Peirong Jiao

Avian-origin H3N2 canine influenza A viruses in Southern China

This study reports four sporadic cases of H3N2 canine influenza in Southern China, which were identified from sick dogs from May 2006 to October 2007. The evolutionary analysis showed that all eight segments of these four viruses are avian-origin and phylogenetically close to the H3N2 canine influenza viruses reported earlier in South Korea. Systematic surveillance is required to monitor the disease and evolutionary behavior of this virus in canine populations in China.

Detection of expression of influenza virus receptors in tissues of BALB/c mice by histochemistry.

Infection of host cells with the influenza virus is mediated by specific interactions between the viral hemagglutinin and its cell receptor, oligosaccharides containing sialic acid (SA) residues. Avian and human influenza viruses preferentially bind to α-2, 3-linked and α-2, 6-linked sialic acids, respectively. Therefore, differential expression of these receptors may be crucial to influenza virus infection. To date, the distribution of these two receptors has never been investigated in the tissues of BALB/c mice, which is the routine animal model for influenza research. Here, the expression pattern of alpha-2,3 and alpha-2,6 sialic acid-linked receptors in various organs (respiratory tract, gastrointestinal tract, brain, cerebellum, spleen, liver, kidney and heart) of BALB/c mice were determined. Histochemical staining of mouse tissue sections was performed by using biotinylated Maackia amurensis lectin II (MAAII), and Sambucus nigra agglutinin (SNA) were performed to detect the alpha-2,3 and alpha-2,6 sialic acid-linked receptors, respectively. The results showed that the alpha-2,3 and alpha-2,6 sialic acid-linked receptors were both expressed on trachea, lung, cerebellum, spleen, liver and kidney. Only the epithelial cells of cecum, rectum and blood vessels in the heart express the alpha-2,6 sialic acid-linked receptors. The distribution patterns of the two receptors may explain why this model animal can be infected by the AIV and HuIV and the pathological changes when infection occurred. These data can account for the multiple organ involvement observed in influenza infection and should assist investigators in interpreting results obtained when analyzing AIV or HuIV in the mouse model of disease.

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.

Molecular Basis of Efficient Replication and Pathogenicity of H9N2 Avian Influenza Viruses in Mice

H9N2 subtype avian influenza viruses (AIVs) have shown expanded host range and can infect mammals, such as humans and swine. To date the mechanisms of mammalian adaptation and interspecies transmission of H9N2 AIVs remain poorly understood. To explore the molecular basis determining mammalian adaptation of H9N2 AIVs, we compared two avian field H9N2 isolates in a mouse model: one (A/chicken/Guangdong/TS/2004, TS) is nonpathogenic, another one (A/chicken/Guangdong/V/2008, V) is lethal with efficient replication in mouse brains. In order to determine the basis of the differences in pathogenicity and brain tropism between these two viruses, recombinants with a single gene from the TS (or V) virus in the background of the V (or TS) virus were generated using reverse genetics and evaluated in a mouse model. The results showed that the PB2 gene is the major factor determining the virulence in the mouse model although other genes also have variable impacts on virus replication and pathogenicity. Further studies using PB2 chimeric viruses and mutated viruses with a single amino acid substitution at position 627 [glutamic acid (E) to lysine, (K)] in PB2 revealed that PB2 627K is critical for pathogenicity and viral replication of H9N2 viruses in mouse brains. All together, these results indicate that the PB2 gene and especially position 627 determine virus replication and pathogenicity in mice. This study provides insights into the molecular basis of mammalian adaptation and interspecies transmission of H9N2 AIVs.

Duck MDA5 functions in innate immunity against H5N1 highly pathogenic avian influenza virus infections.

Melanoma differentiation-associated gene 5 (MDA5) is an important intracellular receptor that recognizes long molecules of viral double-stranded RNA in innate immunity. To understand the mechanism of duck MDA5-mediated innate immunity, we cloned the MDA5 cDNA from the Muscovy duck (Cairina moschata). Quantitative real-time PCR analysis indicates that duck MDA5 mRNA was constitutively expressed in all sampled tissues. A significant increase of MDA5 mRNA was detected in the brain, spleen and lungs of ducks after infection with an H5N1 highly pathogenic avian influenza virus (HPAIV). We investigated the role of the predicted functional domains of MDA5. The results indicate the caspase activation and recruitment domain (CARD) of duck MDA5 had a signal transmission function through IRF-7-dependent signaling pathway. Overexpression of the CARD strongly activated the chicken IFN-β promoter and upregulated the mRNA expression of antiviral molecules (such as OAS, PKR and Mx), proinflammatory cytokines (such as IL-2, IL-6, IFN-α and IFN-γ, but not IL-1β and IL-8) and retinoic acid-inducible gene I (RIG-I)-like receptors (RLR) (RIG-I and LGP2) without exogenous stimulation. We also demonstrate the NS1 of the H5N1 HPAIV inhibited the duck MDA5-mediated signaling pathway in vitro. These results suggest that duck MDA5 is an important receptor for inducing antiviral activity in the host immune response of ducks.

A Single E627K Mutation in the PB2 Protein of H9N2 Avian Influenza Virus Increases Virulence by Inducing Higher Glucocorticoids (GCs) Level

While repeated infection of humans and enhanced replication and transmission in mice has attracted more attention to it, the pathogenesis of H9N2 virus was less known in mice. PB2 residue 627 as the virulent determinant of H5N1 virus is associated with systemic infection and impaired TCR activation, but the impact of this position in H9N2 virus on the host immune response has not been evaluated. In this study, we quantified the cellular immune response to infection in the mouse lung and demonstrate that VK627 and rTsE627K infection caused a significant reduction in the numbers of T cells and inflammatory cells (Macrophage, Neutrophils, Dendritic cells) compared to mice infected with rVK627E and TsE627. Further, we discovered (i) a high level of thymocyte apoptosis resulted in impaired T cell development, which led to the reduced amount of mature T cells into lung, and (ii) the reduced inflammatory cells entering into lung was attributed to the diminished levels in pro-inflammatory cytokines and chemokines. Thereafter, we recognized that higher GCs level in plasma induced by VK627 and rTsE627K infection was associated with the increased apoptosis in thymus and the reduced pro-inflammatory cytokines and chemokines levels in lung. These data demonstrated that VK627 and rTsE627K infection contributing to higher GCs level would decrease the magnitude of antiviral response in lung, which may be offered as a novel mechanism of enhanced pathogenicity for H9N2 AIV.

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.

Development and strategies of cell-culture technology for influenza vaccine

Influenza is a pandemic contagious disease and causes human deaths and huge economic destruction of poultry in the world. In order to control and prevent influenza, mainly type A, influenza vaccine for human and poultry were available since the 1940s and 1920s, respectively. In the development of vaccine production, influenza viruses were cultured originally from chicken embryos to anchorage-dependent cell lines, such as MDCK and Vero. The anchorage-independent lines have also been used to produce influenza virus, such as PER.C6 and engineering modified MDCK and Vero. During the process of influenza vaccine production, the common problem faced by all producers is how to improve the titer of influenza virus. This paper focuses on the developments of cell culture for influenza virus vaccine production, limitations of cell culture, and relative strategies for improvement virus yields in cell-culture systems.

Genetic characterization and evolutionary analysis of 4 Newcastle disease virus isolate full genomes from waterbirds in South China during 2003-2007.

Complete genomes of four Newcastle disease virus (NDV) strains, isolated from ducks and wild birds in Guangdong province of China from 2003 to 2007, were sequenced and analyzed in this study. Pathogenicity tests in chicken embryos and chickens illustrate that D3 and R8 are lentogenic, and W4 and P4 are mesogenic strains. Phylogenetic analysis using all six genes provides a high resolution profile for genotype designation as genotype I for D3 and R8 strains and genotype VI for W4 and P4 strains. In addition, molecular dating based on different genes suggests that D3 and R8 diverged from their common ancestor at around 1998; W4 and P4 diverged from their common ancestor at around 1999. Subsequent selective pressure analysis displayed specific traits of genes evolution in all 4 strains since their divergence from the recent common ancestor. Furthermore, the geographic origins of 4 strains were deduced to be from Europe via two independent introduction events by phylogeographical analysis. This provides insights to the potential influence of waterfowl migration on NDV epidemiology.