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For the first time, we describe phylogenomic signatures of an epidemic lineage of vesicular stomatitis Indiana virus (VSIV). We applied multiple evolutionary analyses to a dataset of 87 full-length genome sequences representing the circulation of an epidemic VSIV lineage in the US between 2019 and 2020. Based on phylogenetic analyses, we predicted the ancestral relationship of this lineage with a specific group of isolates circulating in the endemic zone of Chiapas, Mexico. Subsequently, our findings indicate that the lineage diversified into at least four different subpopulations during its circulation in the US. We identified single nucleotide polymorphisms (SNPs) that differentiate viral subpopulations and assessed their potential relevance using comparative phylogenetic methods, highlighting the preponderance of synonymous mutations during the differentiation of these populations. Purifying selection was the main evolutionary force favoring the conservation of this epidemic phenotype, with P and G genes as the main drivers of the evolution of this lineage. Our analyses identified multiple codon sites under positive selection and the association of these sites with specific functional domains at P, M, G, and L proteins. Based on ancestral reconstruction analyses, we showed the potential relevance of some of the sites identified under positive selection to the adaptation of the epidemic lineage at the population level. Finally, using a representative group of viruses from Colorado, we established a positive correlation between genetic and geographical distances, suggesting that positive selection on specific codon positions might have favored the adaptation of different subpopulations to circulation in specific geographical settings. Collectively, our study reveals the complex dynamics that accompany the evolution of an epidemic lineage of VSIV in nature. Our analytical framework provides a model for conducting future evolutionary analyses. The ultimate goal is to support the implementation of an early warning system for vesicular stomatitis virus in the US, enabling early detection of epidemic precursors from Mexico.

Bluetongue virus (BTV) is a prevalent midge-borne pathogen that infects ruminant species worldwide. BTV infections range from asymptomatic to lethal, with mechanisms that determine the severity of infection remaining largely undefined. Although it is relatively poorly understood, the immune response to BTV infection is thought to be critical for both the propagation of disease as well as the resolution of infection. To bridge this gap in knowledge, we infected cohorts of sheep and muntjac deer with two serotypes of BTV (BTV10 and BTV17) for longitudinal analysis (30 days). Interestingly, species-specific differences were observed. Circulating virus was detected early and remained detectable for the duration of the sheep study, while infections in muntjac showed faltering detection of BTV10 at 3 weeks post infection. The magnitude of the immune response was subdued in the muntjac when compared to the sheep cohorts, though similar responses were observed. We also assessed midge viral uptake and the ability to replicate BTV. Midges successfully fed on both species, yet those that fed on sheep resulted in more efficient BTV transmission. Our findings demonstrate that differences in BTV infections, immune responses, and vector competence across host species and serotypes will impact global BTV emergence and strategies for mitigation.

Bluetongue (BT) is a Culicoides midge-borne hemorrhagic disease affecting cervids and ruminant livestock species, resulting in significant economic losses from animal production and trade restrictions. Experimental animal infections using the α/β interferon receptor knockout IFNAR mouse model and susceptible target species are critical for understanding viral pathogenesis, virulence, and evaluating vaccines. However, conducting experimental vector-borne transmission studies with the vector itself are logistically difficult and experimentally problematic. Therefore, experimental infections are induced by hypodermic injection with virus typically derived from baby hamster kidney (BHK) cells. Unfortunately, for many U.S. BTV serotypes, it is difficult to replicate the severity of the disease seen in natural, midge-transmitted infections by injecting BHK-derived virus into target host animals. Using the IFNAR BTV murine model, we compared the virulence of traditional BHK cell-derived BTV-17 with C. sonorensis midge (W8) cell-derived BTV-17 to determine whether using cells of the transmission vector would provide an in vitro virulence aspect of vector-transmitted virus. At both low and high doses, mice inoculated with W8-BTV-17 had an earlier onset of viremia, earlier onset and peak of clinical signs, and significantly higher mortality compared to mice inoculated with BHK-BTV-17. Our results suggest using a Culicoides W8 cell-derived inoculum may provide an in vitro vector-enhanced infection to more closely represent disease levels seen in natural midge-transmitted infections while avoiding the logistical and experimental complexity of working with live midges.

ABSTRACTCourse-based undergraduate research experiences (CUREs) provide opportunities for undergraduate students to engage in authentic research and generally increase the participation rate of students in research. Students’ participation in research has a positive impact on their science identity and self-efficacy, both of which can predict integration of students in Science, Technology, Engineering, and Math (STEM), especially for underrepresented students. The main goal of this study was to investigate instructor-initiated CUREs implemented as upper-level elective courses in the Biomedical Sciences major. We hypothesized that these CUREs would (i) have a positive impact on students’ scientific identity and self-efficacy and (ii) result in gains in students’ self-assessed skills in laboratory science, research, and science communication. We used Likert-type surveys developed by Estrada et al. (14) under the Tripartite Integration Model of Social Influence to measure scientific identity, self-efficacy, and scientific value orientation. When data from all CUREs were combined, our results indicate that students’ self-efficacy and science identity significantly increased after completion. Students’ self-assessment of research and lab-related skills was significantly higher after completion of the CUREs. We also observed that prior to participation in the CUREs, students’ self-assessment of molecular and bioinformatic skills was low, when compared with microbiological skills. This may indicate strengths and gaps in our curriculum that could be explored further.

The SARS-CoV-2 pandemic showed limitations in human outbreak testing. Veterinary diagnostic laboratories (VDLs) possess capabilities to bolster emergency test capacity. Surveys from 26 participating VDLs found human SARS-CoV-2 testing was mutually beneficial, including One Health benefits. VDLs indicated testing >3.8 million human samples during the pandemic, which included some challenges.

Bluetongue virus (BTV) is an economically important pathogen of ruminant species with worldwide prevalence. While many BTV infections are asymptomatic, animals with symptomatic presentation deteriorate quickly with the sickest succumbing to disease within one week. Animals that survive the infection often require months to recover. The immune response to BTV infection is thought to play a central role in controlling the disease. Key to understanding BTV disease is profiling vertebrate host immunological cellular and cytokine responses. Studies to characterize immune responses in ruminants have been limited by a lack of species-specific reagents and assay technology. Here we assess the longitudinal immunological response to experimental BTV-17-California (CA) infection in sheep using the most up to date assays. We infected a cohort of sheep with BTV-17-CA and longitudinally monitored each animal for clinical disease, viremia and specific immunological parameters (B cells, T cells, monocytes) by RT-qPCR, traditional flow cytometry and/or fluorescent based antibody arrays. BTV-inoculated sheep exhibited clinical signs characteristic of bluetongue virus disease. Circulating virus was demonstrated after 8 days post inoculation (DPI) and remained detectable for the remainder of the time course (24 DPI). A distinct lymphopenia was observed between 7 and 14 DPI that rebounded to mock-inoculated control levels at 17 DPI. In addition, we observed increased expression of pro-inflammatory cytokines after 8 DPI. Taken together, we have established a model of BTV infection in sheep and have successfully monitored the longitudinal vertebrate host immunological response and viral infection progression using a combination of traditional methods and cutting-edge technology.

Bluetongue virus (BTV) is a segmented, double-stranded RNA orbivirus listed by the World Organization for Animal Health and transmitted by Culicoides biting midges. Segmented viruses can reassort, which facilitates rapid and important genotypic changes. Our study evaluated reassortment in Culicoides sonorensis midges coinfected with different ratios of BTV-10 and BTV-17. Midges were fed blood containing BTV-10, BTV-17, or a combination of both serotypes at 90:10, 75:25, 50:50, 25:75, or 10:90 ratios. Midges were collected every other day and tested for infection using pan BTV and cox1 (housekeeping gene) qRT-PCR. A curve was fit to the ∆Ct values (pan BTV Ct—cox1 Ct) for each experimental group. On day 10, the midges were processed for BTV plaque isolation. Genotypes of the plaques were determined by next-generation sequencing. Pairwise comparison of ∆Ct curves demonstrated no differences in viral RNA levels between coinfected treatment groups. Plaque genotyping indicated that most plaques fully aligned with one of the parental strains; however, reassortants were detected, and in the 75:25 pool, most plaques were reassortant. Reassortant prevalence may be maximized upon the occurrence of reassortant genotypes that can outcompete the parental genotypes. BTV reassortment and resulting biological consequences are important elements to understanding orbivirus emergence and evolution.

Bluetongue virus (BTV) is a segmented, double-stranded RNA virus transmitted by Culicoides biting midges. Infection of domestic and wild ruminants with BTV can result in a devastating disease and significant economic losses. As a virus with a segmented genome, reassortment among the BTV serotypes that have co-infected a host may increase genetic diversity, which can alter BTV transmission dynamics and generate epizootic events. The objective of this study was to determine the extent of dissemination and characterize the tropism of BTV serotypes 10 and 17 in co-infected Culicoides sonorensis. Midges were exposed to both BTV serotypes via blood meal and processed for histologic slides 10 days after infection. An in situ hybridization approach was employed using the RNAscope platform to detect the nucleic acid segment 2 of both serotypes. Observations of the mosaic patterns in which serotypes did not often overlap suggest that co-infection at the cellular level may not be abundant with these two serotypes in C. sonorensis. This could be a consequence of superinfection exclusion. Understanding BTV co-infection and its biological consequences will add an important dimension to the modeling of viral evolution and emergence.

During early phases of the SARS-CoV-2 epidemic, many research laboratories repurposed their efforts towards developing diagnostic testing that could aid public health surveillance while commercial and public diagnostic laboratories developed capacity and validated large scale testing methods. Simultaneously, the rush to produce point-of-care and diagnostic facility testing resulted in FDA Emergency Use Authorization with scarce and poorly validated clinical samples. Here, we review serologic test results from 186 serum samples collected in early phases of the pandemic (May 2020) from skilled nursing facilities tested with six laboratory-based and two commercially available assays. Serum neutralization titers were used to set cut-off values using positive to negative ratio (P/N) analysis to account for batch effects. We found that laboratory-based receptor binding domain (RBD) binding assays had equivalent or superior sensitivity and specificity compared to commercially available tests. We also determined seroconversion rate and compared with qPCR outcomes. Our work suggests that research laboratory assays can contribute reliable surveillance information and should be considered important adjuncts to commercial laboratory testing facilities during early phases of disease outbreaks.