| Porcine reproductive and respiratory syndrome (PRRS) is characterized by late-term abortions and delivery of increase number of stillbirths or premature and weak piglets in sows and severe respiratory disease in neonatal and growing pigs. Now the disease is endemic in most areas around the world and recognized as one of most important diseases of swine. In May 2006, a severe disease, initially called'pig high fever syndrome'initially occurred in some provinces in south of China and then spread widely in China, resulting in huge economic losses in pig industry of China. Now a highly pathogenic porcine reproductive and respiratory syndrome virus (HP-PRRSV) variant has been confirmed to be the main pathogen of the severe outbreaks. Pigs of all age were clinically sick and dead in the severe infection. However, whether the virulence and in vivo pathogenicity and clinical features of the new isolates of PRRSV to pigs of all age were similar to those observed in the field, the pathogenesis and immune response of the virus and the immune efficacy of new vaccines for protection against the highly pathogenic porcine reproductive and respiratory syndrome (HP-PRRS) are still unclear. Animal experimentally infection needs to be done to answer the questions.To develop a reliable conventional pig model to study the HP-PRRS, three groups of 30-, 60- and 180-day-old pigs were infected with 3×104 50% tissue culture infective dose (TCID50) per pig of a representative HP-PRRSV strain, 5th-passage HuN4 (HuN4-F5) intramuscularly and intranasally. The results showed that the HP-PRRSV HuN4 could induce the typical PRRS in all the conventional pigs and the clinical features were similar to those observed in the field. Among them, 30-day-old and 60-day-old pigs exhibited severe clinical signs with 100% morbidity and 100% and 60% mortality, respectively. Persistently high fever (41.0～42.0°C), dyspnoea, gross lesions of consolidation of lungs,enlargement and/or haemorrhage of lymph nodes and histopathological lesions of interstitial pneumonia, the inflammation of the lymphoid tissue and encephalitis were observed in the two exposed groups. However, the clinical signs, gross lesions and microscopic lesions of the infected 180-day-old pigs were slight compared with other infected groups. So the results demonstrated that the 30 and 60-day-old conventional pigs are suitable to be the infection model for the HP-PRRSV HuN4 strain.To further investigate the in vivo pathogenicity and virulence and immune responses of the newly emerging PRRSV, 3 groups of 60-day-old conventional piglets were inoculated intranasally with a representative strain of the HP-PRRSV variant, HuN4 with 3 different infection doses(3 103～3 105 TCID50). The clinical features, humoral immune responses, viremia, the viral load in different tissues and levels of IFN-γand IL-10 cytokine production in serum in this study were evaluated. The results revealed that 3 different infection doses of virus could induce severe disease in piglets and the infected pigs in the least infection dose group exhibited a little mild clinical signs than those of other infection dose groups. The significantly and consistently clinical characteristics of the PRRSV-exposed groups consisted of persistently high fever (41.0～41.9℃) and high morbidity and mortality (60～100%), the marked clinical signs of PRRS and severe histopathologic damages in multiple organs. It induced rapid and intense humoral immune responses and seroconversion was detected in most of infected pigs at 7 days post-infection (DPI).The virus rapidly and vigorously replicated in vivo. The virus was isolated from the serum of most of infected pigs at 3 DPI and the viremia was detected in all the infected pigs by 21 DPI days. The highest average titer of viremia peaked near 9.7logs copies/ml serum at 7 DPI. The viral load in different tissue reached 4.66～9.05 logs copies/g. Elevated levels of IFN-γand IL-10 cytokine production in serum and an unbalanced profile of Th1 (IFN-γ) /Th2 (IL-10) responses in this study were also observed. Taken together, our results demonstrated that the HP-PRRSV variant HuN4 strain is a highly pathogenic for piglets and suitable to be a reference strain of highly virulent PRRSV for evaluating the efficacy of the new vaccines to prevent the HP-PRRS. Additionally, according to the experiment data, we initially made a standard of high virulence of the HuN4 for the 60-day-old conventional pigs and a clinical standard of HP-PRRS.To establish a fluorescent quantitative reverse transcription-polymerase chain reaction (RT-PCR) assay for the detection of HP-PRRSV and its attenuated virus and the classical NA-type PRRSV in China,the specific primers and fluorogenetic probe based on the ORF7 gene of PRRSV were designed and a quantitative TaqMan RT-PCR for detecting PRRSV had been developed. The established technique was time-saving, highly sensitive and specific and had reliable reproducibility. The assay for the quantitation of PRRSV template was found to be in the 101～107copies/μL linear dynamic range with excellent linearity. It is more sensitive than conventional RT-PCR and could detect the least template of 10 copies and 1 TCID50 virus. Using the technique, serum and tissue samples from pigs experimentally infected with HP-PRRSV HuN4-F5, and the sixty-fifth passage attenuated virus (HuN4-F65) were collected to detect viral load. The results demonstrated the established technique is suitable for detection PRRSV in early infection, replication and persistent infection in vivo, development and evaluation the efficacy of HP-PRRSV attenuated virus vaccines.The recombinant porcine interferon-γ(IFN-γ) can inhibit the replication of PRRSV in vitro and the IFN-γcould be detected from the lymph node, lung and peripheral blood monocytes (PBMC) of PRRSV infected pigs. To establish a fluorescent quantitative RT-PCR assay for the detection of porcine IFN-γ, the specific primers and fluorogenetic probes based on the gene of porcine IFN-γand housekeeping gene cyclophilin A (CyPA) were designed, by using the recombinant plasmids pMD18-T-IFN-γand pMD18-T-CyPA as standard products, a real-time quantitative TaqMan reverse transcription-polymerase chain reaction (RT-PCR) was performed to construct the standard curves of porcine IFN-γand CyPA. The results indicated that the assay for the quantitation of porcine IFN-γtemplate could work well in the range of 101～107copies/μL with excellent linearity, the coefficient correlation R squared values reached 0.999 and amplification efficiency was higher than 99.0%. The sensitivity was sufficient to detect the least template of 100 copies. IFN-γmRNA in tissue samples from the spleen, lung and lymph node in the euthanatized pigs infected with the HuN4-F5 virus at 21DPI were collected and quantitatively detected by the established assay. The results showed that the expression of IFN-γmRNA in infected pigs is very low with a range of 6.44×102～1.04×104 copies/μL cDNA. Therefore, The currently established porcine IFN-γspecific real time RT-PCR was time-saving, highly sensitive and specific, and had reliable reproducibility and suitable to detect the clinical specimens. |
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