Liping Yan

An infectious disease of ducks caused by a newly emerged Tembusu virus strain in mainland China.

During investigations into an outbreak of egg production decline, retarded growth, and even death among ducks in Southeast China, a novel Tembusu virus strain named Tembusu virus Fengxian 2010 (FX2010) was isolated. This virus replicated in embryonated chicken eggs and caused embryo death. In cross-neutralization tests, antiserum to the partial E protein of Tembusu virus Mm1775 strain neutralized FX2010, whereas antiserum to Japanese encephalitis virus did not. FX2010 is an enveloped RNA virus of approximately 45-50 nm in diameter. Sequence analysis of its E and NS5 genes showed that both genes share up to 99.6% nucleotide sequence identity with Baiyangdian virus, and up to 88% nucleotide sequence identity with their counterparts in Tembusu virus. FX2010 was transmitted without mosquito, and caused systemic infection and lesions in experimentally infected ducks. These results indicate that FX2010 and BYD virus are newly emerged Tembusu virus strains that cause an infectious disease in ducks.

Genetic diversity of H9N2 influenza viruses from pigs in China: a potential threat to human health?

Pandemic strains of influenza A virus might arise by genetic reassortment between viruses from different hosts. Pigs are susceptible to both human and avian influenza viruses and have been proposed to be intermediate hosts or mixing vessels, for the generation of pandemic influenza viruses through reassortment or adaptation to the mammalian host. In this study, we summarize and report for the first time the coexistence of 10 (A-J) genotypes in pigs in China by analyzing the eight genes of 28 swine H9N2 viruses isolated in China from 1998 to 2007. Swine H9N2 viruses in genotype A and B were completely derived from Y280-like and Shanghai/F/98-like viruses, respectively, which indicated avian-to-pig interspecies transmission of H9N2 viruses did exist in China. The other eight genotype (C-J) viruses might be double-reassortant viruses, in which six genotype (E-J) viruses possessed 1-4 H5-like gene segments indicating they were reassortants of H9 and H5 viruses. In conclusion, genetic diversity of H9N2 influenza viruses from pigs in China provides further evidence that avian to pig interspecies transmission of H9N2 viruses did occur and might result in the generation of new reassortant viruses by genetic reassortment with swine H1N1, H1N2 and H3N2 influenza viruses, therefore, these swine H9N2 influenza viruses might be a potential threat to human health and continuing to carry out swine influenza virus surveillance in China is of great significance.

Genetic diversity of the ORF5 gene of porcine reproductive and respiratory syndrome virus isolates in China from 2006 to 2008

Since April 2006, swine herds have experienced the outbreaks of a highly pathogenic porcine reproductive and respiratory syndrome (PRRS) in China. To explore the possible mechanism of the emergence of the highly pathogenic PRRS and more fully understand the extent of genetic diversity of PRRSV in China, we analyzed the ORF5 gene sequences of 159 representative PRRSV isolates in 16 provinces from 2006 to 2008. Sequence and phylogenetic analyses showed that all these 159 isolates belonged to the North American genotype and were further divided into six subgenotypes; 140 of 159 isolates were closely related to the highly pathogenic PRRSV with 98.5-100% nucleotide and 98.3-100% amino acid sequence identities and belonged to Subgenotype I; and 3, 8, 4, 3, 1 of 159 isolates were part of Subgenotypes II-VI, respectively. Amino acid analysis of the GP5 protein revealed that all the isolates in Subgenotypes I-III were found to be highly variable in the primary neutralizing epitope; most of the isolates in Subgenotypes I and IV had more glycosylation sites than those in Subgenotypes II, III, V and VI; and 1, 5, and 9 unique amino acid mutations were observed in Subgenotypes I, IV and VI, respectively. In conclusion, our study provides the evidence of coexistence of six different subgenotype isolates in pigs in China from 2006 to 2008, and emphasizes the importance of reinforcing PRRSV surveillance, especially after the emergence of highly pathogenic PRRS in China.

Further evidence for infection of pigs with human-like H1N1 influenza viruses in China.

Classical swine and avian-like H1N1 influenza viruses were reported widely in swine population worldwide, but human-like H1N1 swine viruses were reported occasionally. In 2006, a human-like H1N1 swine virus (A/swine/Guangdong/96/06) was isolated from pigs in Guangdong province, which was reported in China for the first time. To get further evidence for infection of pigs with human-like H1N1 influenza viruses, we analyzed eight gene segments of three human-like swine H1N1 viruses (A/swine/Guangdong/96/06, A/swine/Tianjin/01/04 and A/swine/Henan/01/06) isolated in China. All the eight genes of the three viruses are highly homologous to recent (about 2000) and early (1980s) human H1N1 influenza viruses, respectively. Phylogenetic analyses revealed that A/Swine/Guangdong/96/06 was directly derived from about 2000 human H1N1 influenza viruses, while A/swine/Tianjin/01/04 and A/swine/Henan/01/06 seemed to be descendants of human H1N1 viruses circulating in 1980s. Seroprevalence of our isolate (A/swine/Guangdong/96/06) confirmed the presence of human-like H1N1 virus in pigs in China. Existence of these influenza viruses, especially older viruses (A/swine/Tianjin/01/04 and A/swine/Henan/01/06), indicates that human-like H1N1 influenza viruses may remain invariant for long periods in pigs and provides the evidence that pigs serve as reservoirs of older influenza viruses for human pandemics.

Establishing a TaqMan-Based Real-Time PCR Assay for the rapid detection and quantification of the newly emerged duck tembusu virus

To establish an accurate, rapid, and a quantifiable method for the detection of the newly emerged duck Tembusu virus (DTMUV) that recently caused a widespread infectious disease in ducks in China, we developed a TaqMan-based real-time PCR assay by using E gene-specific primers and a TaqMan probe. This real-time PCR assay was 100 times more sensitive than the conventional PCR. The reproducibility and specificity of the real-time PCR assay were confirmed using plasmids containing E genes or RNAs and DNAs extracted from well-known viruses causing duck diseases. The reliability of this real-time PCR assay was confirmed in 19 of the 24 swab samples, 22 of the 24 tissue samples collected from experimentally infected ducks, as well as 15 of the 21 clinical samples collected from sick ducks since they were verified as DTMUV-positive. The results reveal that the newly established real-time PCR assay might be a useful diagnostic method for epidemiologically investigating and closely observing the newly emerged DTMUV.

Identification of immunodominant T-cell epitopes in membrane protein of highly pathogenic porcine reproductive and respiratory syndrome virus.

The development of cell-mediated immunity has been known extremely important in clearing porcine reproductive and respiratory syndrome virus (PRRSV) in infected pigs. However, the PRRS immunology regarding the interaction of T-cells and PRRSV proteins is poorly understood. To identify the T-cell immunodominant epitopes on the membrane (M) protein of PRRSV, a series of 31 overlapping pentadecapeptides covering the entire M protein were designed and synthesized. These peptides were screened by ELIspot analysis for their capabilities to elicit interferon-gamma (IFN-γ) responses in the peripheral blood mononuclear cells (PBMCs), which were collected from pigs immunized with attenuated PRRSV HuN4-F112 strain and challenged with highly pathogenic HuN4 strain. After three rounds of screening, 4 peptides (M3, M6, M8 and M12) were shown to elicit high expression of IFN-γ. The stimulation of high IFN-γ transcription in PBMCs by these 4 peptides was further confirmed in real-time PCR. The sequence alignment revealed that the epitope represented by peptide M6 was fully conserved in all of examined 42 North American genotype II PRRSV isolates and the epitopes represented by peptides M3, M8 and M12 showed 2-4 amino acid replacements. The finding of 4 T-cell immunodominant epitopes in the M protein of PRRSV will be beneficial to the understanding of the development of cell-mediated immunity.

Development of a live attenuated vaccine candidate against duck Tembusu viral disease

Duck Tembusu virus (DTMUV) is a newly emerging pathogenic flavivirus that is causing massive economic loss in the Chinese duck industry. To obtain a live vaccine candidate against the disease, the DTMUV isolate FX2010 was passaged serially in chicken embryo fibroblasts (CEFs). Characterization of FX2010-180P revealed that it was unable to replicate efficiently in chicken embryonated eggs, nor intranasally infect mice or shelducks at high doses of 5.5log10 tissue culture infectious doses (TCID50). FX2010-180P did not induce clinical symptoms, or pathological lesions in ducks at a dose of 5.5log10TCID50. The attenuation of FX2010-180P was due to 19 amino acid changes and 15 synonymous mutations. Importantly, FX2010-180P elicited good immune responses in ducks inoculated at low doses (3.5log10TCID50) and provided complete protection against challenge with a virulent strain. These results indicate that FX2010-180P is a promising candidate live vaccine for prevention of duck Tembusu viral disease.

Generation of an infectious clone of HuN4-F112, an attenuated live vaccine strain of porcine reproductive and respiratory syndrome virus

BackgroundNowadays, PRRS has become one of the most economically important infectious diseases of pig worldwide. To better characterize and understand the molecular basis of PRRSV virulence determinants, it would be important to develop the infectious cDNA clones. In this regard, HuN4-F112, a live-attenuated North-American-type PRRSV vaccine strain, could serve as an excellent model.ResultsIn the study, genomic sequence of HuN4-F112, an attenuated vaccine virus derived from the highly pathogenic porcine reproductive and respiratory syndrome virus (PRRSV) HuN4 strain, was determined and its full-length cDNA was cloned. Capped RNA was transcribed in vitro from the cDNA clone and transfected into BHK-21 cells. The supernatant from transfected monolayers were serially passaged in Marc-145 cells. The rescued virus exhibited a similar growth pattern to its parental virus in Marc-145 cells with peak titers at 48 h post-infection.ConclusionIn conclusion, we rescued virus from an infectious cDNA clone of attenuated vaccine. It is possible in the future that a new attenuated PRRSV vaccine with broader specificity and good immunogenicity can be designed in vitro via an infectious cDNA clone platform coupled with validated information on virulence determinants.

Effective inhibition of Japanese encephalitis virus replication by small interfering RNAs targeting the NS5 gene.

Japanese encephalitis virus (JEV), a mosquito-borne flavivirus, causes an acute infection of the central nervous system resulting in encephalitis of humans and many kinds of animals. NS5, the largest and most conserved flavivirus protein, is homologous to methyltransferase and RNA-dependent RNA polymerase. RNA interference is an effective anti-viral strategy to inhibit viral replication in vitro. In this study, four short hairpin RNA (shRNA) expression vectors (pS4.1-NS5-201, pS4.1-NS5-455, pS4.1-NS5-699, and pS4.1-NS5-804) targeting the NS5 gene of JEV were employed to target and destroy JEV transcripts. The four shRNAs expression plasmids were individually co-transfected into 293T cells with the plasmid pNS5-EGFP expressing NS5 fused to enhanced green fluorescent protein. The expression level of NS5 was evaluated by fluorescence microscopy, flow cytometry, real time RT-PCR, and Western blot. The four shRNA expression plasmids were also transfected into BHK-21 cells to examine their inhibition of viral replication by indirect immunofluorescence, real time RT-PCR, and Western blot. The results provided strong evidence that shRNAs targeting the NS5 gene could specifically and efficiently inhibit JEV replication. Three out of four plasmids were highly efficient at inhibiting viral replication, including pS4.1-NS5-455, pS4.1-NS5-699, and pS4.1-NS5-804. This was especially true for pS4.1-NS5-699, which reduced the levels of virus RNA and protein the most. Our data suggest that shRNAs could be used as a tool to inhibit JEV replication in vivo.

Comparison of real-time reverse transcription loop-mediated isothermal amplification and real-time reverse transcription polymerase chain reaction for duck Tembusu virus.

Duck Tembusu virus (DTMUV) has caused huge losses to the poultry industry in China since the spring of 2010. The development of a rapid, convenient, and reliable method to diagnose this emerging duck infectious disease is critical. In the present study, a real-time reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay was compared with the real-time reverse transcription polymerase chain reaction (RT-PCR) for detection of DTMUV. The sensitivity of real-time RT-LAMP was equal to that of the real-time RT-PCR, with a detection limit of 0.01 ELD(50) (50% egg lethal dose). The specificity of the real-time RT-LAMP and real-time RT-PCR was confirmed using RNAs and DNAs extracted from related viruses which cause duck infections. The reproducibility of the real-time RT-PCR assay was better than that of the real-time RT-LAMP. Only three results from 96 tissue samples differed between the real-time RT-LAMP and this real-time RT-PCR, confirming the reliability of these methods. This study indicated that the real-time RT-LAMP is simpler, less time-consuming, and more convenient than the real-time RT-PCR. With its high sensitivity, specificity, and convenience, the real-time RT-LAMP is a practical molecular diagnostic method for rapid and quantitative detection of DTMUV infection in a resource-limited setting.