Kaijian Luo

Development of a Loop-Mediated Isothermal Amplification Assay for Rapid Detection of Subgroup J Avian Leukosis Virus

ABSTRACT Infection of breeder flocks in China with subgroup J avian leukosis virus (ALV-J) has increased recently. In this study, we have developed a loop-mediated isothermal amplification (LAMP) assay for rapid detection of ALV-J from culture isolates and clinical samples. The ALV-J-specific LAMP assay efficiently amplified the target gene within 45 min at 63°C using only a simple laboratory water bath. To determine the specificity of the LAMP assay, various subgroup ALVs and other related viruses were detected. A ladder pattern on gel electrophoresis was observed for ALV-J isolates but not for other viruses. To evaluate the sensitivities of the LAMP assay and conventional PCR, the NX0101 isolate plasmid DNA was amplified by them. The detection limit of the LAMP assay was 5 target gene copies/reaction, which was up to 20 times higher than that of conventional PCR. To evaluate the application of the LAMP assay for detection of ALV-J in clinical samples, 49 samples suspected of ALV infection from breeder flocks were tested by the LAMP assay and PCR. Moreover, virus isolation from these samples was also performed using cell culture. The positive-sample ratios were 21/49 (43%) by conventional PCR, 26/49 (53%) by the LAMP assay, and 19/46 (41%) by virus isolation. Additionally, a positive LAMP reaction can be visually ascertained by the observation of turbidity or a color change after addition of SYBR green I dye. Consequently, the LAMP assay is a simple, rapid, and sensitive diagnostic method and can potentially be developed for rapid detection of ALV-J infection in the field.

Genetic characterization of H5N1 avian influenza viruses isolated in southern China during the 2003–04 avian influenza outbreaks

Summary.The recent H5N1 avian influenza outbreaks in Asia spread over more than 8 countries. It has caused enormous economic loss and grand challenges for the public health. During these breakouts we isolated three strains of H5N1 Avian Influenza Virus (AIV) from chickens and one from duck in different farms of Southern China. We completely sequenced these four AIVs. Molecular characterization demonstrated that these strains retain the reported H5N1 AIV sequence properties relevant to virus virulence and host adaptation. Phylogeny results demonstrated that three of these isolates (except A/Chicken/Guangdong/174/04) were closely linked to other H5N1 AIVs isolated from the recent H5N1 outbreaks in Asia. Six of 8 segments (except PA and M) of A/Chicken/Guangdong/174/04 also shares a close linkage to other H5N1 AIVs isolated from the recent H5N1 outbreaks. However, the PA gene of A/Chicken/Guangdong/174/04 and another H5N1 strain forms a distinct subgroup along with an H6N1 AIV, and the M gene of A/Chicken/Guangdong/174/04 shows a close linkage to some H5N1 AIVs from aquatic species in China. Our findings suggest that a new genotype of AIV (in addition to previous reported ones) was present during the 2003–04 Asian bird flu outbreaks and that continuing virus surveillance of AIVs be conducted to monitor the evolutionary paths of the A/Chicken/Guangdong/174/04-like AIVs.

Pathogenicity in quails and mice of H5N1 highly pathogenic avian influenza viruses isolated from ducks.

In our study, the pathogenicity of H5N1 influenza A viruses circulating in waterfowls in Southern China was investigated. Three H5N1 highly pathogenic avian influenza (HPAI) viruses isolated from ducks, A/Duck/Guangdong/383/2008(DK383), A/Duck/Guangdong/378/2008(DK378) and A/Duck/Guangdong/212/2004(DK212) were inoculated at 10(6) fifty-percent egg infectious doses (EID(50)) into ducks, quails and mice and showed varying levels of pathogenicity. In ducks, the mortality rates ranged from 0 to 60% and the mean death time (MDT) was 0-6.7 days post-inoculation (DPI). While the viruses were highly pathogenic in quails, resulting in 83.3-100% mortality and the MDT of 2.3-3 DPI, they were completely lethal in mice (100% mortality). The viruses replicated in many organs of ducks and quails and were found in the brain, and kidney, lung and spleen of the mice. Phylogenetic analysis revealed that DK383 and DK378 viruses of clade 2.3.2 belonged to genotype 11, while DK212 virus of clade 9 was genotype 3. Our study illustrated H5N1 influenza viruses within Clade 2.3.2 and 9 from duck in Southern China had very highly pathogenicity to Japanese quails and BALB/c mice, but viruses within Clade 2.3.2 had more highly lethality than those of clade 9 to Muscovy ducks. Therefore, they had posed a continued challenge for disease control and public health.

Complete Genome Sequence of an H10N8 Avian Influenza Virus Isolated from a Live Bird Market in Southern China

ABSTRACT An H10N8 avian influenza virus (AIV), designated A/Duck/Guangdong/E1/2012 (H10N8), was isolated from a duck in January 2012. This is first report that this subtype of AIV was isolated from a live bird market (LBM) in Guangdong Province in southern China. Furthermore, the complete genome of this strain was analyzed. The availability of genome sequences is helpful to further investigations of epidemiology and molecular characteristics of AIV in southern China.

Complete Genome Sequence of an H5N2 Avian Influenza Virus Isolated from a Parrot in Southern China

ABSTRACT We report the complete genome sequence of an H5N2 avian influenza virus (AIV) that was first isolated from a parrot in Guangdong in southern China in 2004. Genomic sequence and phylogenetic analyses showed that it was highly homologous with the North American H5N2 viruses and all eight genes of this virus belonged to the North American gene lineage. These data will help in the investigation of the epidemiology and host range of AIVs in southern China.

An ALV-J isolate is responsible for spontaneous haemangiomas in layer chickens in China

Subgroup J avian leukosis virus (ALV-J), first isolated in 1989, mainly induces tumours of myeloid leukosis (ML) in meat-type chickens. In 2006, ALV-J strain SCAU-HN06 was isolated from commercial layer hens with spontaneous haemangiomas in China. To confirm its role in the induction of haemangioma, we constructed a full-length copy of the proviral genome from SCAU-HN06, recovered virus from DF-1 cells detected by enzyme-linked immunosorbent assay, characterized its growth property and investigated its pathogenicity. The recovered virus appeared to be identical to SCAU-HN06 analysed by both blast gene sequences and indirect immunofluorescence assay. It also showed similarities in growth to the parental wild-type virus in vitro. The pathogenicity of the rescued and parental virus in specific-pathogen-free White Leghorn chickens was investigated. Both SCAU-HN06 and rSCAU-HN06 could induce haemangioma, with incidence of 52% and 42.8% respectively. Overall, our findings indicated that the ALV-J strain SCAU-HN06 was the causal agent inducing haemangiomas rather than ML in certain layer chickens.

D701N mutation in the PB2 protein contributes to the pathogenicity of H5N1 avian influenza viruses but not transmissibility in guinea pigs.

H5N1 highly pathogenic avian influenza virus (HPAIV) of clade 2.3.2 has been circulating in waterfowl in Southern China since 2003. Our previous studies showed that certain H5N1 HPAIV isolates within clade 2.3.2 from Southern China had high pathogenicity in different birds. Guinea pigs have been successfully used as models to evaluate the transmissibility of AIVs and other species of influenza viruses in mammalian hosts. However, few studies have reported pathogenicity and transmissibility of H5N1 HPAIVs of this clade in guinea pigs. In this study, we selected an H5N1 HPAIV isolate, A/duck/Guangdong/357/2008, to investigate the pathogenicity and transmissibility of the virus in guinea pigs. The virus had high pathogenicity in mice; additionally, it only replicated in some tissues of the guinea pigs without production of clinical signs, but was transmissible among guinea pigs. Interestingly, virus isolates from co-caged guinea pigs had the D701N mutation in the PB2 protein. These mutant viruses showed higher pathogenicity in mice and higher replication capability in guinea pigs but did not demonstrate enhanced the transmissibility among guinea pigs. These findings indicate the transmission of the H5N1 virus between mammals could induce virus mutations, and the mutant viruses might have higher pathogenicity in mammals without higher transmissibility. Therefore, the continued evaluation of the pathogenicity and transmissibility of avian influenza virus (AIVs) in mammals is critical to the understanding of the evolutionary characteristics of AIVs and the emergence of potential pandemic strains.

Complete Genomic Sequence of an H5N1 Influenza Virus from a Parrot in Southern China

ABSTRACT An H5N1 avian influenza virus (AIV) designated A/Parrot/Guangdong/C99/2005 (H5N1) was first isolated from a sick parrot in Guangdong in southern China in 2005. The complete genome of this strain was analyzed. Genome sequence analysis showed that all 8 gene segments of the virus nucleotide had 99.0% homology to A/chicken/Henan/12/2004 (H5N1). Phylogenetic analysis demonstrated that all 8 gene segments of the virus were derived from the Eurasian lineage. The availability of genome sequences is useful to investigate the host range and genetic evolution of the H5N1 avian influenza virus in Southern China.

Full Genome Sequence of a Recombinant H5N1 Influenza Virus from a Condor in Southern China

ABSTRACT In this study, we report the first genomic information on an H5N1 avian influenza virus (AIV) isolated from a condor in Guangdong Province in southern China in 2003. Full genome sequencing and phylogenetic analyses show that it is a recombinant virus containing genome segments derived from the Eurasia and North America gene pools. This will be useful for analyses of the evolution of H5N1 AIV in southern China.

The genetic and phylogenetic analysis of a highly pathogenic influenza A H5N6 virus from a heron, southern China, 2013

The H5N6 highly pathogenic avian influenza viruses (HPAIVs) have circulated within poultry in China since 2013. Infections of H5N6 in wild birds were reported since 2014. In order to investigate the infection history of H5N6 in wild birds, we conducted a retrospective analysis of H5 positive wild bird samples collected in 2013, the year H5N6 was discovered in poultry. We isolated a new HPAI H5N6 virus from a dead heron collected in 2013. The virus had high identity in all eight gene sequences to those collected from poultry in 2013 (for example, A/chicken/Shenzhen/1845/2013, 99.1%-99.7%). Our findings revealed that H5N6 HPAIVs infected wild birds in southern China since the emergence of H5N6 in poultry in 2013. The co-circulation of H5N6 between wild birds and poultry is very close, and should raise our attention more.