The outbreaks of pseudorabies have been frequently reported in Bartha-K61-vaccinated farms in China since 2011. To study the pathogenicity and evolution of the circulating pseudorabies viruses in Fujian Province, mainland China, we isolated and sequenced the whole genome of a wild-type pseudorabies virus strain named “FJ-2012.” We then conducted a few downstream bioinformatics analyses including phylogenetic analysis and pathogenic analysis and used the virus to infect 6 pseudorabies virus-free piglets. FJ-2012-infected piglets developed symptoms like high body temperature and central nervous system disorders and had high mortality rate. In addition, we identified typical micropathological changes such as multiple gross lesions in infected piglets through pathological analysis and conclude that the FJ-2012 genome is significantly different from known pseudorabies viruses, in which insertions, deletions, and substitutions are observed in multiple immune and virulence genes. In summary, this study shed lights on the molecular basis of the prevalence and pathology of the pseudorabies virus strain FJ-2012. The genome of FJ-2012 could be used as a reference to study the evolution of pseudorabies viruses, which is critical to the vaccine development of new emerging pseudorabies viruses.

We isolated an influenza strain named A/Swine/Fujian/F1/2010 (H1N2) from a pig suspected to be infected with swine flu. The results of electron microscopy, hemagglutination (HA) assay, hemagglutination inhibition (HI) assay, and whole genome sequencing analysis suggest that it was a reassortant virus of swine (H1N1 subtype), human (H3N2 subtype), and avian influenza viruses. To further study the genetic evolution of A/Swine/Fujian/F1/2010 (H1N2), we cloned its whole genome fragments using RT-PCR and performed phylogenetic analysis on the eight genes. As a result, the nucleotide sequences of HA, NA, PB1, PA, PB2, NP, M, and NS gene are similar to those of A/Swine/Shanghai/1/2007(H1N2) with identity of 98.9%, 98.9%, 99.0%, 98.6%, 99.0%, 98.9%, 99.3%, and 99.3%, respectively. Similar to A/Swine/Shanghai/1/2007(H1N2), we inferred that the HA, NP, M, and NS gene fragments of A/Swine/Fujian/F1/2010 (H1N2) strain were derived from classical swine influenza H3N2 subtype, NA and PB1 were derived from human swine influenza H3N2 subtype, and PB2 and PA genes were derived from avian influenza virus. This further validates the role of swine as a “mixer” for influenza viruses.

This study described a TaqMan based real-time fluorescent quantitative PCR (qPCR) method to detect porcine cytomegalovirus (PCMV) infection, targeting the conserved region of the DNA polymerase (DPOL) gene. The standard curve showed a linear regression relationship with a coefficient of 0.999 and a slope of y = −3.249x + 38.958 corresponding to the amplification efficiency at 99.8%. The limit of the qPCR method was 51.9 copies/μl. The established qPCR method showed excellent specificity, with no cross-reaction observed with common porcine pathogens. The coefficient of variation for intra-assay and interassay variability ranged up to 1.51% and 2.24%, respectively. PCMV positive signals can be found in semen using this qPCR method, which suggested that we should pay more attention to PCMV contamination in semen in order to eliminate PCMV infection in artificial insemination and xenotransplantation.

An emerging pseudorabies virus (PRV) variant has been reported on Bartha-K61-vaccinated farms since 2011, causing great economic losses to China’s swine-feeding industry. In this study, two vaccines, FJ-2012ΔgE/gI-GEL02 and FJ-2012ΔgE/gI-206VG, were administered to piglets for immune efficacy investigation. Humoral immunity response, clinical signs, survival rate, tissue viral load, and pathology were assessed in piglets. The results showed that both vaccines were effective against the PRV FJ-2012 challenge, the piglets all survived while developing a high level of gB-specific antibody and neutralizing antibody, the virus load in tissue was alleviated, and no clinical PR signs or pathological lesions were displayed. In the unimmunized challenged group, typical clinical signs of pseudorabies were observed, and the piglets all died at 7 days post-challenge. Compared with commercial vaccines, the Bartha-K61 vaccine group could not provide full protection, which might be due to a lower vaccine dose; the inactivated vaccine vPRV* group piglets survived, displaying mild clinical signs. The asterisk denotes inactivation. These results indicate that FJ-2012ΔgE/gI-GEL02 and FJ-2012ΔgE/gI-206VG were effective and could be promising vaccines to control or eradicate the new PRV epidemic in China.

This study aimed to characterize two novel mcr-1 variants, mcr-1.35 and mcr-1.36, which originated from Moraxella spp. that were isolated from diseased pigs in China. The Moraxella spp. carrying novel mcr-1 variants were subjected to whole-genome sequencing (WGS) and phylogenetic analysis based on the 16S rRNA gene. The mcr-1 variants mcr-1.35 and mcr-1.36 were characterized using phylogenetic analysis, a comparison of genetic environments, and protein structure prediction. The WGS indicated that two novel mcr-1 variants were located in the chromosomes of three Moraxella spp. with a genetic environment of mcr-1-pap2. In addition to the novel colistin resistance genes mcr-1.35 and mcr-1.36, the three Moraxella spp. contained other antimicrobial resistance genes, including aac(3)-IId, tet(O), sul2, floR, and blaROB-3. A functional cloning assay indicated that either the mcr-1.35 or mcr-1.36 gene could confer resistance to colistin in Escherichia coli DH5α and JM109. The nucleotide sequences of mcr-1.35 and mcr-1.36 presented 95.33 and 95.33% identities, respectively, to mcr-1.1. The phylogenetic analysis showed that mcr-1.35 and mcr-1.36 were derived from Moraxella spp. that belonged to subclades that were different from those of the mcr-1 variants (mcr-1.1 to mcr-1.34 except mcr-1.10) originating from Enterobacteriaceae. The deduced amino acid sequences of MCR-1.35 (MCR-1.36) showed 96.67% (96.49%), 82.59% (82.04%), 84.07% (83.52%), 55.52% (55.17%), 59.75% (59.57%), and 61.88% (61.69%) identity to MCR-1.10, MCR-2.2, MCR-6.1, MCR-LIN, MCR-OSL, and MCR-POR, respectively, that originated from Moraxella sp. Notably, protein structure alignment showed only a few changes in amino acid residues between MCR-1.1 and MCR-1.35, as well as between MCR-1.1 and MCR-1.36. In conclusion, this study identified Moraxella spp. carrying two novel mcr-1 variants, mcr-1.35 and mcr-1.36, conferring resistance to colistin, which were isolated from pig farms in China. In addition, mcr-like variants were observed to be located in the chromosomes of some species of Moraxella isolated from pig samples.

Pseudorabies virus (PRV), as a neuroherpes virus, leads to heavy economic losses in the pig industry worldwide. This study was designed to establish recombinant PRV glycoprotein B (gB), C, and D proteins as PRV diagnostic antigens. The gB/C, gC/D, and gB/C/D fusion sequences were synthesized and inserted into pET‐28a+ vector to generate the recombinant plasmids. The identified positive recombinant plasmids were transformed into BL21 Escherichia coli. The results of the polymerase chain reaction and enzyme digestion showed that the gB/C, gC/D, and gB/C/D fusion proteins were successfully expressed. An indirect sandwich ELISA was developed with the gB/C, gC/D, and gB/C/D as coating antigens. The results of indirect enzyme‐linked immunosorbent assay (ELISA) analysis of 184 PRV‐positive porcine sera showed that the positive coincidence rates of three recombinant proteins ELISAs relative to IDEXX kit were 98.25%, 95.32%, and 98.83%, and the negative coincidence rates were 85.71%, 75% and 100%, respectively. The inter and intra batch repeatability tests showed that the coefficient of variations of our kits were all less than 5%. Especially, the gB/C/D‐ELISA has the highest specificity and sensitivity among the ELISA methods developed in this study. We established a series expression system of gB/C, gC/D, and gB/C/D antigen epitope genes and Recombinant protein‐based indirect ELISA, providing new ideas for PV diagnosis and vaccine development.

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Study on the antibody level differences of PED IgG and IgA, TGE IgG and PoR IgG after immunization with different porcine viral diarrhea vaccine combinations. Kafkas Univ Vet Fak Derg, 25 (5): 589-596, 2019. AbstractTo prevent porcine viral diarrhea, a vaccine combination that can provide good antibody levels needs to be determined. In this study, we screened 30 pregnant sows divided into six experimental groups, namely, five immunized groups and one control group, to investigate the antibody level differences of different vaccine combinations on porcine epidemic diarrhea (PED), porcine transmissible gastroenteritis (TGE), porcine rotavirus (PoR) IgG, and PED IgA. The antibody level was detected by ELISA. Results showed that the antibody levels of PED and TGE IgG in serum and PED IgA in breast milk of the "PT+PT*" vaccine combination group were higher than those of the other groups, and vaccine combination including "PTR" could stimulate the sows to produce PoR IgG antibody. These findings revealed that the vaccine combination of "PT+PT*" is optimal for preventing porcine viral diarrhea, and "PTR+PT*" may be an alternative option in areas under PoRV infection risk. This study suggested that pig farms should select suitable immunization on the basis of the local epidemic situation of porcine viral diarrhea.