mouse-adapted H5N6 viruses were named P1 and P10, respectively. P1 and P10 displayed higher pathogenicity in mice than their parent strain. P10 showed significantly higher replication capability in vivo and could be detected in the brains of mice, whereas P1 displayed higher replication efficiency in their lungs but was not detectable in the brain. Similar to its parent strain, P10 remained no transmissible between guinea pigs. Using genome sequencing and alignment, multiple amino acid substitutions, including PB2 E627K, PB2 T23I, PA T97I, and HA R239H, were found in the adaptation of H5N6 to mice. In summary, we identified amino acid changes that are associated with H5N6 adaptation to mice.