A novel triplex real-time PCR assay for the differentiation of lumpy skin disease virus, goatpox virus, and sheeppox virus.

Introduction: Three members of Capripoxvirus (CaPV) genus, including lumpy skin disease virus (LSDV), goatpox virus (GTPV), and sheeppox virus (SPPV), are mentioned as notifiable forms by World Organization for Animal Health. These viruses have negatively impacted ruminant farming industry worldwide, causing great economic losses. Although SPPV and GTPV cause more severe clinical disease in only one animal species, they can transfer between sheep and goats. Both homologous and heterologous immunization strategies are used to protect animals against CaPVs. However, development of accurate and rapid methods to distinguish these three viruses is helpful for the early detection, disease surveillance, and control of CaPV infection. Therefore, we developed a novel triplex real-time PCR (qPCR) for the differentiation of LSDV, GTPV, and SPPV. Methods: Universal primers were designed to detect pan-CaPV sequences. Species-specific minor groove binder (MGB)-based probes were designed, which were labeled with FAM for LSDV, HEX for GTPV, and ROX for SPPV. The sensitivity, specificity, reproducibility, and ability of detecting mixed infections were evaluated for the triplex qPCR. Further, 226 clinical samples of the infection and negative controls were subjected to the triplex qPCR, and the results were verified using PCR-restriction fragment length polymorphism (PCR-RFLP) and sequencing methods for PRO30 gene. Results: The triplex qPCR could successfully distinguish LSDV, GTPV, and SPPV in one reaction, and the assay sensitivity was 5.41, 27.70, and 17.28 copies/µL, respectively. No cross-reactivity was observed with other viruses causing common ruminant diseases, including des petits ruminants virus, foot-and-mouth disease virus, bluetongue virus, ovine contagious pustular dermatitis virus, infectious bovine rhinotracheitis virus, and bovine viral diarrhea-mucosal disease virus. Inter-and intra-assay variabilities were < 2.5%. The results indicated that the triplex qPCR was highly specific, sensitive, and reproducible. Simulation experiments revealed that this assay could successfully distinguish two or three viruses in case of mixed infections without any cross-reaction. For clinical samples, the results were completely consistent with the results of PCR-RFLP and sequencing. This demonstrated that the assay was reliable for clinical application. Discussion: The triplex qPCR is a robust, rapid, and simple tool for identifying various types of CaPV as it can successfully distinguish LSDV, GTPV, and SPPV in one reaction. Furthermore, the assay can facilitate more accurate disease diagnosis and surveillance for better control of CaPV infection.

Summary of the Current Status of African Swine Fever Vaccine Development in China.

African swine fever (ASF) is a highly lethal and contagious disease of domestic pigs and wild boars. There is still no credible commercially available vaccine. The only existing one, issued in Vietnam, is actually used in limited quantities in limited areas, for large-scale clinical evaluation. ASF virus is a large complex virus, not inducing full neutralizing antibodies, with multiple genotypes and a lack of comprehensive research on virus infection and immunity. Since it was first reported in China in August 2018, ASF has spread rapidly across the country. To prevent, control, further purify and eradicate ASF, joint scientific and technological research on ASF vaccines has been carried out in China. In the past 4 years (2018-2022), several groups in China have been funded for the research and development of various types of ASF vaccines, achieving marked progress and reaching certain milestones. Here, we have provided a comprehensive and systematic summary of all of the relevant data regarding the current status of the development of ASF vaccines in China to provide a reference for further progress worldwide. At present, the further clinical application of the ASF vaccine still needs a lot of tests and research accumulation.

A systematic review of genotypes and serogroups of African swine fever virus.

African swine fever virus (ASFV) is the causative agent of African swine fever (ASF). The virus causes an acute highly hemorrhagic disease in domestic pigs, with high mortality. Although the overall genome mutation rate of ASFV, a large DNA virus, is relatively low, ASFV exhibits genetic and antigenic diversity. ASFV can be classified into 24 genotypes on the basis of the B646L gene. Cross-protected ASFV strains can be divided into eight serogroups on the basis of antibody-mediated hemadsorption inhibition. Here, we review research progress on ASFV genotyping and serogrouping, and explain how this information assists in the rapid identification of virus origin during ASF outbreaks and will aid in the development of ASF vaccines.

Genome comparison of African swine fever virus China/2018/AnhuiXCGQ strain and related European p72 Genotype II strains.

African swine fever was introduced into China in August 2018 and led to high mortality in domestic pigs. We reported the genome characterization of the China/2018/AnhuiXCGQ strain mainly based on next-generation sequencing and comparison with related European p72 Genotype II strains. The genome was 189,393 bp long, encoding 181 open reading frames. Pair-wise genome sequence comparison revealed 54-107 variation sites between China/2018/AnhuiXCGQ and the other genotype II virulent strains contributing to the change of expression or alteration of amino acid residues in 10-38 genes. China/2018/AnhuiXCGQ strain shared the highest similarity with POL/2015/Podlaskie strain. Phylogenetic analysis based on a 125 kb long conserved central region revealed that the China/2018/AnhuiXCGQ strain and four European genotype II strains were grouped into three clusters. This study expanded our knowledge on the genetic diversity and evolution of ASFV and provided valuable information for diagnosis improvement and vaccine development.

Immunoproteomic analysis of Brucella melitensis and identification of a new immunogenic candidate protein for the development of brucellosis subunit vaccine.

In order to screen immunogenic candidate antigens for the development of a brucellosis subunit vaccine, an immunoproteomic assay was used to identify immunogenic proteins from Brucella melitensis 16 M soluble proteins. In this study, a total of 56 immunodominant proteins were identified from the two-dimensional electrophoresis immunoblot profiles by liquid chromatography tandem mass spectrometry (LC-MS/MS). Two proteins of interest, riboflavin synthase alpha chain (RS-α) and Loraine synthase (LS-2), which are both involved in riboflavin synthesis, were detected by two-dimensional immunoblots using antisera obtained from Brucella-infected human and goats. LS-2, however, is an already well-known vaccine candidate. Therefore, we focussed our studies on the novel vaccine candidate RS-α. B. melitensis RS-α and LS-2 were then expressed in Escherichia coli as fusion proteins with His tag. The humoral and cellular immune responses to the recombinant (r)RS-α was characterized. In response to in vitro stimulation by rRS-α, splenocytes from mice vaccinated with rRS-α were able to produce γ-interferon (IFN-γ) and interleukin (IL)-2 but not interleukin (IL)-4 and interleukin (IL)-10. Furthermore, rRS-α or rLS-2-vaccinated mice were partially protected against B. melitensis infection. Our results suggested that we have developed a high-throughout, accurate, rapid and highly efficient method for the identification of candidate antigens by a combination of immunoproteomics with immunisation and bacterial challenge and rRs-α could be a useful candidate for the development of subunit vaccines against B. melitensis.

[Bone marrow mesenchymal stem cell transplantation aggravates postangioplasty aortic restenosis in rats].

OBJECTIVE: To observe the effect of bone marrow mesenchymal stem cell transplantation on postangioplasty aortic restenosis in rats. METHODS: 48 SD rats were randomly divided into normal control group, balloon injury group, balloon injury and MSCs transplantation group. MSCs were pre-labeled by DAPI (25 microg/ml) and then infused into aorta through the balloon catheter (MSCs 2 x 10(6)/animal). Thoracic aorta were taken for histological examination (frozen and paraffin sections) at 1, 2, 6 weeks post angioplasty, respectively. DAPI labeled MSCs were detected under immunofluorescence microscopy. Expressions of c-kit, proliferating cell nuclear antigen (PCNA), smooth muscle alpha-actin (alpha-SMA) in aorta were determined by immunocytochemistry using related antibodies. RESULTS: The DAPI-labeled MSCs could be detected on impaired intimae and alpha-SMA expression was seen in these cells 1 weeks after MSCs transplantation. Similar weak c-kit expression in neointima was found in both injury and transplantation group at 2 weeks (P > 0.05). Expressions of PCNA and alpha-SMA in the neointima were significantly higher in transplantation group than in injury group at 2 weeks. Intima/tunica media area ratio and luminal stenosis ratio were significantly increased in transplantation group than injury group at 6 weeks (all P < 0.05). CONCLUSION: Bone marrow MSCs transplanted post aortic angioplasty could home to serious wounded aortic intima, differentiate into smooth muscle like cells, promote neointima cellular proliferation and aggravate postangioplasty aortic restenosis in rats.