Immune Responses to Influenza D Virus in Calves Previously Infected with Bovine Viral Diarrhea Virus 2.

Bovine viral diarrhea virus (BVDV) induces immunosuppression and thymus depletion in calves. This study explores the impact of prior BVDV-2 exposure on the subsequent immune response to influenza D virus (IDV). Twenty 3-week-old calves were divided into four groups. Calves in G1 and G3 were mock-treated on day 0, while calves in G2 and G4 received BVDV. Calves in G1 (mock) and G2 (BVDV) were necropsied on day 13 post-infection. IDV was inoculated on day 21 in G3 calves (mock + IDV) and G4 (BVDV + IDV) and necropsy was conducted on day 42. Pre-exposed BVDV calves exhibited prolonged and increased IDV shedding in nasal secretions. An approximate 50% reduction in the thymus was observed in acutely infected BVDV calves (G2) compared to controls (G1). On day 42, thymus depletion was observed in two calves in G4, while three had normal weight. BVDV-2-exposed calves had impaired CD8 T cell proliferation after IDV recall stimulation, and the α/ß T cell impairment was particularly evident in those with persistent thymic atrophy. Conversely, no difference in antibody levels against IDV was noted. BVDV-induced thymus depletion varied from transient to persistent. Persistent thymus atrophy was correlated with weaker T cell proliferation, suggesting correlation between persistent thymus atrophy and impaired T cell immune response to subsequent infections.

Viability of Veterinary-Relevant Viruses in Decomposing Tissues over a 90-Day Period Using an In-Vitro System.

Depopulation is frequently employed during outbreaks of high-impact animal diseases. Security breaches in sites managing mortality may jeopardize pathogen control efforts as infected carcasses can serve as an infection source. This study evaluated the viability and nucleic acid detection of veterinary-relevant viruses or their surrogates in decomposing tissues. The used viruses were: Senecavirus A1 (SVA), feline calicivirus (FCV), bovine viral diarrhea virus (BVDV), porcine epidemic diarrhea virus (PEDV), bovine alphaherpesvirus 1 (BoHV-1), and swinepox virus (SwPV). Viruses were spiked in three decomposing tissues (swine bone marrow and spleen, and bovine bone marrow) and maintained for 90 days. Samples were kept under two temperature conditions resembling the average soil temperature in central Oklahoma, US, during the winter and summer (5.5 °C and 29.4 °C). At 5.5 °C, SVA and FCV remained viable over the 90 days of the study, followed by BVDV (75 days), BoHV-1 and SwPV (60 days), and PEDV (10 days). At 29.4 °C, SVA remained viable for 45 days, followed by BVDV and BoHV-1 (14 days). SwPV was viable for 10 days, whereas FCV and PEDV were viable for 5 days. Overall, viral nucleic acid detection was not significantly altered during the study. These findings support decision-making and risk management in sites overseeing animal mortality.

Pathogenesis and Host Responses to Viral Diseases in Livestock Species.

Infectious diseases in livestock species are responsible for significant economic losses worldwide and constantly threaten food security [...].

HoBi-like Pestivirus Is Highly Prevalent in Cattle Herds in the Amazon Region (Northern Brazil).

Pestiviruses are globally distributed and cause substantial economic losses to the cattle industry. In Brazil, the country with the world's largest cattle population, pestivirus infections are well described in some regions, such as in the south, where a high frequency of BVDV-2 is described and contrasts with the high prevalence of HoBi-like pestivirus (HoBiPeV) in the northeast. However, there is a lack of information about pestiviruses in the Amazon Region, in northern Brazil, with a cattle population estimated at 55.7 million head, which has a significant impact on the international livestock market. Therefore, this study investigated the seroprevalence and genetic variability of ruminant pestiviruses in 944 bovine serum samples from four states in northern Brazil: Pará (PA), Amapá (AP), Roraima (RR), and Amazonas (AM). Our results showed that 45.4% of the samples were seropositive (19.8% for BVDV-1, 14.1% for BVDV-2, and 20.9% for HoBiPeV). All samples were tested by RT-qPCR, and three were positive and classified as HoBiPeV in a phylogenetic analysis. These serological and molecular results contrast with those from other regions of the world, suggesting that the northern Brazilian states have a high prevalence of all bovine pestiviruses including HoBiPeV.

Evaluation of Ultraviolet Type C Radiation in Inactivating Relevant Veterinary Viruses on Experimentally Contaminated Surfaces.

Many swine farms employ UVC treatment in employees' personal belongings and small tools entering farms as part of the biosecurity protocol to decrease the risk of pathogen introduction into the operation. However, the UVC efficacy in some veterinary viruses is not fully evaluated. This study evaluated the efficacy of ultraviolet type C (UVC) radiation in inactivating seven relevant veterinary viruses: Swine Poxvirus (SwPV), Porcine Reproductive and Respiratory Syndrome Virus (PRRSV), Porcine Epidemic Diarrhea Virus (PEDV), Swine Influenza Virus (SIV), Bovine Viral Diarrhea Virus (BVDV), Porcine Parvovirus (PPV), and Senecavirus A (SVA). The experimentally contaminated materials included polystyrene and filter paper. The samples were exposed to UVC for 5 min (total dose of 360 mJ/cm2). The UVC treatment caused a decrease over 4 log10 in SwPV titer on the polystyrene surface, whereas it consistently reduced about 5 log10 in PPV and SVA samples. No viable virus was recovered from PRRSV, PEDV, SIV, and BVDV samples. In filter paper, conversely, the efficacy was reduced. This study provides essential information on the inactivation effectiveness of a specific dose of UVC on important veterinary viruses, further supporting the rational application and strategic guidance for UVC radiation use to disinfect materials.

Genome sequence and experimental infection of calves with bovine gammaherpesvirus 4 (BoHV-4).

Bovine gammaherpesvirus 4 (BoHV-4) is ubiquitous in cattle worldwide, and it has been detected in animals exhibiting broad clinical presentations. The virus has been detected in the United States since the 1970s; however, its clinical relevance remains unknown. Here, we determined the complete genome sequences of two contemporary BoHV-4 isolates obtained from respiratory (SD16-38) or reproductive (SD16-49) tract specimens and assessed clinical, virological, and pathological outcomes upon intranasal (IN) inoculation of calves with the respiratory BoHV-4 isolate SD16-38. A slight and transient increase in body temperature was observed in BoHV-4-inoculated calves. Additionally, transient viremia and virus shedding in nasal secretions were observed in all inoculated calves. BoHV-4 DNA was detected by nested PCR in the tonsil and regional lymph nodes (LNs) of calves euthanized on day 5 post-inoculation (pi) and in the lungs of calves euthanized on day 10 pi. Calves euthanized on day 35 pi harbored BoHV-4 DNA in the respiratory tract (turbinates, trachea, lungs), regional lymphoid tissues, and trigeminal ganglia. Interestingly, in situ hybridization revealed the presence of BoHV-4 DNA in nerve bundles surrounding the trigeminal ganglia and retropharyngeal lymph nodes (day 35 pi). No histological changes were observed in the respiratory tract (turbinate, trachea, and lung), lymphoid tissues (tonsil, LNs, thymus, and spleen), or central nervous tissues (olfactory bulb and trigeminal ganglia) sampled throughout the animal studies (days 5, 10, and 35 pi). This study contributes to the understanding of the infection dynamics and tissue distribution of BoHV-4 following IN infection in calves. These results suggest that BoHV-4 SD16-38 used in our study has low pathogenicity in calves upon intranasal inoculation.

A Serosurvey for Ruminant Pestivirus Exposure Conducted Using Sera From Stray Mexico Origin Cattle Captured Crossing Into Southern Texas.

The US Department of Agriculture (USDA), Animal Plant Health Inspection Service (APHIS), Cattle Fever Tick Eradication Program (CFTEP) monitor a quarantine zone along the Texas border to prevent the introduction of stray livestock carrying cattle fever ticks entering the United States from Mexico. Stray cattle collected by CFTEP are checked for ticks and several infectious disease-causing pathogens, but not for bovine viral diarrhea virus (BVDV). BVDV is one of the most economically impactful viruses affecting US cattle producers. BVDV is present in all parts of the world, but it has been demonstrated that another distantly related pestivirus, HoBi-like pestivirus (HoBiPev), can also cause BVD. To date, HoBiPev has not been detected in the United States, but is commonly found in Brazil, and sporadically in Europe and Asia. The objective of the current study was to evaluate the seroprevalence of pestiviruses, with a specific focus on HoBiPev, in stray cattle. Virus neutralization (VN) assay was used to determine seroprevalence (or antibody titers) of BVDV-1, BVDV-2, and HoBiPev. Approximately 50% (67 of 134) of the samples were seropositive for pestiviruses; all 67 positive samples were positive (50%) for BVDV-1, 66 samples of the 67 were positive (49.3%) for BVDV-2, and the same 66 samples of the 67 were also positive (49.3%) for HoBiPev. Due to the antigenic cross-reactivity among Pestiviruses, the comparative antibody against each pestivirus was calculated from all VN-positive samples. Titers were clearly higher against BVDV-1, and only one sample had a titer clearly higher against BVDV-2. No sample had an antibody titer higher for HoBiPev, and while this does not prove the absence of HoBiPev, it does provide evidence that the prevalence of HoBiPev is less predominant than BVDV-1. Additionally, data from these samples provide evidence on the susceptibility of animals that may enter into the United States, with ~50% of the animals seronegative for bovine pestiviruses. This cattle population provides a unique opportunity to evaluate and monitor changes in seroprevalence of economically important cattle diseases affecting the cattle industry.

Prolonged Viability of Senecavirus A in Exposed House Flies (Musca domestica).

House flies (Musca domestica) are often present in swine farms worldwide. These flies utilize animal secretions and waste as a food source. House flies may harbor and transport microbes and pathogens acting as mechanical vectors for diseases. Senecavirus A (SVA) infection in pigs occurs via oronasal route, and animals shed high virus titers to the environment. Additionally, SVA possesses increased environmental resistance. Due to these reasons, we investigated the tenacity of SVA in house flies. Five groups of flies, each composed of ten females and ten males, were exposed to SVA, titer of 109.3 tissue culture infectious dose (TCID50/mL). Groups of male and female flies were collected at 0, 6, 12, 24, and 48 h post-exposure. For comparison purposes, groups of flies were exposed to Swinepox virus (SwPV). Infectious SVA was identified in all tested groups. Successful isolation of SVA demonstrated the titers varied between 106.8 and 102.8 TCID50/mL in female groups and varied from 105.85 to 103.8 TCID50/mL in male groups. In contrast, infectious SwPV was only detected in the female group at 6 h. The significant SVA infectious titer for prolonged periods of time, up to 48 h, indicates a potential role of flies in SVA transmission.

Virus viability in spiked swine bone marrow tissue during above-ground burial method and under in vitro conditions.

The emergence of high consequence animal diseases usually requires managing significant mortality. A desirable aspect of any carcass management method is the ability to contain and inactivate the target pathogen. The above-ground burial (AGB) technique was recently developed and proposed as an alternative carcass management method. Here, we investigate the tenacity of swinepox virus (SwPV), as a surrogate model for African swine fever virus (ASFV) in swine carcasses during the AGB process. For this, SwPV was inoculated intrafemorally in 90 adult swine carcasses, which were subsequently disposed under AGB conditions. Bone marrow samples were recovered periodically throughout 12 months and virus viability was assessed by virus isolation (VI), whereas the presence of SwPV DNA was evaluated by quantitative polymerase chain reaction (qPCR). Additionally, an in vitro study assessed the inactivation rate of SwPV, Senecavirus A (SVA), and bovine viral diarrhoea virus (BVDV). Viral suspensions were mixed with bone marrow material and maintained at 21-23°C for 30 days. Virus viability was assessed by VI and viral titration. In the field study, SwPV remained viable only in 11 (55%) bone marrow samples collected on day 7; only viral DNA (and not infectivity) was detected afterwards. SwPV inactivation was estimated to have occurred by day 11. The in vitro testing revealed a variable tenacity of the studied viruses. The viability period was estimated in 28, 80, and 118 days, respectively, for BVDV, SwPV, and SVA. Overall, these findings indicate that the AGB technique was effective in quickly inactivating SwPV. Additionally, the SwPV inactivation rate is comparable to ASFV under field studies and poses a potential model for preliminary ASFV inactivation studies with reduced biosecurity requirements. Moreover, this study contributes to understanding the inactivation kinetics of viruses under specific conditions, which is critical when designing and applying countermeasures in case of biosecurity breaches in sites managing animal mortality.

Virome Characterization in Commercial Bovine Serum Batches-A Potentially Needed Testing Strategy for Biological Products.

Bovine serum has been widely used as a universal supplement in culture media and other applications, including the manufacture of biological products and the production of synthetic meat. Currently, commercial bovine serum is tested for possible viral contaminants following regional guidelines. Regulatory agencies' established tests focused on detecting selected animal origin viruses and are based on virus isolation, immunofluorescence, and hemadsorption assays. However, these tests may fail to detect new or emerging viruses in biological products. High-throughput sequencing is a powerful option since no prior knowledge of the viral targets is required. In the present study, we evaluate the virome of seven commercial batches of bovine serum from Mexico (one batch), New Zealand (two batches), and the United States (four batches) using a specific preparation and enrichment method for pooled samples and sequencing using an Illumina platform. A variety of circular replicase-encoding single-stranded (CRESS) DNA families (Genomoviridae, Circoviridae, and Smacoviridae) was identified. Additionally, CrAssphage, a recently discovered group of bacteriophage correlated with fecal contamination, was identified in 85% of the tested batches. Furthermore, sequences representing viral families with single-stranded DNA (Parvoviridae), double-stranded DNA (Polyomaviridae and Adenoviridae), single-stranded RNA (Flaviviridae, Picornaviridae, and Retroviridae), and double-stranded RNA (Reoviridae) were identified. These results support that high-throughput sequencing associated with viral enrichment is a robust tool and should be considered an additional layer of safety when testing pooled biologicals to detect viral contaminants overlooked by the current testing protocols.

Epidemiology of Pestivirus H in Brazil and Its Control Implications.

Along with viruses in the Pestivirus A (Bovine Viral Diarrhea Virus 1, BVDV1) and B species (Bovine Viral Diarrhea Virus 2, BVDV2), members of the Pestivirus H are mainly cattle pathogens. Viruses belonging to the Pestivirus H group are known as HoBi-like pestiviruses (HoBiPev). Genetic and antigenic characterization suggest that HoBiPev are the most divergent pestiviruses identified in cattle to date. The phylogenetic analysis of HoBiPev results in at least five subgroups (a-e). Under natural or experimental conditions, calves infected with HoBiPev strains typically display mild upper respiratory signs, including nasal discharge and cough. Although BVDV1 and BVDV2 are widely distributed and reported in many South American countries, reports of HoBiPev in South America are mostly restricted to Brazil. Despite the endemicity and high prevalence of HoBiPev in Brazil, only HoBiPev-a was identified to date in Brazil. Unquestionably, HoBiPev strains in BVDV vaccine formulations are required to help curb HoBiPev spread in endemic regions. The current situation in Brazil, where at this point only HoBiPev-a seems present, provides a more significant opportunity to control these viruses with the use of a vaccine with a single HoBiPev subtype. Despite the lack of differentiation among bovine pestiviruses by current BVDV tests, the reduced genetic variability of HoBiPev in Brazil may allow reliable identification of cases within the region. On the other hand, introducing foreign ruminants, biologicals, and genetic material to South America, especially if it originated from other HoBiPev-endemic countries, should consider the risk of introducing divergent HoBiPev subtypes.

Identification and genetic characterization of an isolate of bovine adenovirus 7 from the United States, a putative member of a new species in the genus Atadenovirus.

The bovine adenovirus 7 (BAdV-7) isolate SD18-74 was recovered from lung tissue of calves in South Dakota. The 30,043-nucleotide (nt) genome has the typical organization of Atadenovirus genus members. The sequence shares over 99% nt sequence identity with two Japanese BAdV-7 sequences, followed by 74.9% nt sequence identity with the ovine adenovirus 7 strain OAV287, a member of the species Ovine atadenovirus D. SD18-74 was amplified in both bovine and ovine primary nasal turbinate cells, demonstrating greater fitness in bovine cells. The genomic and biological characteristics of BAdV-7 SD18-74 support the inclusion of the members of the BAdV-7 group in a new species in the genus Atadenovirus.

Virome characterization in serum of healthy show pigs raised in Oklahoma demonstrated great diversity of ssDNA viruses.

In the United States, show pigs are raised to compete in agricultural events. These animals are usually raised in small herds with extensive human, domestic, and wild animal contact. Therefore, pathogen monitoring in this animal category is critical for improved disease surveillance and preparedness. This study characterized the virome of healthy show pigs using high-throughput sequencing using pooled serum samples from 2018 or 2019 (200 samples each pool). Results demonstrated the presence of DNA viral families (Parvoviridae, Circoviridae, and Herpesviridae) and RNA families (Arteriviridae, Flaviviridae, and Retroviridae). Twenty-three viral species were identified, including the first detection of porcine bufavirus in the US. Moreover, important swine pathogens identified included porcine reproductive and respiratory syndrome virus, atypical porcine pestivirus, and porcine circovirus (PCV). Additionally, complete coding genomes of 17 viruses from the Parvoviridae, Anelloviridae, and Circoviridae families were retrieved and included the first near full-length genomes of US Ungulate bocaparvovirus 3 species.

Sequential exposure to bovine viral diarrhea virus and bovine coronavirus results in increased respiratory disease lesions: clinical, immunologic, pathologic, and immunohistochemical findings.

Bovine coronaviruses (BoCVs) have been found in respiratory tissues in cattle and frequently associated with bovine respiratory disease (BRD); however, pathogenesis studies in calves are limited. To characterize the pathogenesis and pathogenicity of BoCV isolates, we used 5 different BoCV strains to inoculate colostrum-deprived calves, ~ 2-5 wk of age. Later, to determine if dual viral infection would potentiate pathogenicity of BoCV, calves were inoculated with BoCV alone, bovine viral diarrhea virus (BVDV) alone, or a series of dual-infection (BVDV-BoCV) schemes. A negative control group was included in all studies. Clinical signs and body temperature were monitored during the study and samples collected for lymphocyte counts, virus isolation, and serology. During autopsy, gross lesions were recorded and fixed tissues collected for histopathology and immunohistochemistry; fresh tissues were collected for virus isolation. Results suggest increased pathogenicity for isolate BoCV OK 1776. Increased body temperature was found in all virus-inoculated groups. Lung lesions were present in calves in all dual-infection groups; however, lesions were most pronounced in calves inoculated with BVDV followed by BoCV inoculation 6 d later. Lung lesions were consistent with mild-to-moderate interstitial pneumonia, and immunohistochemistry confirmed the presence of BoCV antigen. Our studies demonstrated that BVDV-BoCV dual infection may play an important role in BRD pathogenesis, and timing between infections seems critical to the severity of lesions.

Serosurvey for Influenza D Virus Exposure in Cattle, United States, 2014-2015.

Influenza D virus has been detected predominantly in cattle from several countries. In the United States, regional and state seropositive rates for influenza D have previously been reported, but little information exists to evaluate national seroprevalence. We performed a serosurveillance study with 1,992 bovine serum samples collected across the country in 2014 and 2015. We found a high overall seropositive rate of 77.5% nationally; regional rates varied from 47.7% to 84.6%. Samples from the Upper Midwest and Mountain West regions showed the highest seropositive rates. In addition, seropositive samples were found in 41 of the 42 states from which cattle originated, demonstrating that influenza D virus circulated widely in cattle during this period. The distribution of influenza D virus in cattle from the United States highlights the need for greater understanding about pathogenesis, epidemiology, and the implications for animal health.

Persistent Infection and Transmission of Senecavirus A from Carrier Sows to Contact Piglets.

Senecavirus A (SVA) is a picornavirus that causes acute vesicular disease (VD), that is clinically indistinguishable from foot-and-mouth disease (FMD), in pigs. Notably, SVA RNA has been detected in lymphoid tissues of infected animals several weeks following resolution of the clinical disease, suggesting that the virus may persist in select host tissues. Here, we investigated the occurrence of persistent SVA infection and the contribution of stressors (transportation, immunosuppression, or parturition) to acute disease and recrudescence from persistent SVA infection. Our results show that transportation stress leads to a slight increase in disease severity following infection. During persistence, transportation, immunosuppression, and parturition stressors did not lead to overt/recrudescent clinical disease, but intermittent viremia and virus shedding were detected up to day 60 postinfection (p.i.) in all treatment groups following stress stimulation. Notably, real-time PCR and in situ hybridization (ISH) assays confirmed that the tonsil harbors SVA RNA during the persistent phase of infection. Immunofluorescence assays (IFA) specific for double-stranded RNA (dsRNA) demonstrated the presence of double-stranded viral RNA in tonsillar cells. Most importantly, infectious SVA was isolated from the tonsil of two animals on day 60 p.i., confirming the occurrence of carrier animals following SVA infection. These findings were supported by the fact that contact piglets (11/44) born to persistently infected sows were infected by SVA, demonstrating successful transmission of the virus from carrier sows to contact piglets. Results here confirm the establishment of persistent infection by SVA and demonstrate successful transmission of the virus from persistently infected animals.IMPORTANCE Persistent viral infections have significant implications for disease control strategies. Previous studies demonstrated the persistence of SVA RNA in the tonsil of experimentally or naturally infected animals long after resolution of the clinical disease. Here, we showed that SVA establishes persistent infection in SVA-infected animals, with the tonsil serving as one of the sites of virus persistence. Importantly, persistently infected carrier animals shedding SVA in oral and nasal secretions or feces can serve as sources of infection to other susceptible animals, as evidenced by successful transmission of SVA from persistently infected sows to contact piglets. These findings unveil an important aspect of SVA infection biology, suggesting that persistently infected pigs may function as reservoirs for SVA.

Analysis of tRNA halves (tsRNAs) in serum from cattle challenged with bovine viral diarrhea virus.

Acute infections of bovine viral diarrhea virus (BVDV) lead to a range of clinical presentations. Laboratory tests for detection depend on collection of samples during a short viremia. Acutely infected animals remain largely undiagnosed. Transfer RNA halves (tsRNAs) are hypothesized to function like microRNAs to regulate gene expression during an immune response. The objective of this study was to identify tsRNAs in cattle that had been challenged with a non-cytopathic field strain of BVDV. Colostrum-deprived neonatal Holstein calves were either challenged with BVDV (n=5) or mock challenged (n=4). Sera was collected prior to challenge and days 4, 9, and 16 post challenge. RNA was extracted and read counts of small non-coding RNAs were assessed using next-generation sequencing. A total of 87,838,207 reads identified 41 different tsRNAs. Two 5' tsRNAs, tsRNAProAGG and tsRNAValAAC, differed across time. Two 5' tsRNAs, tsRNAGlyCCC and tsRNAGlyGCC, differed between treatment groups across time. Four days post challenge, 5' tsRNAGlyCCC and tsRNAGlyGCC were significantly lower in the challenged group than the control group. Further studies are needed to identify the importance and function of 5' tsRNAGlyCCC and tsRNAGlyGCC in serum samples of cattle challenged with BVDV.

Identification and genetic characterization of a porcine hepe-astrovirus (bastrovirus) in the United States.

Here we describe the identification and genetic characterization of a porcine hepe-astrovirus, or bastrovirus, obtained from feces from pigs in the United States. The genome of the new bastrovirus is 5,955 nt long and contains two open reading frames (ORFs). ORF1 encodes a protein containing three domains, viral methyltransferase, RNA helicase and RNA-dependent RNA polymerase (RdRp), and is closely related to the RdRp of hepatitis E virus. The ORF2 protein shares similarities with the astrovirus capsid precursor protein. Although structural features of bastroviruses may resemble those of astroviruses, distinct characteristics of the newly identified bastrovirus include the presence of an RNA helicase domain in ORF1 and the lack of ORF1b. In addition to genetic characterization, screening of 368 porcine samples (oral fluids, oral swabs or fecal swabs) collected in the United States (US) using a porcine-bastrovirus-specific real-time PCR assay revealed that 31% of those samples were positive. These results suggest a broad distribution of bastroviruses in the swine population in the US. This represents the first description of bastrovirus in swine in the US.