Jianli Shi

Concurrent highly pathogenic porcine reproductive and respiratory syndrome virus infection accelerates Haemophilus parasuis infection in conventional pigs.

This study was aimed at determining the effect of highly pathogenic porcine reproductive and respiratory syndrome virus (HP-PRRSV) on Haemophilus parasuis (HPS) in co-infection. A quantitative real-time PCR targeting infB gene, which is conserved among different HPS serotypes, was developed to improve the accuracy and speed of the detection of HPS. A total of 32 four-week-old conventional pigs were distributed randomly into four groups: pigs in group I were intranasally infected with HP-PRRSV first, and were then intraperitoneally inoculated with HPS on 5 days after HP-PRRSV infection; pigs in group II were intranasally inoculated with HP-PRRSV alone; pigs in group III were intraperitoneally inoculated with HPS alone; pigs in group IV were intraperitoneally inoculated with physiological saline. The amount of HPS in serum on 0, 3, 6, 9 and 12 days post-inoculation (dpi) with HPS were detected using the established quantitative real-time PCR. Clinical signs, pathological changes and histopathological lesions were observed. The amount of HPS in serum reached 10(6)copies/μl at 3 dpi with HPS in pigs of group I, while it arrived 10(5.7)copies/μl at 9 dpi with HPS in pigs of group III. The HPS loads in hearts and lungs were much higher than in other tissues. The study showed that HP-PRRSV was able to accelerate HPS infection and loads.

A mouse model to study infection against porcine circovirus type 2: viral distribution and lesions in mouse

BackgroundLittle information is known about viral distribution and transmission of porcine circovirus type 2 (PCV2) in species other than swine. It is still a debated topic whether the PCV2 could be infected and caused clinical lesions. Our study is aimed to estimate the susceptibility of Kunming mouse to PCV2. Forty-eight, 6-week-old Kunming mice were randomly divided into four groups. Group A (C1-C12) was inoculated with PK-15 cell culture as a control group. Group B (sPCV1-12) was inoculated orally and intramuscularly with PCV2 (106.2TCID50/ml). Group C (mPCV1-12) was inoculated orally and intramuscularly with PCV2 (106.2TCID50/ml) and a booster inoculation at days 14 and 28 after the first inoculation. Group D (MixPCV1-12) was unvaccinated but released into Group C. Each group was sacrificed at 7, 14, 28, and 42 days post-inoculation, respectively. Necropsy was checked on every mouse. Sera samples were collected for the test of PCV2 specific antibody. Tissues were collected for histopathology study and polymerase chain reaction (PCR).ResultsThe results showed that viral replication, seroconversion, and microscopic lesions were found in inoculated mice. Continuous existence of PCV2 viruses in lymph nodes have been confirmed by PCR, which took at least seven days for the virus to be transferred into other organs from the primary interface, and the diffusion to thymus had been retarded for seven days. Special PCV2 antibody could be found in PCV2 inoculation mice and was significantly higher than that in the control. Further more, microscopic lesions and the main target of PCV2 focused in the lymph nodes with a characteristic depletion and occasional necrosis of lymphocytes in the cortex and paracortex were found in inoculated mice.ConclusionsThe Kunming mouse could be infected by PCV2 virus and used as a PCV2 infected experimental model.

Loop-mediated isothermal amplification for detection of porcine circovirus type 2

BackgroundPorcine circovirus type 2 (PCV2) is the primary causative agent of the emerging swine disease known as postweaning multisystemic wasting syndrome (PMWS). Nowadays, polymerase chain reaction (PCR) is still the most widespread technique in pathogen detection. Loop-mediated isothermal amplification (LAMP), a novel nucleic acid amplification method developed in 2000, will possibly replace PCR in the field of detection. To establish a LAMP method for rapid detection of PCV2, two pairs of primers were designed specially from the open reading frame 2 (ORF2) sequences of PCV2. A LAMP method for rapid detection of PCV2 was established. To compare with PCR, sensitivity and specificity of LAMP were evaluated using the optimized reaction system. The LAMP products could be determined by agarose gel electrophoresis or adding SYBR Green I dye.ResultsThe amplification of LAMP could be obtained at 63°C for 60 min. The detection limit was nearly 1 copy of DNA plasmid, more sensitive than PCR. There was no cross-reaction with porcine circovirus type 1 (PCV1), porcine pseudorabies virus (PRV) and porcine parvovirus (PPV) under the same conditions.ConclusionsLAMP is an useful rapid detection method with high sensitivity and specificity for PCV2.

Evaluation of a DNA vaccine candidate co-expressing GP3 and GP5 of porcine reproductive and respiratory syndrome virus (PRRSV) with interferon α/γ in immediate and long-lasting protection against HP-PRRSV challenge

Porcine reproductive and respiratory syndrome virus (PRRSV) has become one of the most economically important diseases to the global pork industry. Current vaccination strategies only provide a limited protective efficacy. In this study, a DNA vaccine, pVAX1©-α-γ-GP35, co-expressing GP3 and GP5 of PRRSV with interferon α/γ was constructed, and its immediate and long-lasting protection against highly pathogenic PRRSV (HP-PRRSV) challenge were examined in pigs. For immediate protection, the results showed that pVAX1©-α-γ-GP35 could provide partially protective efficacy, which was similar to the pVAX1©-α-γ (expressing interferon α/γ). For long-lasting protection, pigs inoculated with pVAX1©-α-γ-GP35 developed significantly higher PRRSV-specific antibody response, T cell proliferation, IFN-γ, and IL-4, than those vaccinated with pVAX1©-GP35 (expressing GP3 and GP5 of PRRSV). Following homologous challenge with HP-PRRSV strain SD-JN, pigs inoculated with pVAX1©-α-γ-GP35 showed almost no clinical signs, no lung lesions, and significantly lower viremia, as compared to those in pVAX1©-GP35 group. It indicated that pVAX1©-α-γ-GP35 could induce enhanced immune responses and provide both immediate and long-lasting protection against HP-PRRSV challenge in pigs. The DNA vaccine pVAX1©-α-γ-GP35 might be an attractive candidate vaccine for the prevention and control of HP-PRRSV infections.

Highly Efficient Expression of Interleukin-2 under the Control of Rabbit β-Globin Intron II Gene Enhances Protective Immune Responses of Porcine Reproductive and Respiratory Syndrome (PRRS) DNA Vaccine in Pigs

Highly pathogenic porcine reproductive and respiratory syndrome virus (HP-PRRSV) had caused catastrophic losses in swine industry in China. The current inactivated vaccine provided only limited protection, and the attenuated live vaccine could protect piglets against the HP-PRRSV but there was a possibility that the attenuated virus returned to high virulence. In this study, the eukaryotic expression vector pVAX1© was modified under the control of rabbit β-globin intron II gene and the modified vector pMVAX1© was constructed. Porcine interleukin-2 (IL-2) and GP3-GP5 fusion protein of HP-PRRSV strain SD-JN were highly expressed by pMVAX1©. Mice inoculated with pMVAX1©-GP35 developed significantly higher PRRSV-specific antibody responses and T cell proliferation than those vaccinated with pVAX1©-GP35. pMVAX1©-GP35 was selected as PRRS DNA vaccine candidate and co-administrated with pVAX1©-IL-2 or pMVAX1©-IL-2 in pigs. pMVAX1©-IL-2+pMVAX1©-GP35 could provide enhanced PRRSV-specific antibody responses, T cell proliferation, Th1-type and Th2-type cytokine responses and CTL responses than pMVAX1©-GP35 and pVAX1©-IL-2+pMVAX1©-GP35. Following homologous challenge with HP-PRRSV strain SD-JN, similar with attenuated PRRS vaccine group, pigs inoculated with pMVAX1©-IL-2+pMVAX1©-GP35 showed no clinical signs, almost no lung lesions and no viremia, as compared to those in pMVAX1©-GP35 and pVAX1©-IL-2+pMVAX1©-GP35 groups. It indicated that pMVAX1©-IL-2 effectively increases humoral and cell mediated immune responses of pMVAX1©-GP35. Co-administration of pMVAX1©-IL-2 and pMVAX1©-GP35 might be attractive candidate vaccines for preventing HP-PRRSV infections.

Emergence of Different Recombinant Porcine Reproductive and Respiratory Syndrome Viruses, China

Epidemiological investigations were conducted on recently emerging porcine reproductive and respiratory syndrome virus (PRRSV) strains in Shandong province in 2014–2015. The proportion of the NADC30 strain identified by ORF7 sequence alignment has been gradually increasing. Three emerging PRRSV strains were successfully isolated, and the complete genomic sequences were determined. Our results indicate the importance of recombinant strains in Shandong province, China. There was a varied degree of recombination of two or three strains (classical, HP-PRRSV and/or NADC30). Moreover, the recombination strains affected the pathogenicity of newly emerged strains.

Immunogenicity of porcine circovirus type 2 nucleic acid vaccine containing CpG motif for mice

BackgroundThis study aimed at reseaching the immune effect of porcine circovirus type 2 (PCV2) DNA vaccine containing CpG motif on mice.MethodsA total of 40 6-week-old female BALB/c mice were randomly divided into four groups which were immunized by 18CpG-pVAX1-ORF2, pVAX1-ORF2, pVAX1 and PBS, respectively, and immunized again 2 weeks later. All mice were challenged with 0.2 mL PCV2 cells virulent strain SD (106.0 TCID50/mL) after 4 weeks. Average daily gain, blood antibody levels, microscopic changes and viremia were detected to estimate the effect of DNA vaccine.Results and DiscussionThe results showed that compared to those of the control mice, groups immunized with pVAX1-ORF2 and 18CpG-pVAX1-ORF2 could induce PCV2-specific antibodies. The PCV2-specific antibodies level of 18 CpG-pVAX1-ORF2 groups was higher significantly than other groups and decreased slowly along with time. There was no distinct pathological damage and viremia occurring in mice that inoculated with CpG motif DNA vaccines. The results demonstrated that the DNA vaccine containing 18 CpG could build up resistibility immunity and reduce immune organ damage on mice.

The integrity of PRRSV nucleocapsid protein is necessary for up-regulation of optimal interleukin-10 through NF-κB and p38 MAPK pathways in porcine alveolar macrophages

Porcine reproductive and respiratory syndrome (PRRS), a highly contagious disease, has been constantly causing huge economic losses all over the world. PRRS virus (PRRSV) infection results in immunosuppression and IL-10 up-regulation. The relationship between them is still in dispute. Previous studies demonstrated the protein of PRRSV nucleocapsid (N) protein is able to up-regulate IL-10, yet the underlying molecular mechanisms remain unknown. In this study, the expression kinetics of IL-10 up-regulation induced by PRRSV N protein were analyzed in immortalized porcine alveolar macrophages (PAMs). N protein induced IL-10 expression in a time- and dose-dependent manner. Inhibition experiments of signaling pathways suggested NF-κB and p38 MAPK pathways are both involved in N protein-induced IL-10 up-regulation. Besides, the integrity of N protein is essential for significant IL-10 up-regulation. This research is beneficial for further understanding of the interplay between PRRSV and host immune system.

Complete Genome Sequence of Porcine Circovirus 2b Strain Shandong

ABSTRACT Shandong is a porcine circovirus 2b (PCV2b) strain that was isolated and purified from tissue samples from pigs with postweaning multisystemic wasting syndrome (PMWS) in the Shandong Province of China. Here, we report the complete genome sequence of strain Shandong, which may aid in understanding the molecular characteristics of this strain.

The BALB/c mouse infection model for improving the Haemophilus parasuis serotyping scheme

The use of BALB/c mouse as an alternative model to study Haemophilus parasuis (HPS) infections was evaluated, supplying the serotyping scheme by comparing the pathogenicity of different serovar HPS in pigs and mice challenge using statistical analysis. Results showed that the pathogenicity of different serovar HPS in mouse was consistent with in pigs, proving that this model is a viable alternative to pigs. It provides a convenient methodology for determining the virulence of HPS strains.