Development and Evaluation of Vaccine Candidates for Senecavirus A

Senecavirus A (SVA) has been associated with several outbreaks of vesicular disease (VD) since 2014 in major swine-producing countries around the world. The virus causes VD that is clinically indistinguishable from foot-and-mouth disease (FMD) in pigs. Currently there are no commercial vaccines available for SVA. Here, we developed a live attenuated SVA strain by mutating a wild type SVA strain SD15-26 (wt SVA). The live attenuated virus, rSVA mSacII, replicated less efficiently in cell cultures in vitro and expressed lower amounts of VP1 and VP2 capsid proteins than wt SVA. Most importantly, the rSVA mSacII virus failed to induce clinical signs in experimentally infected pigs. Additionally, animals inoculated with rSVA mSacII presented lower levels of viremia, virus shedding and viral load in tissues than wt SVA. Notably, despite its attenuated phenotype animals inoculated with rSVA mSacII developed similar virus neutralizing antibody responses to those animals inoculated with the wt SVA. This led us to assess the immunogenicity and protective efficacy of the rSVA mSacII virus. For this, the rSVA mSacII was either inactivated with aziridine compound binary ethyleneamine (BEI) to be used as an inactivated or used as a live vaccine administered via the intramuscular (IM) or intranasal (IN) routes. An animal experiment was conducted consisting of four groups each containing 6 pigs: control group was sham-immunized with plain RPMI-1640; inactivated group was immunized with 106 TCID50 of BEI inactivated virus; live IM group was immunized with 106 TCID50 of live rSVA mSacII via IM route; and live IN group was immunized with 106 TCID50 of live rSVA mSacII via the IN route. None of the immunized animals developed clinical signs nor lesions of SVA. RT-qPCR was performed on serum, oral, nasal and rectal swabs to assess viremia and virus shedding. Live immunized animals were viremic and shed virus, decreasing gradually after 7-14 days post-immunization. SVA specific virus neutralizing antibodies began to appear 3-5 days pi in live group and 14 days pi in inactivated group. The peak titer of inactivated vaccine was, however, lower than live vaccinated group even after a booster immunization at 21 days pi. After PBMC recall stimulation by UV inactivated SVA and recombinant SVA-VP2 protein, we observed higher proliferation of CD4+, CD8+ and CD4+CD8+ T cells in live vaccinated groups than other groups. At 42 days pi, challenge with 108.5 TCID50 of a heterologous SVA strain was performed and the protective efficacy of the rSVA mSacII virus evaluated. We observed clinical signs and vesicular lesions characteristic of SVA in control and inactivated group four days post-challenge. None of the animals in live vaccinated group displayed any clinical signs. Both live vaccinated groups presented lower levels of viremia and shed less virus than control group, however, level the of viremia and virus shedding in inactivated group was similar to control group. Viral load in tissues including tonsil, mediastinal and mesenteric lymph nodes were also assessed. Similar to virological findings in serum and secretions, viral loads in tissues were also lower in live vaccine groups when compared to control and inactivated groups. A negative correlation was observed between viremia/virus shedding with neutralizing antibody titer and with percentage proliferative CD4+, CD8+ and CD4+/CD8+ T cells. Similar negative correlation was observed between viral load in tissues with neutralizing antibody titer and with percentage proliferative CD8+ and CD4+/CD8+ T cells. These findings show that live vaccination by rSVA mSacII protects against SVA challenge and the vaccine candidate has potential as a tool to control SVA infection

Sero-prevalence of Toxoplasma gondii in sheep in different geographical regions of Nepal

The present study was conducted to investigate the prevalence of Toxoplasma gondii in sheep in Nepal. Blood samples were collected from 235 sheep from three districts of three different eco-zones namely, mountainous Jumla (88), hilly Pokhara (62) and plain/terai Chitwan (85). The samples were tested by using commercial ELISA kit. The overall prevalence of T. gondii infection in sheep was 36.17% (CI: 30.29–42.49%). The region wise prevalence showed highest in Chitwan (57.65%; CI: 47.04–67.60%), followed by Pokhara (32.94%; CI: 23.88–43.48%) and Jumla (9.41%; CI: 4.85–17.49%). Prevalence of T. gondii in Jumla was significantly lower than Pokhara and Chitwan (p 0.05). Similarly, no significant difference (p > 0.05) in prevalence of T. gondii was found in sex and age groups (p > 0.05). The result showed that T. gondii parasite is widely spread in the studied geographical regions of Nepal. Keywords: ELISA, Seroprevalence, Toxoplasma gondii, Nepa

RNA N⁶-Methyladenosine Affects Copper-Induced Oxidative Stress Response in <i>Arabidopsis thaliana</i>

Recently, post-transcriptional regulation of mRNA mediated by N6-methyladenosine (m6A) has been found to have profound effects on transcriptome regulation during plant responses to various abiotic stresses. However, whether this RNA modification can affect an oxidative stress response in plants has not been studied. To assess the role of m6A modifications during copper-induced oxidative stress responses, m6A-IP-seq was performed in Arabidopsis seedlings exposed to high levels of copper sulfate. This analysis revealed large-scale shifts in this modification on the transcripts most relevant for oxidative stress. This altered epitranscriptomic mark is known to influence transcript abundance and translation; therefore we scrutinized these possibilities. We found an increased abundance of copper-enriched m6A-containing transcripts. Similarly, we also found increased ribosome occupancy of copper-enriched m6A-containing transcripts, specifically those encoding proteins involved with stress responses relevant to oxidative stressors. Furthermore, the significance of the m6A epitranscriptome on plant oxidative stress tolerance was uncovered by assessing germination and seedling development of the mta (N6-methyladenosine RNA methyltransferase A mutant complemented with ABI3:MTA) mutant exposed to high copper treatment. These analyses suggested hypersensitivity of the mta mutant compared to the wild-type plants in response to copper-induced oxidative stress. Overall, our findings suggest an important role for m6A in the oxidative stress response of Arabidopsis