Biology subjects

Khatiwada, S.

Publications and source records attributed to Khatiwada, S..

5 recordsLinked to original sources

bioRxiv2026

Novel recombinant bovine papular stomatitis virus expressing foot and mouth disease virus-like particles elicits protective levels of neutralizing antibodies in cattle

Foot-and-mouth disease (FMD) remains a major burden in endemic regions, where inactivated vaccines are constrained by cost, short duration of immunity, cold-chain dependence, and high-containment manufacturing. Here, we engineered a recombinant bovine papular stomatitis virus (rBPSV) expressing the FMDV A24 Cruzeiro capsid precursor P1-2A together with an attenuated 3C protease (3Cpro L127P). Infection of ovine fetal turbinate (OFTu) cells resulted in robust capsid protein expression and assembly of abundant 25-30 nm icosahedral virus-like particles (VLPs). Intramuscular immunization of two BPSV-seropositive calves with rBPSV (107 TCID50 on days 0, 21 and 35) induced strong anti-FMDV humoral responses. By day 28, liquid-phase blocking ELISA (LPBE) titers exceeded laboratory defined protective threshold ([≥]1.9 log10), and homologous neutralizing titers against A24 Cruzeiro surpassed the threshold associated with protection ([≥]1.36 log10). Intra-serotypic cross-neutralization was observed against A/Argentina/2001, whereas no neutralization was detected against heterologous serotype O1 Campos. Pre-existing anti-BPSV antibodies did not prevent induction of neutralizing responses. These findings establish first proof-of-concept that BPSV can serve as a cattle-adapted vector platform for delivery of FMDV VLPs and other heterologous antigens.

immunology↗

bioRxiv2024

A porcine chronic hepatitis E virus (HEV) infection model exhibits HEV replication in male accessory reproductive glands and immune-mediated reproductive damage

Hepatitis E virus (HEV) is an expanding zoonotic viral disease threat. Although HEV causes acute viral hepatitis, it is increasingly being recognized as a systemic pathogen with detection and damage in extrahepatic tissues. The presence of HEV RNA in the semen of chronically infected human patients in the absence of viremia and fecal shedding and presence of HEV in the sperm head underscores the need to understand the interaction of HEV within the male reproduction system. Male accessory glands secrete biofluids necessary for sperm nourishment and to neutralize the acidity of the vagina. The role of male accessory glands in the dissemination and persistence of HEV infection have not been studied. Using an immunosuppressed pig model for chronic HEV infection, we demonstrate infectious HEV in mature sperm cells altering the sperm motility and morphology. HEV isolated from sperm cells remained infectious in human hepatoma cells. Spermatic fluid contained lower virus titers than the sperm cells from chronically infected pigs highlighting that the sperm cells themselves can associate with the virus. Evaluation of the male accessory glands demonstrated viral replication, infiltration of CD45 leukocytes, and apoptosis associated with HEV infection. A decrease in serum testosterone levels was evident in the HEV infected pigs. Even though a lower viral RNA titer was seen in serum and feces of chronically infected, immunosuppressed and ribavirin treated pigs, high viral RNA and infectious particles in sperm is a concern. Our findings necessitate further studies defining the mechanism of sperm cell invasion by HEV, length of HEV survival in sperm cells during chronic HEV infection, and risk of sexual transmission of HEV during both acute and chronic phases of infection. Author SummaryHepatitis E virus, a leading cause of acute viral hepatitis, causes both acute and chronic infection in humans. Recent advances within the HEV field have demonstrated extrahepatic diseases associated with HEV. More recent findings have revealed infectious HEV in the vagina, Sertoli cells, and ejaculate of humans, and sperm cells of pigs. We demonstrate that the male accessory sex glands may have a role in the persistence of HEV infection during chronic infections. We utilized an established immunosuppressed pig model and treated pigs with ribavirin to study the presence of virus in the sperm cells. We demonstrated high viral RNA loads and infectious particles associated with sperm cells. Our study further highlights the importance of the testis, as an immune privileged site, in the maintenance of chronic HEV infection. New studies to evaluate the mechanisms by which HEV associates with sperm cells, the length of HEV survival in sperm cell fractions, and consideration of the testes as a potential HEV reservoir are necessary.

microbiology↗

bioRxiv2023

Rat hepatitis E virus (HEV) cross-species infection and transmission in pigs

Rocahepevirus ratti, an emerging hepatitis E virus (HEV), has recently been found to be infectious to humans. Rats are a primary reservoir of the virus; thus, it is referred to as "rat HEV". Rats are often found on swine farms in close contact with pigs. Our goal was to determine whether swine may serve as a transmission host for rat HEV by characterizing an infectious cDNA clone of a zoonotic rat HEV, strain LCK-3110, in vitro and in vivo. RNA transcripts of LCK-3110 were constructed and assessed for their replicative capacity in cell culture and in gnotobiotic pigs. Fecal suspension from rat HEV-positive gnotobiotic pigs was inoculated into conventional pigs cohoused with naive pigs. Our results demonstrated that capped RNA transcripts of LCK-3110 rat HEV replicated in vitro and successfully infected conventional pigs that transmit the virus to cohoused animals. The infectious clone of rat HEV may afford an opportunity to study the genetic mechanisms of rat HEV cross-species infection and tissue tropism. Significance StatementNew zoonotic strains of Rocahepevirus ratti (rat HEV) have emerged infecting both immunocompetent and immunosuppressed people through unknown transmission sources. Pigs are a primary source of transmission for human HEV strains and could be serving a similar role for rat HEV transmission as rats are a common pest found on swine farms worldwide. Rats could be transmitting rat HEV to pigs which could then be transmitted to humans. Determining susceptibility of pigs to emerging zoonotic rat HEV strains can define potentially new transmission routes to inform public health policy and could provide pathology models for rat HEV disease.

microbiology↗

bioRxiv2022

Genomic Variation, Population Structure, and Gene Flow across Asian Pikas

Asian pikas have one of the most complex systematics and evolutionary history. The Himalayas is an important habitat for Asian pikas as it hosts 23 - 25% of pikas global diversity and has provided the ancestral training ground for local adaptation to high altitudes. They are one of the most abundant species in the Himalayas and Qinghai Tibetan Plateau (QTP), however genetic studies to explore their population structure and evolution are limited. Here, we utilize a population genomics approach using ~28,000 genome-wide SNP markers obtained from restriction-site associated DNA sequencing in six species of Asian Pikas (Ochotona spp.) that are distributed across the Himalayas and neighboring high-altitude mountains. We examined intra- and inter-species genetic diversity, population structure, phylogenetic history and explored processes that shaped the current genetic diversity of Pikas across the Himalayas. We identified low nucleotide diversity and high inbreeding coefficient across all species which possibly indicated decreasing population size in these species. We also identified extensive evidence of gene flow (both historic and contemporary) across these species. Our findings indicate that inter-species gene flow is a key evolutionary process that has been countering the negative effect of low genetic diversity among Asian pikas.

evolutionary biology↗

bioRxiv2020

African Swine Fever Virus CD2v protein promotes β-Interferon expression and apoptosis in swine cells

African swine fever (ASF) is a disease of swine characterized by massive lymphocyte depletion in lymphatic tissues due to apoptosis of B and T cells, most likely triggered by proteins or factors secreted by infected adjacent macrophages. Here we describe a role for the ASF virus (ASFV) protein CD2v in apoptosis induction in lymphocytes. CD2v is a viral homolog of host CD2 that has been implicated in viral virulence and immunomodulation in vitro; however, its actual function remains unknown. We show that CD2v is secreted into culture medium of CD2v-expressing swine cells; and expression of-or treatment with CD2v led to significant induction of IFN-{beta}/ISGs transcription and antiviral state. CD2v expression led to enhanced NF-{kappa}B-p65 nuclear translocation in these cultures and incubation with a NF-{kappa}B inhibitor reduced CD2v-induced NF-{kappa}B-p65 nuclear translocation and IFN-{beta} transcription. We show that CD2v binds CD58, the natural CD2 ligand, and that CD58 siRNA knock-down results in significant reduction in NF-{kappa}B-p65 nuclear translocation and IFN-{beta} transcription. Treatment of swine PBMC with purified CD2v led to enhanced NF-{kappa}B-p65 nuclear translocation and induction of IFN-{beta} transcription. Further, induction of caspase-3 and PARP1 cleavage was observed in these swine PBMC at later times, providing a mechanism for CD2v-induced apoptosis of lymphocytes. Finally, IFN-{beta} induction and NF-{kappa}B activation was inhibited in swine PBMC treated with purified CD2v pre-incubated with antibodies against CD2v. Overall, our results indicate that ASFV CD2v is an immunomodulatory protein that, by promoting lymphocyte apoptosis, may contribute to bystander lymphocyte depletion observed during ASFV infection in pigs. IMPORTANCEASF, a severe hemorrhagic disease of domestic swine, represents a significant economic threat to swine industry worldwide. One critical pathological event observed in pigs infected with virulent isolates is an extensive destruction of lymphoid tissue and massive lymphocyte depletion. However, viral factor/s involved in this event are yet to be identified. Here we show that, by inducing NF-{kappa}B-dependent IFN signaling, ASFV CD2v is able to promote apoptosis in swine PBMC. We propose that CD2v released by ASFV-infected macrophages contributes to the massive depletion of lymphocytes observed in lymphoid tissues of infected pigs. Results here improve our understanding of ASFV pathogenesis and will encourage novel intervention approaches.

microbiology↗