Evaluation of antibody responses against the whole virions of goatpox and sheeppox viruses after subcutaneous immunization of rabbits.

Antibody development is the integral process of generating and characterizing an antibody. It commences by inoculating the antigen of interest into laboratory animals, allowing the immune system develops large quantities of antibodies. This was aimed at developing antibodies against the virion of Goatpox and Sheeppox virus vaccines. The ability of Goatpox and Sheeppox vaccines was assessed. Regarding this study, the antibody titers against both Goatpox and Sheeppox viruses was increased in the same manner. The amount of IgG was determined to be 2.29 µg/µl and 2.18 µg/µl against virions of Goatpox virus and Sheeppox respectively. The purified IgG was analyzed by SDS-PAGE. Different bands of the purified antibodies were clearly visualized, and the molecular weight of IgG was estimated to be 67 kDa and 25 kDa. Additionally, antigen/antibody binding was confirmed by Western blot using GTPV A27 antigen. No significant differences in antibody titers were observed between the two groups (p < 0, 05).

Aichivirus C isolate is a diarrhoea-causing pathogen in goats.

Aichivirus C is an emerging virus in goats, but its biological significance remains unknown. In this study, 18 diarrheic and 16 non-diarrheic faecal samples of kids were collected from a farm with an on-going diarrheic outbreak in Sichuan Province, China in May 2021. Of these samples, 77.8% (14/18) of diarrheic samples were detected as Aichivirus C positive by RT-PCR, which was significantly higher than that of non-diarrheic faces (0%, p < .001); meanwhile, other common diarrhoea-causing pathogens in goats were not detected in diarrheic samples, except for two samples that were detected as caprine enterovirus positive, suggesting that Aichivirus C was associated with goat diarrhoea. Furthermore, five Aichivirus C strains were successfully isolated from positive samples using Vero cell lines and two isolates were further plaque-purified, named SWUN/F5/2021(10-6.7 TCID50 /0.1 mL) and SWUN/F6/2021(10-7 TCID50 /0.1 mL). Interestingly, Aichivirus C strain could cause systemic infection in experimental kids via oral administration, with the main clinical manifestation being severe watery diarrhoea. Histopathological changes observed in the duodenum and jejunum were characteristic, with shedding of mucosal epithelial cells. In addition, the virus was detected in tissues of diarrhoea kids naturally infected with Aichivirus C, exhibiting pathological changes similar to those of experimental infections. Overall, this study first isolated Aichivirus C and confirmed its pathogenicity in kids, with further study needed to better understand the virus pathogenicity. As Aichivirus C has been detected in South Korea, Italy and the USA and widely prevalent in southwest China, the results obtained here have significant implications for the diagnosis and control of diarrhoea in goats.

Identification of a novel Aichivirus D in sheep.

A novel kobuvirus was found in diarrheal fecal samples of Tibetan sheep using a viral metagenomics approach, and a full kobuvirus genome was successfully obtained by RT-PCR from a diarrheal fecal sample. The full genomic sequence was 8485 nucleotides (nt) in length with a standard picornavirus genome organization. The novel genome shares 62.9% and 77.8% nt homology with Aichivirus D1 genotype strain 1-22-KoV, and Aichivirus D2 genotype strain 2-44-KoV, respectively. According to the species classification criteria of the International Committee on Taxonomy of Viruses (ICTV), the new kobuvirus belongs to Aichivirus species D. Interestingly, compared with 2 known Aichivirus D genotype strains, the novel Aichivirus D has unique amino acid substitutions in the 5'untranslated region (-UTR), VP0, VP3, and VP1, with a recombination event in the 2C region.These characteristics make the novel Aichivirus D cluster into an independent branch in the phylogenetic tree, suggesting that strain may represent a novel genotype in Aichivirus D. Moreover, the novel Aichivirus D was detected in 9.2% (18/195) of the sheep diarrheal fecal samples from 4 farms in 3 counties of the Qinghai Tibet Plateau in China. In addition, full-length VP0, VP3, and VP1 genes were successfully obtained from 12 samples from 4 farms, and phylogenetic analysis based on these genes revealed a unique evolutionary pattern for this novel Aichivirus D strain. This study identified a novel Aichivirus D that is circulating in sheep in Qinghai Tibet Plateau in China and these findings provide a better understanding of the epidemiologic and genetic evolution of kobuviruses.

First detection and molecular characteristics of caprine kobuvirus in goats in China.

Caprine kobuvirus (CKoV), a member of the genus Kobuvirus, has only been identified in South Korea and Italy until now. In this study, 24 goat diarrheic fecal samples were collected from 3 farms in Sichuan province, China, and 87.5% (21/24) samples were detected as CKoV positive by RT-PCR. Meanwhile, full-length VP0, VP3, and VP1 genes were simultaneously cloned from 17 clinical samples. Phylogenetic analysis showed that all CKoV strains were most closely related to porcine kobuvirus based on amino acid (aa) sequences of VP0 and VP3 proteins, but CKoV strains were closely related to with Aichivirus B strains (ferret, bovine, and sheep kobuvirus) based on aa sequences of the VP1 protein. Interestingly, compared with known CKoV strains in the GenBank database, Chinese CKoV strains have unique amino acid changes in VP0 and VP1 proteins. Moreover, the first Chinese CKoV nearly complete genome was successfully obtained from a diarrheic fecal sample, named SWUN/F11/2019. Compared with the two known CKoV strains, five aa mutations (S60A, L252I, V267T, I, V 306 L, V331I) were found in the VP0 gene and 7 aa mutations (S57N, G, T243A, V244I, T, A248V, L, S251A, R252H, and M255L) were found in VP1 in the SWUN/F11/2019 genome. This was the first report of the detection and molecular characteristics of CKoV from goats in China, which could be helpful for improving the understanding of the prevalence and genetic evolution of CKoV.

The cGas-Sting Signaling Pathway Is Required for the Innate Immune Response Against Ectromelia Virus.

Activation of the DNA-dependent innate immune pathway plays a pivotal role in the host defense against poxvirus. Cyclic GMP-AMP synthase (cGAS) is a key cytosolic DNA sensor that produces the cyclic dinucleotide cGMP-AMP (cGAMP) upon activation, which triggers stimulator of interferon genes (STING), leading to type I Interferons (IFNs) production and an antiviral response. Ectromelia virus (ECTV) has emerged as a valuable model for investigating the host-Orthopoxvirus relationship. However, the role of cGas-Sting pathway in response to ECTV is not clearly understood. Here, we showed that murine cells (L929 and RAW264.7) mount type I IFN responses to ECTV that are dependent upon cGas, Sting, TANK binding kinase 1 (Tbk1), and interferon regulatory factor 3 (Irf3) signaling. Disruption of cGas or Sting expression in mouse macrophages blocked the type I IFN production and facilitated ECTV replication. Consistently, mice deficient in cGas or Sting exhibited lower type I IFN levels and higher viral loads, and are more susceptible to mousepox. Collectively, our study indicates that the cGas-Sting pathway is critical for sensing of ECTV infection, inducing the type I IFN production, and controlling ECTV replication.

Analysis of the CDR3 length repertoire and the diversity of T cell receptor α and ß chains in swine CD4+ and CD8+ T lymphocytes.

The T cell receptor (TCR) is a complex heterodimer that recognizes fragments of antigens as peptides and binds to major histocompatibility complex molecules. The TCR α and ß chains possess three hypervariable regions termed complementarity determining regions (CDR1, 2 and 3). CDR3 is responsible for recognizing processed antigen peptides. Immunoscope spectratyping is a simple technique for analyzing CDR3 polymorphisms and sequence length diversity, in order to investigate T cell function and the pattern of TCR utilization. The present study employed this technique to analyze CDR3 polymorphisms and the sequence length diversity of TCR α and ß chains in porcine CD4+ and CD8+ T cells. Polymerase chain reaction products of 19 TCR α variable regions (AV) and 20 TCR ß variable regions (BV) gene families obtained from the CD4+ and CD8+ T cells revealed a clear band following separation by 1.5% agarose gel electrophoresis, and each family exhibited >8 bands following separation by 6% sequencing gel electrophoresis. CDR3 spectratyping of all identified TCR AV and BV gene families in the sorted CD4+ and CD8+ T cells by GeneScan, demonstrated a standard Gaussian distribution with >8 peaks. CDR3 in CD4+ and CD8+ T cells demonstrated different expression patterns. The majority of CDR3 recombined in frame and the results revealed that there were 10 and 14 amino acid discrepancies between the longest and shortest CDR3 lengths in specific TCR AV and TCR BV gene families, respectively. The results demonstrated that CDR3 polymorphism and length diversity demonstrated different expression and utilization patterns in CD4+ and CD8+ T cells. These results may facilitate future research investigating the porcine TCR CDR3 gene repertoire as well as the functional complexity and specificity of the TCR molecule.

Comparison of Host Gene Expression Profiles in Spleen Tissues of Genetically Susceptible and Resistant Mice during ECTV Infection.

Ectromelia virus (ECTV), the causative agent of mousepox, has emerged as a valuable model for investigating the host-Orthopoxvirus relationship as it relates to pathogenesis and the immune response. ECTV is a mouse-specific virus and causes high mortality in susceptible mice strains, including BALB/c and C3H, whereas C57BL/6 and 129 strains are resistant to the disease. To understand the host genetic factors in different mouse strains during the ECTV infection, we carried out a microarray analysis of spleen tissues derived from BALB/c and C57BL/6 mice, respectively, at 3 and 10 days after ECTV infection. Differential Expression of Genes (DEGs) analyses revealed distinct differences in the gene profiles of susceptible and resistant mice. The susceptible BALB/c mice generated more DEGs than the resistant C57BL/6 mice. Additionally, gene ontology and KEGG pathway analysis showed the DEGs of susceptible mice were involved in innate immunity, apoptosis, metabolism, and cancer-related pathways, while the DEGs of resistant mice were largely involved in MAPK signaling and leukocyte transendothelial migration. Furthermore, the BALB/c mice showed a strong induction of interferon-induced genes, which, however, were weaker in the C57BL/6 mice. Collectively, the differential transcriptome profiles of susceptible and resistant mouse strains with ECTV infection will be crucial for further uncovering the molecular mechanisms of the host-Orthopoxvirus interaction.

αß T-cell receptor bias in disease and therapy (Review).

The diversity and specificity of T cell receptors (TCR), the characteristics of T-cell surface marker, are central to the adaptive immunity. TCR variability is required for successful immunization coverage because this structural foundation is indispensable for the valid identification of short antigen peptides (derived from degraded antigens) that are presented by major histocompatibility molecules on the surfaces of antigen-presenting cells. Despite the vast T-cell repertoire, biased αß TCR has become a common theme in immunology. To date, numerous examples of TCR bias have been observed in various diseases. Immunotherapy strategies that are based on αß T cell responses are also emerged as a prominent component of clinical treatment. In the present review, we briefly summarize the current knowledge regarding basic structural information and the molecular mechanisms underlying TCR diversity. Moreover, we outline the role of TCR repertoire bias in some diseases, and its application for therapeutic interventions, as these play significant roles in disease progression, even with patients with a good prognosis.

Infection of mouse bone marrow-derived immature dendritic cells with classical swine fever virus C-strain promotes cells maturation and lymphocyte proliferation.

In this study, the interactions of classical swine fever virus (CSFV) C-strain and the virulent GSLZ strain with mouse bone marrow-derived immature dendritic cells (BM-imDCs) were investigated for the first time. Both the C-strain and the virulent GSLZ strain could effectively infect and replicate in mouse BM-imDCs. C-strain-infected BM-imDCs showed a greatly enhanced degree of maturation, and could effectively promote the expansion and proliferation of allogeneic naive T cells. The C-strain induced a stronger Th1 response. Infection with the virulent GSLZ strain had no obvious influence on cell maturation or lymphocyte proliferation, and failed to induce any obvious immune response. The results of this study provided initial information for research of the immunologic mechanisms of CSFV using mouse DCs as the model cells.

[Progress on host range factors and their mechanisms of poxvirus].

Poxvirus is one of the most serious zoonosis pathogens, which has largest genome and broadest host spectrum. With the development of molecular biology, functional genomics, and immunology-related technology, the interactions between pathogen and the host, particularly a large array of host range factors and their functions have been increasingly discovered. These findings provide references for the molecular basis of poxvirus tissue tropism and host specificity. This review focus on the introduction of host range factors in major members of Chordopoxvirinae to highlight the understanding of the mechanisms of molecular genetic evolution, the host tropism, and cross-species infection of poxviruses.

[Modulation of the host toll-like receptor signaling pathways by virus infection].

Toll-like receptors (TLRs) are germline-encoded pattern recognition receptors (PRRs) that play a central role in host cell recognition and responses to virus infection, leading to the production of interferons (IFNs) and proinflammatory cytokines. In parallel, in order to establish an infection, viruses have to develop exclusively strategies to interfere with TLRs signaling, particularly some important adaptors activation such as MyD88, NF-kappaB, TRIF and IRFs, and suppress or escape host's antiviral immune response. In this paper, we review the latest findings on the various strategies used by viruses to modulate TLRs-mediated innate immune response, with special emphasis on immune evasion mechanism of VACV, HCV and HIV. By highlighting recent progress in these areas, we hope to convey a greater understanding of how viruses hamper TLRs signaling and how to overcome viral infection.

[Investigation on echinococcosis in animals in Gannan Tibetan Autonomous Prefecture].

OBJECTIVE: To make an investigation on echinococcosis among animals in Gannan Tibetan Autonomous Prefecture. METHODS: 21 villages from Maqu and Luqu counties were selected for the survey in August of 2004-September of 2007. Rodents were trapped in the field. Sheep and yak livers, hearts and lungs were collected from the local slaughterhouses for pathological examination. Domestic dogs (shepherd dogs) were de-wormed by 15% arecoline to receive adult worms and stray dogs were shot for dissection. RESULTS: The prevalence of alveolar echinococcosis (AE) in Ochotona dahurica was 1.2% (1/87), and 2.3% (3/132) in Myospalax fontaniere, but no infection was found in Marmota himalayana, Ochotona tibetana and Mus musculus. 113 out of 1021 (11.1%) sheep were found infected with cystic echinococcosis (CE), and 3 (0.3%) with AE. 126 out of 634 (19.9%) yaks were infected with CE, and 2 yaks (0.3%) with AE. 17 out of 74 (23.0%) dogs were infected with Echinococcus granulosus (Eg), and 4 (5.4%) with Echinococcus multilocularis (Em). CONCLUSION: The results showed that there is a widespread endemic of Echinococcus granulosus in dogs and wild animals in Gannan Tibetan Autonomous Prefecture, with less Echinococcus multilocularis infection.

[Observation on the ultrastructure of Taenia solium oncosphere].

OBJECTIVE: To observe the ultrastructure of Taenia solium oncospheres. METHODS: Patients infected with Taenia solium were de-wormed by decoction arecae and pumpkin seeds to get the mature proglottids and collect eggs. The eggs were treated with sodium hypochlorite to break the eggshells. Oncospheres were collected in Percoll (isoosmotic solution), and activated with artificial intestinal juice. The specimens were prepared with hot agar centrifugation for ultra-thin sections and observed with transmission electron microscopy (TEM) . RESULTS: T. solium oncosphere was in oval shape with a size of (14-17) microm x (10-13) microm. There were some irregular ecphymata or plicae on its surface. The hooks were composed of outer pellet layer, the middle fibrous layer and the central core marrow. Myoblasts, hook-forming cells and cerebral cortex cells were observed in the mature oncospheres. CONCLUSION: The ultrastructure of Taenia solium oncosphere is similar to that of Hymenolepis diminuta, with difference in hooks. There are binucleate cells which play a role in forming epithelium in the development of oncospheres.

[Construction, expression and immunogenicity of eukaryotic vectors based on goat pox virus P32 gene].

The full-length P32 gene and the truncated P32 gene (MP-32) were amplified from the recombinant plasmid pMD-P32 by polymerase chain reaction (PCR) and cloned into pcDNA3. 1(+) and pcDNA3.1-CpG respectively. The recombinant plasmids (pcDNA3.1-P32, pcDNA3.1-CpG-P32 and pcDNA3. 1-CpG-MP32) were transfected into BHK-21 cells by using lipofectin. The expressed P32 protein was confirmed by indirect immunofluorescence assay (IFA). The BALB/c mice were immunized with these recombinant plasmids by intramuscular injection. The specific antibodies aginst CPV were detected by ELISA kit weekly. The murine splenic T lymphocyte subgroups CD4+ and CD8+ were detected by flow cytometry. Results showed that the P32 protein was expressed successfully in vitro. After 2 weeks post im munization, the specific IgG antibodies against CPV were detected in the vaccinated mice. The percentage of CD4+ /CD8+ T cells was significantly higher than that of the control. In conclusion, these constructed eukaryotic vectors could induce humoral and celluar immune responses in mice.

[Expression and analysis of recombinant pIL-18 in Pichia pastoris].

The porcine IL-18 gene was amplified from recombinant plasmid pGEM-IL-18 by PCR, then the pPIC9K-IL-18 of fusion expression vector was constructed by inserting IL-18 fragment,and was transformed to GS115 by electroporation, multi-copy recombinant strains were screened by G418. The expression of recombinant fusion protein was induced by methanol, SDS-PAGE was used to analyze expression product, fusion protein was purified by Sephadex G-100 column, bioactivity of IL-18 was measured by MTT assays. Experiment results show fusion protein of pIL-18 secreted by GS115,expression reaches the secretion peak of 160 mg/L at 72 h. We have expressed and purified successfully the recombinant pIL-18 with obvious biological activity in Pichia pastoris.

[Molecular characteristics of inlB protein of Listeria monocytogenes wild strain and it's expression and purification].

Internalin B of Listeria monocytogenes TA wild strain was amplified by PCR method and sequenced, then subcloned into prokaryotic vector pET28a for expression. The complete length of InlB gene was 1893 bp, encoding 630 amino acids. The front 35 amino acids consisted of signal peptide. There were one cap domain with alpha-helix region, 6 leucine-rich repeat motifs, one immunoglobulin (Ig)-like domain, one B repeat sequence, 3 direct repeat GW domains and 5 N-glycosylation sites. The amino acid of leucine amounted to 10.2 percent in all amino acids of deduced sequence Compared the InlB genes with that of other 18 isolates reported in GenBank, the identities of nucleotide sequence and deduced amino acid sequence were 91.1% to approximately 99.6% and 92.3% to approximatgely 99.8%, respectively. Expressed InlB protein was detected by SDS-PAGE and confirmed by western blot. Recombinant protein was successfully purified by Ni2+ affinity chromatograph column.

[Combined expression of TSO45W-4BX from Taenia solium and porcine CD58 in Escherichia coli].

OBJECTIVE: To express the TSO45W-4BX of Taenia solium in combination with CD58 as a molecular adjuvant for improving the protective efficacy of the TSO45W-4BX recombinant vaccine. METHODS: TSO45W-4BX and porcine CD58 genes were amplified by PCR, using recombinant plasmids pGEM-4B and pGEM-CD58 as template respectively. The CD58 fragment was inserted into the recombinant plasmid pGEX-4T-1 with directly ligated TSO45W-4BX. The transformant was induced with IPTG and followed by identifying the integrity of the recombinant containing TS045W-4BX and porcine CD58 with PCR and sequencing. The products were analyzed by SDS-PAGE and Western blotting. RESULTS: The expression products of Mr 69,000 GST-4BX/CD58 and Mr 41,000 GST-4BX were present mainly in the form of inclusion bodies and soluble substance respectively, and both were recognized by sera of cysticercosis patients. CONCLUSIONS: The TSO45W-4BX co-expressed with porcine CD58 conserves its immune reactivity.

[Adjuvant effect of CpG DNA recombinant plasmid on antigen of Cysticercus cellulosae].

In order to prove the adjuvant effect of CpG DNA recombinant plasmid, the total antibodies and their IgG2a subtype induced by antigen of Cysticercus cellulosae, and content of IL-4 and IFN-gamma secreted from splenic cell of mouse immunized were measured. The recombinant plasmids showed an adjuvant effect, and CpG2 was the best adjuvant among the plasmids. It is proved that the CpG DNA possesses a synergistic effect with AI(OH)3 and 206 adjuvant, and is an effective Th1 type adjuvant in mice.

[Molecular relationship of Eurytrema coelmaticum inferred from 18S rRNA sequence].

OBJECTIVE: To elucidate the taxonomic position of Eurytrema coelmaticum by using molecular technology. METHODS: 18S rRNA fragment was amplified from E. coelmaticum genomic DNA by specific conservative primers and sequenced. Homology and phylogenic tree of 18S rRNA sequences between E. coelmaticum and other Dicrocoeliidae trematodes were analyzed and constructed by DNAStar and MEGA3 respectively, and their evolutionary relationship was determined. RESULTS: E. coelmaticum 18S rRNA sequence was with high homology to those from Dicrocoelium dendriticum, Lyperosomum collurionis and Brachylecithum lobatum. Among them, the diversity of E. coelmaticum from D. dendriticum was 2.42%, and that from L. collurionis was 1.75%; D. dendriticum and B. lobatum were closer in evolution only with 1.09% diversity. CONCLUSION: For Dicrocoeliidae trematodes, classification based on 18S rRNA target is valid and the sequences are highly conservative. E. coelmaticum is evolutionarily closer to L. collurionis than to D. dendriticum and B. lobatum.

[High-level expression of the potential vaccine antigen TSO18 of Taenia solium in Pichia pastoris].

TSO18 gene was subcloned into the Pichia pastoris expression vector pPIC9K. The recombinant plasmid pPIC9K-TSO18 was transformed into P. pastoris GS115 by electroporation so that the plasmid will be integrated with chromosome of P. pastoris. The P. pastoris strains containing multi-copy recombinant were screened by G418 and induced by methanol. The expression product was analyzed by SDS-PAGE, Western blot, deglycosylation, and purified by Sephadex column, and was used to immunize mice. The results indicated that the target protein was efficiently expressed in P. pastoris, and glycosylated moderately, and had immunological activity. In a 5 liter fermentor, the expression level of the target protein was up to 2.54 mg/mL. These results will benefit for the development of genetically engineering vaccine.