Prophylactic Efficacy of Equine Immunoglobulin F(ab')2 Fragments Against Feline Parvovirus.

Feline parvovirus (FPV), a type of parvovirus prevalent worldwide, can cause foetal death and acute enteritis in adult cats with severe leukopenia, and yet there are no effective drugs to prevent or treat FPV. Here, the immune effects of two FPV vaccines on horses were compared. IgG was extracted from FPV-immunized horse sera. Equine F(ab')2 fragments were obtained from pepsin-digested IgG and then purified by protein-G column chromatography. The results showed that the inactivated FPV oil vaccine was more effective than the inactivated FPV propolis vaccine in helping healthy horses to produce hyper-immune serum. Four methods were tested, among which the optimized octanoic acid-ammonium sulphate precipitation method was proved to be the best process for extracting IgG. The optimal condition for preparing F(ab')2 by pepsin digestion was 30 °C for 3.5 h, and the content, purity and recovery of F(ab')2 were 8.64 mg/mL, 90.36% and 93.24%, respectively. Our equine immunoglobulin F(ab')2 fragments effectively neutralized activity in vitro against FPV, alleviated the clinical symptoms of FPV-infected cats, reduced the viral loads in the intestine and had prophylactic effects in FPV-infected cats. These results indicate that the F(ab')2 fragment prepared from inactivated FPV-immunized horses may be used as a prophylactic agent for diseases caused by FPV.

Isolation, genetic analysis of the first Akabane virus from goat in China.

Akabane virus (AKAV) is an important insect-borne virus belonging to the genus Orthobunyavirus, the Peribunyaviridae family. An AKAV defined as GXDH 01 here, was isolated for the first time from blood from a sentinel goat in China in 2016, and its full-length open reading frames (ORFs) were sequenced in this study. Sequence analysis suggested that the isolate GXDH 01 probably had undergone a reassortment incident and acquired L segments from other strain originating from an attenuated vaccine, such as OBE-1. This study aims to provide more understanding as to the origin and epidemiology of AKAV in China.

Characterization of Akabane virus from domestic bamboo rat, Southern China.

To identify the causative agents in 3 large-scale outbreaks of encephalitis and death among farmed bamboo rats (Rhizomys pruinosus). The routine bacterial culture and identification were performed. There were no significant pathogenic bacteria isolated from the brain, heart, liver, spleen, lung, or kidney of diseased bamboo rats. Using PCR-based methods, we excluded the following as causative agent: pox virus, herpesvirus, adenovirus, lymphocytic choriomeningitis virus, rabies virus, and sendai virus. Furthermore, the homogenate from the diseased bamboo rats was subjected to viral metagenomic analysis which revealed 48506 filtered viral reads annotated to Akabane virus (AKAV) with >75% nucleotide identity, suggesting the presence of AKAVs in bamboo rats. Five novel AKAV isolates were successfully isolated and characterized. Furthermore the newly isolated AKAV isolate was used to demonstrate that it can reproduce the severe encephalitic and pneumonic disease in bamboo rats and mice. The findings add to the better understanding of AKAV epidemiology and to the prevention and control of Akabane diseases in China.

Genetic diversity of subgenotype 2.1 isolates of classical swine fever virus.

As the causative agent of classical swine fever, the economically devastating swine disease worldwide, classical swine fever virus (CSFV) is currently classified into the 11 subgenotypes, of which subgenotype 2.1 is distributed worldwide and showing more genetic diversity than other subgenotypes. Prior to this report, subgenotype 2.1 was divided into three sub-subgenotypes (2.1a-2.1c). To further analyze the genetic diversity of CSFV isolates in China, 39 CSFV isolates collected between 2004 and 2012 in two Chinese provinces Guangxi and Guangdong were sequenced and subjected to phylogenetic analysis together with reference sequences retrieved from GenBank. Phylogenetic analyses based on the 190-nt and/or 1119-nt full length E2 gene fragments showed that current CSFV subgenotype 2.1 virus isolates in the world could be divided into 10 sub-subgenotypes (2.1a-2.1j) and the 39 isolates collected in this study were grouped into 7 of them (2.1a-2.1c and 2.1g-2.1j). Among the 10 sub-subgenotypes, 2.1d-2.1j were newly identified. Sub-subgenotype 2.1d isolates were circulated only in India, however the rest 9 sub-subgenotypes were from China with some of them closely related to isolates from European and neighboring Asian countries. According to the temporal and spatial distribution of CSFV subgenotype 2.1 isolates, the newly classified 10 sub-subgenotypes were further categorized into three groups: dominant sub-subgenotype, minor sub-subgenotype and silent sub-subgenotype, and each sub-subgenotype can be found only in certain geographical areas. Taken together, this study reveals the complex genetic diversity of CSFV subgenotype 2.1 and improves our understanding about the epidemiological trends of CSFV subgenotype 2.1 in the world, particularly in China.

Complete Genome Sequence of a Porcine Endogenous Retrovirus Isolated from a Bama Minipig in Guangxi, Southern China.

A porcine endogenous retrovirus (PERV) strain, PERV-A-BM, was isolated from a Bama minipig in Guangxi, China. This is the first entire genome sequence of PERV isolated from Guangxi Bama minipigs. The isolate is closely related to isolates from Wuzhishan miniature pigs and distantly related to isolates from large white pigs.

[Optimal expression and activity detection of recombinant, chimeric and genetically engineered tetravalent anti-human CD22 antibodies in P.pastoris].

AIM: To express the recombinant, chimeric and genetically engineered tetravalent anti-human CD22 antibodies (cRFB4FC and cRFB4CH3) using yeast cells secreting type carrier pPIC9K, and screen for optimal conditions of engineered yeast cells expressing cRFB4FC and cRFB4CH3. METHODS: The genes of cRFB4FC and cRFB4CH3 were cloned into P. pastoris eukaryotic expression vector pPIC9K to construct the recombinant plasmids pPIC9K-cRFB4FC and pPIC9K-cRFB4CH3, then identified by DNA sequencing. The recombinant plasmid pPIC9K-cRFB4FC and pPIC9K-cRFB4CH3 were transfected into P. pastoris SMD1163. The recombinant yeast cell line chosen by G418 resistance was identified by PCR. After methanol induction, the expressed protein products were verified by SDS-PAGE and Western blotting, and biologic activity was identified by ELISA. Finally, orthogonal test was conducted to optimize the expression conditions of the recombinant yeast cell lines so as to increase the expression level of the antibodies. RESULTS: The secretory type yeast carriers pPIC9K-cRFB4FC and pPIC9K-cRFB4CH3 were successfully constructed and chosen by G418 as well as identified by PCR to obtain the highly copied and integrated recombinant yeast cell line. The cRFB4FC and cRFB4CH3 proteins were expressed by yeast cells containing pPIC9K-cRFB4FC and pPIC9K-cRFB4CH3 induced by methanol. The relative molecular mass (M(r);) of cRFB4FC and cRFB4CH3 were about 326 000 and 295 000 Da. They could be identified by goat anti-human IgG(Fc) monoclonal antibody with Western blotting on SDS-PAGE, and higher biologic activity was confirmed by ELISA. Under the optimized expression conditions, the mean expression levels of cRFB4FC and cRFB4CH3 in recombinant yeast increased by 196.4% and 151.7%. CONCLUSION: The recombinant, chimeric and genetically engineered tetravalent anti-human CD22 antibodies (cRFB4FC and cRFB4CH3) proteins can be highly expressed in the P.pastoris SMD1163 expression system, and possess immunological activity.

[Preparation and bioactivity of anti-human red blood cell ScFv and CSFV E2 bifunctional fusion protein].

The aim of this study is to construct a bifunctional fusion protein, which can conjugate both human red blood cells and antibodies against classical swine fever virus (CSFV). We respectively amplified 2E8ScFv and mE2 genes from different recombinant vectors, in which 2E8ScFv gene is the single chain Fv gene against H antigen of human red blood cells, whereas mE2 gene is the main antigen coding region gene of CSFV E2 protein. We used overlap extension PCR to obtain an artificial fusion gene segment 2E8mE2 containing genes of Both 2E8ScFv and mE2, then ligated into the expression vector pET-DsbA and expressed in Escherichia coli BL21(DE3) PlysS host cells, after induced with IPTG the target fusion protein was successfully expressed and identified in inclusion bodies by SDS-PAGE and Western blotting. We purified the fusion protein and renatured it from inclusion bodies to obtain a native state of well biological activity. The Erythrocyte agglutination test results indicated that the fusion protein can conjugate both human red blood cells and antibodies of CSFV.