Immunological characteristics of a recombinant alphaherpesvirus with an envelope-embedded Cap protein of circovirus.

Introduction: Variant pseudorabies virus (PRV) is a newly emerged zoonotic pathogen that can cause human blindness. PRV can take advantage of its large genome and multiple non-essential genes to construct recombinant attenuated vaccines carrying foreign genes. However, a major problem is that the foreign genes in recombinant PRV are only integrated into the genome for independent expression, rather than assembled on the surface of virion. Methods: We reported a recombinant PRV with deleted gE/TK genes and an inserted porcine circovirus virus 2 (PCV2) Cap gene into the extracellular domain of the PRV gE gene using the Cre-loxP recombinant system combined with the CRISPR-Cas9 gene editing system. This recombinant PRV (PRV-Cap), with the envelope-embedded Cap protein, exhibits a similar replication ability to its parental virus. Results: An immunogenicity assay revealed that PRV-Cap immunized mice have 100% resistance to lethal PRV and PCV2 attacks. Neutralization antibody and ELISPOT detections indicated that PRV-Cap can enhance neutralizing antibodies to PRV and produce IFN-γ secreting T cells specific for both PRV and PCV2. Immunological mechanistic investigation revealed that initial immunization with PRV-Cap stimulates significantly early activation and expansion of CD69+ T cells, promoting the activation of CD4 Tfh cell dependent germinal B cells and producing effectively specific effector memory T and B cells. Booster immunization with PRV-Cap recalled the activation of PRV-specific IFN-γ+IL-2+CD4+ T cells and IFN-γ+TNF-α+CD8+ T cells, as well as PCV2-specific IFN-γ+TNF-α+CD8+ T cells. Conclusion: Collectively, our data suggested an immunological mechanism in that the recombinant PRV with envelope-assembled PCV2 Cap protein can serve as an excellent vaccine candidate for combined immunity against PRV and PCV2, and provided a cost-effective method for the production of PRV- PCV2 vaccine.

A chimeric porcine reproductive and respiratory syndrome virus 1 strain containing synthetic ORF2-6 genes can trigger T follicular helper cell and heterologous neutralizing antibody responses and confer enhanced cross-protection.

The prevalence of porcine reproductive and respiratory syndrome virus 1 (PRRSV1) isolates has continued to increase in Chinese swine herds in recent years. However, no effective control strategy is available for PRRSV1 infection in China. In this study, we generated the first infectious cDNA clone (rHLJB1) of a Chinese PRRSV1 isolate and subsequently used it as a backbone to construct an ORF2-6 chimeric virus (ORF2-6-CON). This virus contained a synthesized consensus sequence of the PRRSV1 ORF2-6 gene encoding all the envelope proteins. The ORF2-6 consensus sequence shared > 90% nucleotide similarity with four representative strains (Amervac, BJEU06-1, HKEU16 and NMEU09-1) of PRRSV1 in China. ORF2-6-CON had replication efficacy similar to that of the backbone rHLJB1 virus in primary alveolar macrophages (PAMs) and exhibited cell tropism in Marc-145 cells. Piglet inoculation and challenge studies indicated that ORF2-6-CON is not pathogenic to piglets and can induce enhanced cross-protection against a heterologous SD1291 isolate. Notably, ORF2-6-CON inoculation induced higher levels of heterologous neutralizing antibodies (nAbs) against SD1291 than rHLJB1 inoculation, which was concurrent with a higher percentage of T follicular helper (Tfh) cells in tracheobronchial lymph nodes (TBLNs), providing the first clue that porcine Tfh cells are correlated with heterologous PRRSV nAb responses. The number of SD1291-strain-specific IFNγ-secreting cells was similar in ORF2-6-CON-inoculated and rHLJB1-inoculated pigs. Overall, our findings support that the Marc-145-adapted ORF2-6-CON can trigger Tfh cell and heterologous nAb responses to confer improved cross-protection and may serve as a candidate strain for the development of a cross-protective PRRSV1 vaccine.

Immune cell early activation, apoptotic kinetic, and T-cell functional impairment in domestic pigs after ASFV CADC_HN09 strain infection.

African swine fever (ASF) caused by the African swine fever virus (ASFV) is a fatal and highly contagious disease of domestic pigs characterized by rapid disease progression and death within 2 weeks. How the immune cells respond to acute ASFV infection and contribute to the immunopathogenesis of ASFV has not been completely understood. In this study, we examined the activation, apoptosis, and functional changes of distinct immune cells in domestic pigs following acute infection with the ASFV CADC_HN09 strain using multicolor flow cytometry. We found that ASFV infection induced broad apoptosis of DCs, monocytes, neutrophils, and lymphocytes in the peripheral blood of pigs over time. The expression of MHC class II molecule (SLA-DR/DQ) on monocytes and conventional DCs as well as CD21 expression on B cells were downregulated after ASFV infection, implying a potential impairment of antigen presentation and humoral response. Further examination of CD69 and ex vivo expression of IFN-γ on immune cells showed that T cells were transiently activated and expressed IFN-γ as early as 5 days post-infection. However, the capability of T cells to produce cytokines was significantly impaired in the infected pigs when stimulated with mitogen. These results suggest that the adaptive cellular immunity to ASFV might be initiated but later overridden by ASFV-induced immunosuppression. Our study clarified the cell types that were affected by ASFV infection and contributed to lymphopenia, improving our understanding of the immunopathogenesis of ASFV.

Enhanced antigen-specific CD8 T cells contribute to early protection against FMDV through swine DC vaccination.

Foot-and-mouth disease virus (FMDV) remains a challenge for cloven-hooved animals. The currently licensed FMDV vaccines induce neutralizing antibody (NAb)-mediated protection but show defects in the early protection. Dendritic cell (DC) vaccines have shown great potency in inducing rapid T-cell immunity in humans and mice. Whether DC vaccination could enhance early protection against FMDV has not been elaborately explored in domestic pigs. In this study, we employed DC vaccination as an experimental approach to study the roles of cellular immunity in the early protection against FMDV in pigs. Autologous DCs were differentiated from the periphery blood mononuclear cells of each pig, pulsed with inactivated FMDV (iFMDV-DC) and treated with LPS, and then injected into the original pigs. The cellular immune responses and protective efficacy elicited by the iFMDV-DC were examined by multicolor flow cytometry and tested by FMDV challenge. The results showed that autologous iFMDV-DC immunization induced predominantly FMDV-specific IFN-γ-producing CD4+ T cells and cytotoxic CD8+ T cells (CTLs), high NAb titers, compared to the inactivated FMDV vaccine, and accelerated the development of memory CD4 and CD8 T cells, which was concomitantly associated with early protection against FMDV virulent strain in pigs. Such early protection was associated with the rapid proliferation of secondary T-cell response after challenge and significantly contributed by secondary CD8 effector memory T cells. These results demonstrated that rapid induction of cellular immunity through DC immunization is important for improving early protection against FMDV. Enhancing cytotoxic CD8+ T cells may facilitate the development of more effective FMDV vaccines.IMPORTANCEAlthough the currently licensed FMDV vaccines provide NAb-mediated protection, they have defects in early immune protection, especially in pigs. In this study, we demonstrated that autologous swine DC immunization augmented the cellular immune response and induced an early protective response against FMDV in pigs. This approach induced predominantly FMDV-specific IFN-γ-producing CD4+ T cells and cytotoxic CD8+ T cells, high NAb titers, and rapid development of memory CD4 and CD8 T cells. Importantly, the early protection conferred by this DC immunization is more associated with secondary CD8+ T response rather than NAbs. Our findings highlighted the importance of enhancing cytotoxic CD8+ T cells in early protection to FMDV in addition to Th1 response and identifying a strategy or adjuvant comparable to the DC vaccine might be a future direction for improving the current FMDV vaccines.

Co-administration of chicken IL-2 alleviates clinical signs and replication of the ILTV chicken embryo origin vaccine by pre-activating natural killer cells and cytotoxic T lymphocytes.

IMPORTANCE: Chickens immunized with the infectious laryngotracheitis chicken embryo origin (CEO) vaccine (Medivac, PT Medion Farma Jaya) experience adverse reactions, hindering its safety and effective use in poultry flocks. To improve the effect of the vaccine, we sought to find a strategy to alleviate the respiratory reactions associated with the vaccine. Here, we confirmed that co-administering the CEO vaccine with chIL-2 by oral delivery led to significant alleviation of the vaccine reactions in chickens after immunization. Furthermore, we found that the co-administration of chIL-2 with the CEO vaccine reduced the clinical signs of the CEO vaccine while enhancing natural killer cells and cytotoxic T lymphocyte response to decrease viral loads in their tissues, particularly in the trachea and conjunctiva. Importantly, we demonstrated that the chIL-2 treatment can ameliorate the replication of the CEO vaccine without compromising its effectiveness. This study provides new insights into further applications of chIL-2 and a promising strategy for alleviating the adverse reaction of vaccines.

Characterization of chicken interleukin-9 receptor alpha chain.

Interleukin-9 receptor alpha chain (IL-9Rα) is the ligand-binding subunit of IL-9R that plays roles in IL-9-mediated allergy, inflammation, infection, and tumor immunity. While mammalian IL-9Rαs have been extensively investigated, avian IL-9Rα has not yet been identified and characterized. In this study, we cloned chicken IL-9Rα (chIL-9Rα) and performed a phylogenetic analysis, analyzed its tissue distribution, characterized the expression form of natural chIL-9Rα. Phylogenetic analysis showed that chIL-9Rα has less than 25% amino acid homology with mammalian IL-9Rαs. The chIL-9Rα mRNA was abundantly detected only in heart and mitogen-activated peripheral blood mononuclear cells. Furthermore, 4 monoclonal antibodies (mAbs) against chIL-9Rα were generated using prokaryotic recombinant chIL-9Rα (rchIL-9Rα). Using anti-chIL-9Rα mAbs, natural chIL-9Rα expressed on the splenocytes of chickens was observed by indirect immunofluorescence assay (IFA), and its molecular weight of 51 kDa was identified by Western blotting. Overall, our study reveals for the first time the presence of IL-9Rα in birds, and provides immunological tools for further investigating the roles of chIL-9 in diseases and immunity.

Efficacy of a porcine reproductive and respiratory syndrome virus 1 (PRRSV-1) natural recombinant against a heterologous PRRSV-1 isolate both clustered within the subgroup of BJEU06-1-like isolates.

Porcine reproductive and respiratory syndrome virus 1 (PRRSV-1) has been prevalent in more than 20 provinces of China. However, no PRRSV-1-specific vaccine is commercially available in China. To evaluate the feasibility of using a low virulent PRRSV-1 isolate against potential outbreaks caused by virulent Chinese PRRSV-1 isolates, here we evaluated the efficacy of a low virulent PRRSV-1 HLJB1 strain isolated in 2014 as live vaccine against a virulent PRRSV-1 SD1291 strain isolated in 2022. Genome-based phylogenetic analysis showed that both HLJB1 and SD1291 were grouped within BJEU06-1-like isolates. However, they shared only 85.27% genomic similarity. Piglet inoculation and challenge study showed that HLJB1 inoculation could reduce viremia but did not significantly alleviate clinical signs and tissue lesions. Virus neutralization test indicated that HLJB1 inoculation could induce homologous neutralizing antibodies (NAbs) but no heterologous NAbs at 42 dpi. In addition, flow cytometric analyses showed that no memory T follicular helper (Tfh) cells against SD1291 and SD1291-specific IFN-γ secreting cells were induced by HLJB1 pre-inoculation. These results supported that HLJB1 inoculation only provides partial cross-protection against SD1291 infection even though they are clustered within the same PRRSV-1 subgroup, which is closely related to the failure in conferring cross-protective adaptive immune responses.

A potent CD8 T-cell response may be associated with partial cross-protection conferred by an attenuated Chinese HP-PRRSV vaccine against NADC30-like PRRSV challenge.

Porcine reproductive and respiratory syndrome virus (PRRSV) is one of the most devastating pathogens to the global swine industry. Many commercial PRRSV vaccines, originally designed to provide homologous protection, have shown partial protection against heterologous strains. However, the protective immune mechanisms mediated by these PRRSV vaccines are not fully understood. In this study, we investigated the factors responsible for partial protection conferred by an attenuated Chinese HP-PRRSV vaccine (TJM-F92) against heterologous NADC30-like PRRSV. By analysing peripheral T-cell responses induced by the TJM-F92 vaccine and local and systemic memory responses following challenge with NADC30-like PRRSV (SD17-38 strains) as well as neutralizing antibody response, we found that the TJM-F92 vaccine induced a significant expansion of CD8 T cells but not CD4 T cells or γδ T cells. The expanded CD8 T cells exhibited a phenotype of effector memory T cells and secreted IFN-γ upon restimulation with SD17-38 strains in vitro. In addition, only CD8 T cells in the prior immunized pigs rapidly expanded in the blood and spleen after heterologous challenge, with higher magnitude, compared to the unvaccinated pigs, showing a remarkable memory response. In contrast, no obvious humoral immune response was enhanced in the vaccinated and challenged pigs, and no heterologous neutralizing antibodies were detected throughout the experiment. Our results suggested that CD8 T cells elicited by the TJM-F92 vaccine may be responsible for partial heterologous protection against NADC30-like PRRSV strains and potentially recognize the conserved antigens among PRRSV strains.

Development of Specific Monoclonal Antibodies against Porcine RIG-I-like Receptors Revealed the Species Specificity.

The RIG-I-like receptors (RLRs) play critical roles in sensing and combating viral infections, particularly RNA virus infections. However, there is a dearth of research on livestock RLRs due to a lack of specific antibodies. In this study, we purified porcine RLR proteins and developed monoclonal antibodies (mAbs) against porcine RLR members RIG-I, MDA5 and LGP2, for which one, one and two hybridomas were obtained, respectively. The porcine RIG-I and MDA5 mAbs each targeted the regions beyond the N-terminal CARDs domains, whereas the two LGP2 mAbs were both directed to the N-terminal helicase ATP binding domain in the Western blotting. In addition, all of the porcine RLR mAbs recognized the corresponding cytoplasmic RLR proteins in the immunofluorescence and immunochemistry assays. Importantly, both RIG-I and MDA5 mAbs are porcine specific, without demonstrating any cross-reactions with the human counterparts. As for the two LGP2 mAbs, one is porcine specific, whereas another one reacts with both porcine and human LGP2. Thus, our study not only provides useful tools for porcine RLR antiviral signaling research, but also reveals the porcine species specificity, giving significant insights into porcine innate immunity and immune biology.

Dairy Cows Experimentally Infected With Bovine Leukemia Virus Showed an Increased Milk Production in Lactation Numbers 3-4: A 4-Year Longitudinal Study.

Bovine leukemia virus (BLV) is widespread in global cattle populations, but the effects of its infection on milk quantity and quality have not been clearly elucidated in animal models. In this study, 30 healthy first-lactation cows were selected from ≈2,988 cows in a BLV-free farm with the same criteria of parity, age, lactation number, as well as milk yield, SCS, and composition (fat, protein, and lactose). Subsequently, these cows were randomly assigned to the intervention (n = 15) or control (n = 15) group, and reared in different cowsheds. Cows in the intervention group were inoculated with 1 × phosphate-buffered solution (PBS) resuspended in peripheral blood mononuclear cells (PBMC) from a BLV-positive cow, while the controls were inoculated with the inactivated PBMC from the same individual. From June 2016 to July 2021, milk weight (kg) was automatically recorded by milk sensors, and milk SCS and composition were originated from monthly performed dairy herd improvement (DHI) testing. Fluorescence resonance energy transfer (FRET)-qPCR and ELISA showed that cows in the intervention group were successfully infected with BLV, while cows in the control group were free of BLV for the entire period. At 45 days post-inoculation (DPI), the numbers of whole blood cells (WBCs) (P = 0.010), lymphocytes (LYMs) (P = 0.002), and monocytes (MNCs) (P = 0.001) and the expression levels of IFN-γ (P = 0.013), IL-10 (P = 0.031), and IL-12p70 (P = 0.008) increased significantly in the BLV infected cows compared to the non-infected. In lactation numbers 2-4, the intervention group had significantly higher overall milk yield (P < 0.001), fat (P = 0.031), and protein (P = 0.050) than the control group, while milk SCS (P = 0.038) and lactose (P = 0.036) decreased significantly. Further analysis indicated that BLV infection was associated with increased milk yield at each lactation stage in lactation numbers 3-4 (P = 0.021 or P < 0.001), but not with SCS and milk composition. Together, this 4-year longitudinal study revealed that artificial inoculation of BLV increased the milk yield in cows in this BLV challenge model.

First Characterization of Chicken Interleukin-9.

Interleukin-9 (IL-9) is a pleiotropic cytokine that acts on a variety of cells and tissues, and plays roles in inflammation and infection as well as tumor immunity. While mammalian IL-9s have been widely investigated, avian IL-9 has not yet been identified and characterized. In this study, we cloned chicken IL-9 (chIL-9) and performed a phylogenetic analysis, examined its tissue distribution, characterized the biological functions of recombinant chIL-9 (rchIL-9) and the expression form of natural chIL-9. Phylogenetic analysis showed that chIL-9 has less than 30% amino acid identity with mammalian IL-9s. The chIL-9 mRNA can be abundantly detected only in the testis and thymus, and are significantly up-regulated in peripheral blood mononuclear cells (PBMCs) upon mitogen stimulation. The rchIL-9 was produced by prokaryotic and eukaryotic expression systems and showed biological activity in activating monocytes/macrophages to produce inflammatory cytokines and promoting the proliferation of CD3+ T cells. In addition, four monoclonal antibodies (mAbs) and rabbit polyclonal antibody (pAb) against rchIL-9 were generated. Using anti-chIL-9 mAbs and pAb, natural chIL-9 expressed by the activated PBMCs of chickens with a molecular weight of 25kD was identified by Western-blotting. Collectively, our study reveals for the first time the presence of functional IL-9 in birds and lays the ground for further investigating the roles of chIL-9 in diseases and immunity.

Development of a Monoclonal Antibody to Pig CD69 Reveals Early Activation of T Cells in Pig after PRRSV and ASFV Infection.

The CD69 molecule, as an early activation marker of lymphocytes, is often used to assess the activation of cellular immunity. However, for pigs, an anti-pig CD69 antibody is not yet available for this purpose after infection or vaccination. In this study, a monoclonal antibody (mAb) against pig CD69 was produced by peptide immunization and hybridoma technique. One mAb (5F12) showed good reactivity with pig CD69 that was expressed in transfected-HEK-293T cells and on mitogen-activated porcine peripheral blood mononuclear cells (PBMCs) by indirect immunofluorescence assay and flow cytometry. This mAb did not cross-react with activated lymphocytes from mouse, bovine, and chicken. Epitope mapping showed that the epitope recognized by this mAb was located at amino acid residues 147-161 of pig CD69. By conjugating with fluorochrome, this mAb was used to detect the early activation of lymphocytes in PRRSV- and ASFV-infected pigs by flow cytometry. The results showed that PRRSV infection induced the dominant activation of CD4 T cells in mediastinal lymph nodes and CD8 T cells in the spleen at 14 days post-infection, in terms of CD69 expression. In an experiment on ASFV infection, we found that ASFV infection resulted in the early activation of NK cells, B cells, and distinct T cell subsets with variable magnitude in PBMCs, spleen, and submandibular lymph nodes. Our study revealed an early event of lymphocyte and T cell activation after PRRSV and ASFV infections and provides an important immunological tool for the in-depth analysis of cellular immune response in pigs after infection or vaccination.

Development of specific monoclonal antibodies for the detection of natural chicken tumor necrosis factor-alpha.

Tumor necrosis factor alpha (TNF-α) is an important proinflammatory cytokine and the only known cytokine that can directly kill tumor cells. Unlike mammalian counterparts, chicken TNF-α (chTNF-α) gene has not been identified until very recently due to its high GC content (∼70%) and long GC fragments. The biological functions of this newly-identified cytokine and its detection methods remain to be further investigated. In this study, the extracellular domain of chTNF-α was cloned into prokaryotic vector after codon optimization and recombinant chTNF-α protein was expressed. Subsequently, using recombinant chTNF-ɑ as immunogen, rabbit polyclonal antibody (pAb) and eight clones of mouse anti-chTNF-ɑ monoclonal antibodies (mAbs) were produced, respectively. Both the pAb and mAbs specifically recognized recombinant chTNF-ɑ expressed in E.coli and transfected COS-7 cells. Further mapping the antigenic region showed that all the mAbs recognized a region of amino acid residues 195-285 of chTNF-ɑ. Furthermore, an antigen-capture enzyme-linked immunosorbent assay for the detection of chTNF-ɑ was established using one mAb and the pAb. This assay showed no cross-reactivity with irrelevant Trx-fused antigens and could detect natural chTNF-ɑ expressed by mitogen-activated chicken splenocytes in a dose-dependent manner, with a detection limit of 1 ng/mL. Collectively, our results indicated that the mAbs and pAb against chTNF-α are specific and could be used for the study of the biological functions of chTNF-ɑ and the detection of chTNF-ɑ.

The Evaluation of Cellular Immunity to Avian Viral Diseases: Methods, Applications, and Challenges.

Cellular immune responses play critical roles in the control of viral infection. However, the immune protection against avian viral diseases (AVDs), a major challenge to poultry industry, is yet mainly evaluated by measuring humoral immune response though antibody-independent immune protection was increasingly evident in the development of vaccines against some of these diseases. The evaluation of cellular immune response to avian viral infection has long been neglected due to limited reagents and methods. Recently, with the availability of more immunological reagents and validated approaches, the evaluation of cellular immunity has become feasible and necessary for AVD. Herein, we reviewed the methods used for evaluating T cell immunity in chickens following infection or vaccination, which are involved in the definition of different cellular subset, the analysis of T cell activation, proliferation and cytokine secretion, and in vitro culture of antigen-presenting cells (APC) and T cells. The pros and cons of each method were discussed, and potential future directions to enhance the studies of avian cellular immunity were suggested. The methodological improvement and standardization in analyzing cellular immune response in birds after viral infection or vaccination would facilitate the dissection of mechanism of immune protection and the development of novel vaccines and therapeutics against AVD.

Prevalence and molecular characterization of Wolbachia in field-collected Aedes albopictus, Anopheles sinensis, Armigeres subalbatus, Culex pipiens and Cx. tritaeniorhynchus in China.

Wolbachia are maternally transmitted intracellular bacteria that can naturally and artificially infect arthropods and nematodes. Recently, they were applied to control the spread of mosquito-borne pathogens by causing cytoplasmic incompatibility (CI) between germ cells of females and males. The ability of Wolbachia to induce CI is based on the prevalence and polymorphism of Wolbachia in natural populations of mosquitoes. In this study, we screened the natural infection level and diversity of Wolbachia in field-collected mosquitoes from 25 provinces of China based on partial sequence of Wolbachia surface protein (wsp) gene and multilocus sequence typing (MLST). Among the samples, 2489 mosquitoes were captured from 24 provinces between July and September, 2014 and the remaining 1025 mosquitoes were collected month-by-month in Yangzhou, Jiangsu province between September 2013 and August 2014. Our results showed that the presence of Wolbachia was observed in mosquitoes of Aedes albopictus (97.1%, 331/341), Armigeres subalbatus (95.8%, 481/502), Culex pipiens (87.0%, 1525/1752), Cx. tritaeniorhynchus (17.1%, 14/82), but not Anopheles sinensis (n = 88). Phylogenetic analysis indicated that high polymorphism of wsp and MLST loci was observed in Ae. albopictus mosquitoes, while no or low polymorphisms were in Ar. subalbatus and Cx. pipiens mosquitoes. A total of 12 unique mutations of deduced amino acid were identified in the wsp sequences obtained in this study, including four mutations in Wolbachia supergroup A and eight mutations in supergroup B. This study revealed the prevalence and polymorphism of Wolbachia in mosquitoes in large-scale regions of China and will provide some useful information when performing Wolbachia-based mosquito biocontrol strategies in China.

Chimeric HP-PRRSV2 containing an ORF2-6 consensus sequence induces antibodies with broadly neutralizing activity and confers cross protection against virulent NADC30-like isolate.

Due to the substantial genetic diversity of porcine reproductive and respiratory syndrome virus (PRRSV), commercial PRRS vaccines fail to provide sufficient cross protection. Previous studies have confirmed the existence of PRRSV broadly neutralizing antibodies (bnAbs). However, bnAbs are rarely induced by either natural infection or vaccination. In this study, we designed and synthesized a consensus sequence of PRRSV2 ORF2-6 genes (ORF2-6-CON) encoding all envelope proteins based on 30 representative Chinese PRRSV isolates. The ORF2-6-CON sequence shared > 90% nucleotide identities to all four lineages of PRRSV2 isolates in China. A chimeric virus (rJS-ORF2-6-CON) containing the ORF2-6-CON was generated using the avirulent HP-PRRSV2 JSTZ1712-12 infectious clone as a backbone. The rJS-ORF2-6-CON has similar replication efficiency as the backbone virus in vitro. Furthermore, pig inoculation and challenge studies showed that rJS-ORF2-6-CON is not pathogenic to piglets and confers better cross protection against the virulent NADC30-like isolate than a commercial HP-PRRS modified live virus (MLV) vaccine. Noticeably, the rJS-ORF2-6-CON strain could induce bnAbs while the MLV strain only induced homologous nAbs. In addition, the lineages of VDJ repertoires potentially associated with distinct nAbs were also characterized. Overall, our results demonstrate that rJS-ORF2-6-CON is a promising candidate for the development of a PRRS genetic engineered vaccine conferring cross protection.

An Anti-Tumor Vaccine Against Marek's Disease Virus Induces Differential Activation and Memory Response of γδ T Cells and CD8 T Cells in Chickens.

Marek's disease virus (MDV) is a highly oncogenic alphaherpesvirus that causes deadly T-cell lymphomas and serves as a natural virus-induced tumor model in chickens. The most efficacious vaccine, CVI988/Rispens (CVI988), against MD has been used for several decades. However, the mechanisms leading to protective immunity following vaccination are not fully understood. In this study, employing multi-parameter flow cytometry, we performed a comprehensive analysis of T cell responses in CVI988-vaccinated chickens. CVI988 vaccination induced significant expansion of γδ T cells and CD8α+ T cells but not CD4+ T cells in spleen, lung and blood at early time-points. The expansion of these cells was CVI988-specific as infection with very virulent MDV RB1B did not elicit expansion of either γδ or CD8α+ T cells. Phenotypic analysis showed that CVI988 vaccination elicited preferential proliferation of CD8α+ γδ T cells and CD8αα co-receptor expression was upregulated on γδ T cells and CD8α+ T cells after immunization. Additionally, cell sorting and quantitative RT-PCR showed that CVI988 vaccination activated γδ T cells and CD8α+ T cells which exhibited differential expression of cytotoxic and T cell-related cytokines. Lastly, secondary immunization with CVI988 induced the expansion of CD8+ T cells but not γδ T cells at higher magnitude, compared to primary immunization, suggesting CVI988 did induce memory CD8+ T cells but not γδ T cells in chickens. Our results, for the first time, reveal a potential role of γδ T cells in CVI988-induced immune protection and provide new insights into the mechanism of immune protection against oncogenic MDV.

Isolation and characterization of multidrug-resistant Klebsiella pneumoniae from raw cow milk in Jiangsu and Shandong provinces, China.

Antimicrobials are the most important therapy to bovine mastitis. Bacterial infection and antibiotic treatment of mastitis cycles frequently in dairy farms worldwide, giving rise to concerns about the emergence of multidrug-resistant (MDR) bacteria. In this study, we examined the microbial diversity and antibiotic resistance profiles of bacteria isolated from raw milk from dairy farms in Jiangsu and Shandong provinces, China. Raw milk samples were collected from 857 dairy cattle including 800 apparently healthy individuals and 57 cows with clinical mastitis (CM) and subjected to microbiological culture, antimicrobial susceptibility assay and detection of antibiotic-resistant genes by polymerase chain reaction (PCR) and sequencing. A total of 1,063 isolates belonging to 41 different bacterial genera and 86 species were isolated and identified, of which Pseudomonas spp. (256/1,063, 24.08%), Staphylococcus. spp. (136/1,063, 12.79%), Escherichia coli (116/1,063, 10.91%), Klebsiella spp. (104/1,063, 9.78%) and Bacillus spp. (84/1,063, 7.90%) were most frequently isolated. K. pneumoniae, one of the most prevalent bacteria, was more frequently isolated from the farms in Jiangsu (65/830, 7.83%) than Shandong (1/233, 0.43%) province, and showed a positive association with CM (p < .001). The antimicrobial susceptibility assay revealed that four of the K. pneumoniae isolates (4/66, 6.06%) were MDR bacteria (acquired resistance to ≥three classes of antimicrobials). Furthermore, among 66 isolates of K. pneumoniae, 21.21% (14/66), 13.64% (9/66) and 12.12% (8/66) were resistant to tetracycline, chloramphenicol and aminoglycosides, respectively. However, all K. pneumoniae isolates were sensitive to monobactams and carbapenems. The detection of antibiotic-resistant genes confirmed that the ß-lactamase genes (blaSHV and blaCTX-M ), aminoglycoside modifying enzyme genes [aac(6')-Ib, aph(3')-I and ant(3″)-I], tetracycline efflux pump (tetA) and transposon genetic marker (intI1) were positive in MDR isolates. This study indicated that MDR K. pneumoniae isolates emerged in dairy farms in Jiangsu province and could be a potential threat to food safety and public health.

Establishing a Multicolor Flow Cytometry to Characterize Cellular Immune Response in Chickens Following H7N9 Avian Influenza Virus Infection.

Avian influenza virus (AIV) emerged and has continued to re-emerge, continuously posing great threats to animal and human health. The detection of hemagglutination inhibition (HI) or virus neutralization antibodies (NA) is essential for assessing immune protection against AIV. However, the HI/NA-independent immune protection is constantly observed in vaccines' development against H7N9 subtype AIV and other subtypes in chickens and mammals, necessitating the analysis of the cellular immune response. Here, we established a multi-parameter flow cytometry to examine the innate and adaptive cellular immune responses in chickens after intranasal infection with low pathogenicity H7N9 AIV. This assay allowed us to comprehensively define chicken macrophages, dendritic cells, and their MHC-II expression, NK cells, γδ T cells, B cells, and distinct T cell subsets in steady state and during infection. We found that NK cells and KUL01+ cells significantly increased after H7N9 infection, especially in the lung, and the KUL01+ cells upregulated MHC-II and CD11c expression. Additionally, the percentages and numbers of γδ T cells and CD8 T cells significantly increased and exhibited an activated phenotype with significant upregulation of CD25 expression in the lung but not in the spleen and blood. Furthermore, B cells showed increased in the lung but decreased in the blood and spleen in terms of the percentages or/and numbers, suggesting these cells may be recruited from the periphery after H7N9 infection. Our study firstly disclosed that H7N9 infection induced local and systemic cellular immune responses in chickens, the natural host of AIV, and that the flow cytometric assay developed in this study is useful for analyzing the cellular immune responses to AIVs and other avian infectious diseases and defining the correlates of immune protection.

Revisiting cellular immune response to oncogenic Marek's disease virus: the rising of avian T-cell immunity.

Marek's disease virus (MDV) is a highly oncogenic alphaherpesvirus that causes deadly T-cell lymphomas and serves as a natural virus-induced tumor model in chickens. Although Marek's disease (MD) is well controlled by current vaccines, the evolution of MDV field viruses towards increasing virulence is concerning as a better vaccine to combat very virulent plus MDV is still lacking. Our understanding of molecular and cellular immunity to MDV and its immunopathogenesis has significantly improved, but those findings about cellular immunity to MDV are largely out-of-date, hampering the development of more effective vaccines against MD. T-cell-mediated cellular immunity was thought to be of paramount importance against MDV. However, MDV also infects macrophages, B cells and T cells, leading to immunosuppression and T-cell lymphoma. Additionally, there is limited information about how uninfected immune cells respond to MDV infection or vaccination, specifically, the mechanisms by which T cells are activated and recognize MDV antigens and how the function and properties of activated T cells correlate with immune protection against MDV or MD tumor. The current review revisits the roles of each immune cell subset and its effector mechanisms in the host immune response to MDV infection or vaccination from the point of view of comparative immunology. We particularly emphasize areas of research requiring further investigation and provide useful information for rational design and development of novel MDV vaccines.