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Deletions within the 3A coding region of foot-and-mouth disease virus (FMDV) are associated with decreased virulence in cattle; however, the mechanisms are unknown. We compared experimental infection of cattle with virulent FMDV O1Campos (O1Ca) and a mutant derivative (O1Ca∆3A) lacking residues 87-106 of 3A. Unexpectedly, primary infection of the nasopharyngeal mucosa was similar for both viruses. However, while O1Ca caused viremia and fulminant clinical disease, O1Ca∆3A infection was subclinical and aviremic. There were no differences in expression of anti-viral cytokines in nasopharyngeal tissues between the groups, suggesting attenuation by O1Ca∆3A was a consequence of reduced replication efficiency in bovine cells, rather than a difference in the host response. These results demonstrated that although deletion in 3A of FMDV confers a clinically attenuated phenotype in cattle, the deletion does not prevent subclinical infection. These findings have implications for field scenarios involving outbreaks with apparently host-limited strains of FMDV.

Foot-and-mouth disease (FMD) is a highly contagious and economically important, transboundary viral disease of cloven-hoofed animals. It is known that an asymptomatic, persistent FMD virus (FMDV) infection may occur subsequent to acute or subclinical FMDV infection in adult ruminants. However, virus persistence in young calves has not been studied. In the current investigation, FMDV infection parameters were examined for calves born to FMD-clinically recovered cows (CRC), asymptomatic cows from infected herds (ASC) and cows from with no history of FMD (NHF). The study was conducted in natural condition after FMD outbreaks in two dairy herds in India. No calves described herein had any clinical signs of FMD. Six out of 12 calves born to CRC had detectable FMDV RNA in oesophageal-pharyngeal fluid consistent with asymptomatic FMDV infection. Three of the 12 calves of CRC group had seroreactivity against FMDV non-structural proteins. One calf had detectable FMDV RNA at two consecutive samplings at 2 months apart. However, infectious FMDV was not isolated from any calf in the study. None of the calves in the ASC or NHF groups had any evidence of FMDV infection. Overall, these data are consistent with earlier report on calves having been infected in utero. Further investigation of FMDV persistence in calves under controlled conditions may lead to greater understanding of the viral pathogenesis.

Vesicular stomatitis (VS) is the most common vesicular livestock disease in North America. Transmitted by direct contact and by several biting insect species, this disease results in quarantines and animal movement restrictions in horses, cattle and swine. As changes in climate drive shifts in geographic distributions of vectors and the viruses they transmit, there is considerable need to improve understanding of relationships among environmental drivers and patterns of disease occurrence. Multidisciplinary approaches integrating pathology, ecology, climatology, and biogeophysics are increasingly relied upon to disentangle complex relationships governing disease. We used a big data model integration approach combined with machine learning to estimate the potential geographic range of VS across the continental United States (CONUS) under long-term mean climate conditions over the past 30 years. The current extent of VS is confined to the western portion of the US and is related to summer and winter precipitation, winter maximum temperature, elevation, fall vegetation biomass, horse density, and proximity to water. Comparison with a climate-only model illustrates the importance of current processes-based parameters and identifies regions where uncertainty is likely to be greatest if mechanistic processes change. We then forecast shifts in the range of VS using climate change projections selected from CMIP5 climate models that most realistically simulate seasonal temperature and precipitation. Climate change scenarios that altered climatic conditions resulted in greater changes to potential range of VS, generally had non-uniform impacts in core areas of the current potential range of VS and expanded the range north and east. We expect that the heterogeneous impacts of climate change across the CONUS will be exacerbated with additional changes in land use and land cover affecting biodiversity and hydrological cycles that are connected to the ecology of insect vectors involved in VS transmission.

Potency tests for commercial oil-adjuvanted foot-and-mouth disease (FMD) vaccines are usually carried out in cattle, using a full dose (2 ml) of vaccine and homologous virus challenge. However, in sheep the recommended vaccine dose is half of the cattle dose (1 ml) and most vaccines have not been potency tested for this species, especially with heterologous viruses. To determine the efficacy of a high potency (>6PD50) FMD virus (FMDV) O1Manisa vaccine in sheep, we carried out a study using a heterologous FMDV (FMDV O/SKR/2010 - Mya-98 strain) challenge. Groups of seven animals each were vaccinated with 2×, 1×, 1/2× or 1/4× dose (2 ml, 1 ml, 0.5 ml or 0.25 ml respectively) and challenged at 7 days post vaccination (dpv). Only 3 of the 7 sheep in the group vaccinated with 2 ml were protected. With 2 additional groups, receiving double or single doses and challenged at 14 dpv, 4 of 7 sheep were protected in each group. None of the sheep had measurable neutralising antibodies against the vaccine or challenge virus at 7 dpv. However, all vaccinated animals challenged at 14 dpv had a homologous neutralising response against FMDV O1 Manisa on the day of challenge and all but one animal also had a heterologous response to FMDV O/SKR/2010. Infectious FMDV and viral RNA could be found in nasal swabs between 1 and 6 days post challenge (dpc) in most vaccinated sheep, but those vaccinated with higher doses or challenged at 14 dpv showed significant decreases in the level of FMDV detection. Intermittent virus shedding was noticed between 1 and 35 dpc in all vaccinated groups, but persistent infection could be demonstrated only in 4 sheep (20%). This study showed that at the recommended dose, a high potency (>6 PD50) FMDV O1Manisa vaccine does not protect sheep against a heterologous challenge at 7 dpv. However, partial protection was observed when a double dose was used at 7 dpv or when double or single dose vaccinated sheep were challenged at 14 dpv.

Vesicular stomatitis virus (VSV) is a negative-stranded RNA virus that naturally causes disease in livestock including horses, cattle and pigs. The two main identified VSV serotypes are New Jersey (VSNJV) and Indiana (VSIV). VSV is a rapidly replicating, potently immunogenic virus that has been engineered to develop novel oncolytic therapies for cancer treatment. Swine are a natural host for VSV and provide a relevant and well-established model, amenable to biological sampling to monitor virus shedding and neutralizing antibodies. Previous reports have documented the pathogenicity and transmissibility of wild-type isolates and recombinant strains of VSIV and VSNJV using the swine model. Oncolytic VSV engineered to express interferon-beta (IFNβ) and the sodium iodide symporter (NIS), VSV-IFNβ-NIS, has been shown to be a potent new therapeutic agent inducing rapid and durable tumor remission following systemic therapy in preclinical mouse models. VSV-IFNβ-NIS is currently undergoing clinical evaluation for the treatment of advanced cancer in human and canine patients. To support clinical studies and comprehensively assess the risk of transmission to susceptible species, we tested the pathogenicity and transmissibility of oncolytic VSV-IFNβ-NIS using the swine model. Following previously established protocols to evaluate VSV pathogenicity, intradermal inoculation with 107 TCID50 VSV-IFNβ-NIS caused no observable symptoms in pigs. There was no detectable shedding of infectious virus in VSV-IFNβ-NIS in biological excreta of inoculated pigs or exposed naive pigs kept in direct contact throughout the experiment. VSV-IFNβ-NIS inoculated pigs became seropositive for VSV antibodies, while contact pigs displayed no symptoms of VSV infection, and importantly did not seroconvert. These data indicate that oncolytic VSV is both nonpathogenic and not transmissible in pigs, a natural host. These findings support further clinical development of oncolytic VSV-IFNβ-NIS as a safe therapeutic for human and canine cancer.

The goal of this study was to characterize the properties and duration of the foot-and-mouth disease (FMD) carrier state and associated serological responses subsequent to vaccination and naturally occurring infection at two farms in northern India. Despite previous vaccination of cattle in these herds, clinical signs of FMD occurred in October 2013 within a subset of animals at the farms containing juvenile-yearling heifers and steers (Farm A) and adult dairy cattle (Farm B). Subsequent to the outbreak, FMD virus (FMDV) asymptomatic carriers were identified in both herds by seroreactivity to FMDV non-structural proteins and detection of FMDV genomic RNA in oropharyngeal fluid. Carriers' seroreactivity and FMDV genome detection status were subsequently monitored monthly for 23 months. The mean extinction time of the carrier state was 13.1 ± 0.2 months, with extinction having occurred significantly faster amongst adult dairy cattle at Farm B compared to younger animals at Farm A. The rate of decrease in the proportion of carrier animals was calculated to be 0.07 per month. Seroprevalence against FMDV non-structural proteins decreased over the course of the study period, but was found to increase transiently following repeated vaccinations. These data provide novel insights into viral and host factors associated with the FMDV carrier state under natural conditions. The findings reported herein may be relevant to field veterinarians and governmental regulatory entities engaged in FMD response and control measures.

In our previous transcriptomic studies using DNA microarray analysis, a probe designed from an unknown expressed sequence tag (EST) showed significant differential gene expression in the pharyngeal epithelia. The objectives of this study are to annotate the gene sequence and compare the gene transcription levels among different bovine tissues based on our published microarray data. The gene transcribing the EST contains a 90-amino-acid protein sequence. The results of bioinformatic analyses using comparative genetics, multiple sequence alignments, phylogenetic analysis and promoter sequence analysis indicated that this gene is a novel ELR+ CXCL gene orthologous to mouse CXCL15. The gene is highly conserved in ruminants and exists in many other mammals but not in chickens, primates or pigs. Phylogenetic analysis and gene structures showed that CXCL15 is closer to CXCL8 than to other ELR+ CXCLs. Our microarray data show that bovine CXCL15 expression was higher in laser capture micro-dissected bovine pharyngeal epithelia than in the whole pharyngeal tissues, which agrees with the expression in mice. However, unlike the high expression in the mouse lung, our results showed that the bovine nasal turbinate, dorsal nasopharynx, dorsal soft palate and tongue expressed higher levels of CXCL15 than the lung and skins. Promoter analysis showed that ruminants have more immune-related transcription factor binding sites in the proximal promoters of CXCL15 than mouse. CXCL15 has previously only been reported in mice and has neutrophil chemotactic activity. Given the critical roles of neutrophils in innate immunity, this study provides useful information for further characterization of bovine CXCL15.

We report the full-genome sequence of a New Jersey vesiculovirus strain, commonly referred to as vesicular stomatitis New Jersey virus (VSNJV), obtained from an epithelial lesion of a naturally infected cow in Chiapas, Mexico. This genome is a representative from the zone of endemicity in Mexico, a region of high genetic diversity.

Copyright: This is an open access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication. The role of foot-and-mouth disease virus (FMDV) persistently infected ruminants in initiating new outbreaks remains controversial, and the perceived threat posed by such animals hinders international trade in FMD-endemic countries. In this study we report longitudinal analyses of genetic and antigenic variations of FMDV serotype O/ME-SA/Ind2001d sublineage during naturally occurring, persistent infection in cattle and buffalo at an organised dairy farm in India. The proportion of animals from which FMDV RNA was recovered was not significantly different between convalescent (post-clinical) and sub-clinically infected animals or between cattle and buffalo across the sampling period. However, infectious virus was isolated from a higher proportion of buffalo samples and for a longer duration compared to cattle. Analysis of the P1 sequences from recovered viruses indicated fixation of mutations at the rate of 1.816 x 10-2substitution/site/year (s/s/y) (95% CI 1.362–2.31 x 10−2 s/s/y). However, the majority of point mutations were transitional substitutions. Within individual animals, the mean dN/dS (ω) value for the P1 region varied from 0.076 to 0.357, suggesting the selection pressure acting on viral genomes differed substantially across individual animals. Statistical parsimony analysis indicated that all of the virus isolates from carrier animals originated from the outbreak virus. The antigenic relationship value as determined by 2D-VNT assay revealed fluctuation of antigenic variants within and between carrier animals during the carrier state which suggested that some carrier viruses had diverged substantially from the protection provided by the vaccine strain. This study contributes to understanding the extent of within-host and within-herd evolution that occurs during the carrier state of FMDV.

Vesicular stomatitis (VS) is a vector-borne livestock disease caused by vesicular stomatitis New Jersey virus (VSNJV) or vesicular stomatitis Indiana virus (VSIV). The disease circulates endemically in northern South America, Central America, and Mexico and only occasionally causes outbreaks in the United States. Over the past 20 years, VSNJV outbreaks in the southwestern and Rocky Mountain regions occurred with incursion years followed by virus overwintering and subsequent expansion outbreak years. Regulatory response by animal health officials is deployed to prevent spread from lesioned animals. The 2019 VS incursion was the largest in 40 years, lasting from June to December 2019 with 1144 VS-affected premises in 111 counties in eight states (Colorado, Kansas, Nebraska, New Mexico, Oklahoma, Texas, Utah, and Wyoming) and was VSIV serotype, last isolated in 1998. A subsequent expansion occurred from April to October 2020 with 326 VS-affected premises in 70 counties in eight states (Arizona, Arkansas, Kansas, Missouri, Nebraska, New Mexico, Oklahoma, and Texas). The primary serotype in 2020 was VSIV, but a separate incursion of VSNJV occurred in south Texas. Summary characteristics of the outbreaks are presented along with VSV-vector sampling results and phylogenetic analysis of VSIV isolates providing evidence of virus overwintering.

Foot-and-mouth disease (FMD) remains one of the most devastating livestock diseases around the world. Several serotype-specific vaccine formulations exist, but they require about 5 to 7 days to induce protective immunity. Our previous studies have shown that a constitutively active fusion protein of porcine interferon (IFN) regulatory factors (IRF) 7 and 3 [IRF7/3(5D)] strongly induced type I IFN and antiviral genes in vitro and prevented mortality in an FMD mouse model when delivered with a replication-defective adenoviral vector [Ad5-poIRF7/3(5D)]. Here, we demonstrate that pigs treated with 10 8 , 10 9 , or 10 10 PFU of Ad5-poIRF7/3(5D) 24 h before FMDV challenge were fully protected from FMD clinical signs and did not develop viremia, virus shedding or antibodies against FMDV nonstructural proteins. Pigs treated with Ad5-poIRF7/3(5D) had higher levels of IFN and antiviral activity in serum, and upregulated expression of several IFN-stimulated genes in peripheral blood mononuclear cells, compared to pigs treated with Ad5-Blue vector control. Importantly, treatment of porcine cultured cells with Ad5-poIRF7/3(5D) inhibited the replication of all 7 FMDV serotypes. In vitro experiments using cultured embryonic fibroblasts derived from IFN receptor knockout mice suggested that the antiviral response induced by Ad5-poIRF7/3(5D) was dependent on type I and III IFN pathways; however, experiments with mice demonstrated that a functional type I IFN pathway mediates Ad5-poIRF7/3(5D) protection conferred in vivo . Our studies demonstrate that inoculation with Ad5-poIRF7/3(5D) completely protects swine against FMD by inducing a strong type I IFN response and highlights its potential application to rapidly and effectively prevent FMDV replication and dissemination. IMPORTANCE Foot-and-mouth disease virus (FMDV) causes a fast-spreading disease that affects farm animals, with economically and socially devastating consequences. Our study shows that inoculation with a constitutively active transcription factor, namely, a fusion protein of porcine interferon (IFN) regulatory factors (IRF) 7 and 3 delivered by an adenovirus vector [Ad5-poIRF7/3(5D)], is a new effective treatment to prevent FMD in swine. Animals pretreated with Ad5-poIRF7/3(5D) 1 day before being exposed to FMDV were completely protected from viral replication and clinical disease. It is noteworthy that the doses of Ad5-poIRF7/3(5D) required for protection are lower than those previously reported for similar approaches using Ad5 vectors delivering type I, II, or III IFN, suggesting that this novel strategy would be economically appealing to counteract FMD. Our results also indicate that a dynamic interplay among different components of pigs' innate immune defenses allows potent antiviral effects after Ad5-poIF7/3(5D) administration.

Here, we engineered two FMD viruses with histidine residues inserted into or fused to the FMDV capsid. Both 6xHis viruses exhibited growth kinetics, plaque morphologies and antigenic characteristics similar to wild-type virus. The 6xHis tag allowed one-step purification of the mutant virions by Co(2+) affinity columns. Electron microscopy and biochemical assays showed that the 6xHis FMDVs readily assembled into antigen: adjuvant complexes in solution, by conjugating with Ni(2+)-chelated nanolipoprotein and monophosphoryl lipid A adjuvant (MPLA:NiNLP). Animals Immunized with the inactivated 6xHis-FMDV:MPLA:NiNLP vaccine acquired enhanced protective immunity against FMDV challenge compared to virions alone. Induction of anti-6xHis and anti-FMDV neutralizing antibodies in the immunized animals could be exploited in the differentiation of vaccinated from infected animals needed for the improvement of FMD control measures. The novel marker vaccine/nanolipid technology described here has broad applications for the development of distinctive and effective immune responses to other pathogens of importance.

The pathogenesis of persistent foot-and-mouth disease virus (FMDV) infection was investigated in 46 cattle that were either naive or had been vaccinated using a recombinant, adenovirus-vectored vaccine 2 weeks before challenge. The prevalence of FMDV persistence was similar in both groups (62% in vaccinated cattle, 67% in nonvaccinated cattle), despite vaccinated cattle having been protected from clinical disease. Analysis of antemortem infection dynamics demonstrated that the subclinical divergence between FMDV carriers and animals that cleared the infection had occurred by 10 days postinfection (dpi) in vaccinated cattle and by 21 dpi in nonvaccinated animals. The anatomic distribution of virus in subclinically infected, vaccinated cattle was restricted to the pharynx throughout both the early and the persistent phases of infection. In nonvaccinated cattle, systemically disseminated virus was cleared from peripheral sites by 10 dpi, while virus selectively persisted within the nasopharynx of a subset of animals. The quantities of viral RNA shed in oropharyngeal fluid during FMDV persistence were similar in vaccinated and nonvaccinated cattle. FMDV structural and nonstructural proteins were localized to follicle-associated epithelium of the dorsal soft palate and dorsal nasopharynx in persistently infected cattle. Host transcriptome analysis of tissue samples processed by laser capture microdissection indicated suppression of antiviral host factors (interferon regulatory factor 7, CXCL10 [gamma interferon-inducible protein 10], gamma interferon, and lambda interferon) in association with persistent FMDV. In contrast, during the transitional phase of infection, the level of expression of IFN-λ mRNA was higher in follicle-associated epithelium of animals that had cleared the infection. This work provides novel insights into the intricate mechanisms of FMDV persistence and contributes to further understanding of this critical aspect of FMDV pathogenesis. IMPORTANCE The existence of a prolonged, asymptomatic carrier state is a political impediment for control and potential eradication of foot-and-mouth disease (FMD). When FMD outbreaks occur, they are often extinguished by massive depopulation of livestock due to the fear that some animals may have undiagnosed subclinical infection, despite uncertainty over the biological relevance of FMD virus (FMDV) persistence. The work described here elucidates aspects of the FMDV carrier state in cattle which may facilitate identification and/or abrogation of asymptomatic FMDV infection. The divergence between animals that clear infection and those that develop persistent infection was demonstrated to occur earlier than previously established. The host antiviral response in tissues maintaining persistent FMDV was downregulated, whereas upregulation of IFN-λ mRNA was found in the epithelium of cattle that had recently cleared the infection. This suggests that the clearing of FMDV infection is associated with an enhanced mucosal antiviral response, whereas FMDV persistence is associated with suppression of the host antiviral response.

Background Foot-and-mouth disease (FMD) is an enzootic viral disease affecting livestock in Pakistan. Objectives To determine the seroprevalence of FMD in large ruminants in periurban dairy farms near Islamabad. Methods Serum samples were collected from 636 large ruminants during 2011 to 2012; 584 (92%) were buffaloes (Bos bubalis bubalis) and 52 (18%) were cattle (Bos taurus indicus). The population sampled was mainly adult (n = 514) and female (n = 596). Sera were assayed for antibodies against a nonstructural protein of the FMD virus using a Chekit FMD-3ABC bo-ov enzyme immunoassay Kit (Idexx Laboratories). Data were analyzed using a χ2 test and multiple logistic regression. Results The seroprevalence of FMD in the ruminants was 46% (n = 293, 95% confidence interval (CI); 42.18- 49.95) and was significantly higher in buffaloes (285, 97%; χ2 = 21.46; P < 0.001) than in cattle (8, 3%). The risk of seropositivity increased significantly with age (χ2 = 72.71; P < 0.001); adult animals were 7.05 times more likely (odds ratio (OR) 7.05, 95% CI 3.60-13.79; P < 0.001) to be seropositive after adjusting for the effect of species. Buffaloes were more likely to be seropositive (OR 3.99, 95% CI 1.78-8.92, P = 0.001). Sex was not significantly associated with FMD seropositivity (OR 0.58, 95% CI 0.17-1.95, P = 0.38). Conclusions Large ruminants in periurban dairy farms near Islamabad have a high FMD virus seroprevalence and play a potential role in the persistence and transmission of FMD in Pakistan.

Foot-and-mouth disease virus (FMDV), the causative agent of foot-and-mouth disease, is an Aphthovirus within the Picornaviridae family. During infection with FMDV, several host cell membrane rearrangements occur to form sites of viral replication. FMDV protein 2C is part of the replication complex and thought to have multiple roles during virus replication. To better understand the role of 2C in the process of virus replication, we have been using a yeast two-hybrid approach to identify host proteins that interact with 2C. We recently reported that cellular Beclin1 is a natural ligand of 2C and that it is involved in the autophagy pathway, which was shown to be important for FMDV replication. Here, we report that cellular vimentin is also a specific host binding partner for 2C. The 2C-vimentin interaction was further confirmed by coimmunoprecipitation and immunofluorescence staining to occur in FMDV-infected cells. It was shown that upon infection a vimentin structure forms around 2C and that this structure is later resolved or disappears. Interestingly, overexpression of vimentin had no effect on virus replication; however, overexpression of a truncated dominant-negative form of vimentin resulted in a significant decrease in viral yield. Acrylamide, which causes disruption of vimentin filaments, also inhibited viral yield. Alanine scanning mutagenesis was used to map the specific amino acid residues in 2C critical for vimentin binding. Using reverse genetics, we identified 2C residues that are necessary for virus growth, suggesting that the interaction between FMDV 2C and cellular vimentin is essential for virus replication.

Foot-and-mouth disease (FMD) is a highly contagious viral disease that severely impacts global food security and is one of the greatest constraints on international trade of animal products. Extensive viral population diversity and rapid, continuous mutation of circulating FMD viruses (FMDVs) pose significant obstacles to the control and ultimate eradication of this important transboundary pathogen. The current study investigated mechanisms contributing to within-host evolution of FMDV in a natural host species (cattle). Specifically, vaccinated and non-vaccinated cattle were infected with FMDV under controlled, experimental conditions and subsequently sampled for up to 35 days to monitor viral genomic changes as related to phases of disease and experimental cohorts. Consensus-level genomic changes across the entire FMDV coding region were characterized through three previously defined stages of infection: early, transitional, and persistent. The overall conclusion was that viral evolution occurred via a combination of two mechanisms: emergence of full-genomic minority haplotypes from within the inoculum super-swarm, and concurrent continuous point mutations. Phylogenetic analysis indicated that individuals were infected with multiple distinct haplogroups that were pre-existent within the ancestral inoculum used to infect all animals. Multiple shifts of dominant viral haplotype took place during the early and transitional phases of infection, whereas few shifts occurred during persistent infection. Overall, this work suggests that the establishment of the carrier state is not associated with specific viral genomic characteristics. These insights into FMDV population dynamics have important implications for virus sampling methodology and molecular epidemiology.

Foot-and-mouth disease virus (FMDV) is capable of infecting all cloven-hoofed domestic livestock species, including cattle, pigs, goats, and sheep. However, in contrast to cattle and pigs, the pathogenesis of FMDV in small ruminants has been incompletely elucidated. The objective of the current investigation was to characterize tissue- and cellular tropism of early and late stages of FMDV infection in sheep following three different routes of simulated natural virus exposure. Extensive post-mortem harvest of tissue samples at pre-determined time points during early infection (24 and 48 hours post infection) demonstrated that tissues specifically susceptible to primary FMDV infection included the paraepiglottic- and palatine tonsils, as well as the nasopharyngeal mucosa. Additionally, experimental aerosol inoculation of sheep led to substantial virus replication in the lungs at 24-48 hours post-inoculation. During persistent infection (35 days post infection), the paraepiglottic- and palatine tonsils were the only tissues from which infectious FMDV was recovered. This is strikingly different from cattle, in which persistent FMDV infection has consistently been located to the nasopharyngeal mucosa. Analysis of tissue sections by immunomicroscopy revealed a strict epithelial tropism during both early and late phases of infection as FMDV was consistently localized to cytokeratin-expressing epithelial cells. This study expands upon previous knowledge of FMDV pathogenesis in sheep by providing detailed information on the temporo-anatomic distribution of FMDV in ovine tissues. Findings are discussed in relation to similar investigations previously performed in cattle and pigs, highlighting similarities and differences in FMDV pathogenesis across natural host species.

Transboundary movement of animals is an important mechanism for foot-and-mouth disease virus (FMDV) spread in endemic regions, such as Cameroon. Several transboundary animal trade routes cross the Far North Region of Cameroon, and cattle moved on foot along these routes often come in contact with native (sedentary and transhumant) herds. The purpose of this study was to investigate the role of transboundary trade cattle in the epidemiology of FMDV in the Far North Region of Cameroon. A total of 582 oropharyngeal fluid (OPF) samples were collected from asymptomatic transboundary trade cattle at official border check points and 57 vesicle epithelial tissues were collected from clinically affected native cattle in the Far North Region of Cameroon during 2010-2014. Viral protein 1 (VP1) coding sequences were obtained from 6 OPF samples from transboundary cattle (4 serotype O, 2 serotype SAT2) and 19 epithelial tissue samples from native cattle (7 serotype O, 3 serotype SAT2, 9 serotype A). FMDV serotype O viruses belonged to two topotypes (East Africa-3 and West Africa), and phylogenetic analyses suggested a pattern of continuous transmission in the region. Serotype SAT2 viruses belonged to a single topotype (VII), and phylogenetic analysis suggested a pattern of repeated introductions of different SAT2 lineages in the region. Serotype A viruses belonged to topotype AFRICA/G-IV, and the pattern of transmission was unclear. Spearman rank correlation analysis of VP1 coding sequences obtained in this study from transboundary and native cattle showed a positive correlation between genetic distance and time for serotype O (ρ = 0.71, p = 0.003) and between genetic distance and geographic distance for serotype SAT2 (ρ = 0.54, p = 0.1). These data suggest that transboundary trade cattle participate in the transmission of FMDV in the Far North Region of Cameroon, however the dynamics and direction of transmission could not be determined in this study. Results of this study contribute to the understanding of transboundary FMDV epidemiology in Central Africa and will help to inform control programs in Cameroon and in the region.

This study reports the use of a site-specific recombination cloning technique for rapid development of a full-length cDNA clone that can produce infectious vesicular stomatitis New Jersey virus (VSNJV). The full-length genome of the epidemic VSNJV NJ0612NME6 strain was amplified in four overlapping cDNA fragments which were linked together and cloned into a vector plasmid by site-specific recombination. Furthermore, to derive infectious virus, three supporting plasmid vectors containing either the nucleoprotein (N), phosphoprotein (P) or polymerase (L) genes were constructed using the same cloning methodology. Recovery of recombinant VSNJV was achieved after transfecting all four vectors on into BSR-T7/5 cells, a BHK-derived cell line stably expressing T7 RNA polymerase (PMID: 9847328). In vitro characterization of recombinant and parental viruses revealed similar growth kinetics and plaque morphologies. Furthermore, experimental infection of pigs with the recombinant virus resulted in severe vesicular stomatitis with clinical signs similar to those previously reported for the parental field strain. These results validate the use of site-directed specific recombination cloning as a useful alternative method for rapid construction of stable full-length cDNA clones from vesicular stomatitis field strains. The approach reported herein contributes to the improvement of previously published methodologies for the development of full-length cDNA clones of this relevant virus.

In the event of an intentional or accidental incursion of a transboundary animal disease (TAD) virus into the US, a major concern to the meat industry would be the potential contamination of packing plants by processing infected animals. TAD agents such as foot and mouth disease virus (FMDV), African swine fever virus (ASFV) and classical swine fever virus (CSFV) are found in swine products such as blood and feces and are present in the tissues of infected animals. To test the disinfection of TAD viruses in a pork-packing environment, a previously developed disinfection assay was used to test two biocides currently used by industry sanitarians, against TAD viruses dried on industry relevant surfaces in saline or swine products. With the exception of one virus, both commercial disinfectants tested were effective against the TAD viruses dried on steel, plastic, and sealed concrete surfaces in the absence of the swine products. Disinfectant activity was greatly inhibited in the presence of dried blood and meat juices. The acidic disinfectants were able to inactivate the viruses in swine feces whereas fecal material generally inhibited sodium hypochlorite-based disinfectants. These results highlight the importance of manufacturer-recommended pre-cleaning steps to remove gross soil before surface disinfection. Taken together, these data support the use of acid- and surfactant-containing commercial products for packing plant disinfection during a TAD virus outbreak event.

Type I interferons (IFNs) are key mediators of the innate antiviral response in mammalian cells. Elongation initiation factor 4E binding proteins (4E-BPs) are translational controllers of interferon regulatory factor 7 (IRF-7), the "master regulator" of IFN transcription. Previous studies have suggested that mouse cells depleted of 4E-BPs are more sensitive to IFNβ treatment and had lower viral loads as compared to wild type (WT) cells. However, such approach has not been tested as an antiviral strategy in livestock species. In this study, we tested the antiviral activity of porcine cells depleted of 4E-BP1 by a Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR-associated protein-9 nuclease (Cas9) genome engineering system. We found that 4E-BP1 knockout (KO) porcine cells had increased expression of IFNα and β, IFN stimulated genes, and significant reduction in vesicular stomatitis virus titer as compare to WT cells. No phenotypical changes associated with CRISPR/Cas9 manipulation were observed in 4E-BP1 KO cells. This work highlights the use of the CRISPR/Cas9 system to enhance the antiviral response in porcine cells.

Foot-and-mouth disease (FMD) is a major constraint to transboundary trade in animal products, yet much of its natural ecology and epidemiology in endemic regions is still poorly understood. To address this gap, a multidisciplinary, molecular and conventional epidemiological approach was applied to an investigation of endemic FMD in Vietnam. Within the study space, it was found that 22.3% of sampled ruminants had previously been infected with FMD virus (FMDV), of which 10.8% were persistent, asymptomatic carriers (2.4% of the total population). Descriptive data collected from targeted surveillance and a farm questionnaire showed a significantly lower prevalence of FMDV infection for dairy farms. In contrast, farms of intermediate size and/or history of infection in 2010 were at increased risk of FMD exposure. At the individual animal level, buffalo had the highest exposure risk (over cattle), and there was spatial heterogeneity in exposure risk at the commune level. Conversely, carrier prevalence was higher for beef cattle, suggesting lower susceptibility of buffalo to persistent FMDV infection. To characterize virus strains currently circulating in Vietnam, partial FMDV genomic (VP1) sequences from carrier animals collected between 2012 and 2013 (N = 27) and from FMDV outbreaks between 2009 and 2013 (N = 79) were compared by phylogenetic analysis. Sequence analysis suggested that within the study period, there were two apparent novel introductions of serotype A viruses and that the dominant lineage of serotype O in Vietnam shifted from SEA/Mya-98 to ME-SA/PanAsia. FMDV strains shared close ancestors with FMDV from other South-East Asian countries indicating substantial transboundary movement of the predominant circulating strains. Close genetic relationships were observed between carrier and outbreak viruses, which may suggest that asymptomatic carriers of FMDV contribute to regional disease persistence. Multiple viral sequences obtained from carrier cattle over a 1-year period had considerable within-animal genetic variation, indicating within-host virus evolution.

In this study we describe the adaptive changes fixed on the capsid of several foot-and-mouth disease virus serotype A strains during propagation in cell monolayers. Viruses passaged extensively in three cell lines (BHK-21, LFBK and IB-RS-2) consistently gained positively charged amino acids in the putative heparin-sulfate-binding pocket (VP2 βE-βF loop, VP1 C-terminus and VP3 β-B knob) surrounding the fivefold symmetry axis (VP1 βF-βG loop) and at other discrete sites on the capsid (VP3 βG-βH loop, VP1 C-terminus, VP2 βC strand and VP1 βG-βH loop). A lysine insertion in the VP1 βF-βG loop of two of the BHK-21-adapted viruses supports the biological advantage of positively charged residues acquired in cell culture. The charge transitions occurred irrespective of cell line, suggesting their possible role in ionic interaction with ubiquitous negatively charged cell-surface molecules such as glycosaminoglycans (GAG). This was supported by the ability of the cell-culture-adapted variants to replicate in the integrin-deficient, GAG-positive CHO-K1 cells and their superior fitness in competition assays compared with the lower passage viruses with WT genotypes. Substitutions fixed in the VP1 βG-βH loop (-3, -2 and +2 'RGD' positions) or in the structural element known to be juxtaposed against that loop (VP1 βB-βC loop) suggest their possible role in modulating the efficiency and specificity of interaction of the 'RGD' motif with αv-integrin receptors. The nature and location of the substitutions described in this study could be applied in the rapid cell culture adaptation of viral strains for vaccine production.

Foot-and-mouth disease (FMD) is one of the most contagious and economically important livestock diseases worldwide. Four serotypes of FMD virus (FMDV; O, A, SAT1, SAT2) circulate in Cameroon, and a trivalent inactivated vaccine against the three most common serotypes (O, A, SAT2) was recently introduced in 2014. The objective of this study was to characterize vaccine performance in cattle under natural hyperendemic conditions in the Adamawa region of Cameroon. Vaccinated cattle (n = 50) and non-vaccinated controls (n = 100) were monitored by serum and oropharyngeal fluid (OPF) sample collection through a 12-month period. Anti-FMDV non-structural protein (anti-NSP) seroprevalence increased from 59.3% (89/150) at the beginning of the study to 85.8% (103/120) at the end of the study, and FMDV RNA was found in 28% (42/150) of animals overall, despite detection of clinical signs of FMD in only 6 non-vaccinated animals. Viral sequence analysis indicated that subclinical infections of FMDV serotypes O and A were present within the study herds during the study period, which was reflected by an overall increase of anti-NSP seroprevalence during the study. There was no association between vaccination status and seroconversion or prevalence of FMDV RNA in OPF. Younger cattle had higher odds of detection of FMDV RNA in OPF, but older animals were more likely to be seropositive. This study suggests vaccination of herds previously exposed to FMDV may help to limit clinical signs and reduce economic losses caused by FMDV. These findings also suggest that subclinical circulation of FMDV occurs in hyperendemic regions regardless of vaccination.

Foot-and-mouth disease (FMD) is a severe infection caused by a picornavirus that affects livestock and wildlife. Persistence in ruminants is a well-documented feature of Foot-and-mouth disease virus (FMDV) pathogenesis and a major concern for disease control. Persistently infected animals harbor virus for extended periods, providing a unique opportunity to study within-host virus evolution. This study investigated the genetic dynamics of FMDV during persistent infections of naturally infected Asian buffalo. Using next-generation sequencing (NGS) we obtained 21 near complete FMDV genome sequences from 12 sub-clinically infected buffalo over a period of one year. Four animals yielded only one virus isolate and one yielded two isolates of different serotype suggesting a serial infection. Seven persistently infected animals yielded more than one virus of the same serotype showing a long-term intra-host viral genetic divergence at the consensus level of less than 2.5%. Quasi-species analysis showed few nucleotide variants and non-synonymous substitutions of progeny virus despite intra-host persistence of up to 152 days. Phylogenetic analyses of serotype Asia-1 VP1 sequences clustered all viruses from persistent animals with Group VII viruses circulating in Pakistan in 2011, but distinct from those circulating on 2008–2009. Furthermore, signature amino acid (aa) substitutions were found in the antigenically relevant VP1 of persistent viruses compared with viruses from 2008–2009. Intra-host purifying selective pressure was observed, with few codons in structural proteins undergoing positive selection. However, FMD persistent viruses did not show a clear pattern of antigenic selection. Our findings provide insight into the evolutionary dynamics of FMDV populations within naturally occurring subclinical and persistent infections that may have implications to vaccination strategies in the region.

TThe quantitative relationship between the exposure dose of foot-and-mouth disease virus (FMDV) and subsequent infection dynamics has been demonstrated through controlled inoculation studies in various species. However, similar quantitation of viral doses has not been achieved during contact exposure experiments due to the intrinsic difficulty of measuring the virus quantities exchanged between animals. In the current study, novel modeling techniques were utilized to investigate FMDV infection dynamics in groups of pigs that had been contact-exposed to FMDV-infected donors shedding varying levels of virus, as well as in pigs inoculated via the intra-oropharyngeal (IOP) route. Estimated virus exposure doses were modelled and were found to be statistically significantly associated with the dynamics of FMDV RNA detection in serum and oropharyngeal fluid (OPF), and with the time to onset of clinical disease. The minimum estimated shedding quantity in OPF that defined infectiousness of donor pigs was 6.55 log10 genome copy numbers (GCN)/ml (95% CI 6.11, 6.98), which delineated the transition from the latent to infectious phase of disease which occurred during the incubation phase. This quantity corresponded to a minimum estimated exposure dose of 5.07 log10 GCN/ml (95% CI 4.25, 5.89) in contact-exposed pigs. Thus, we demonstrated that a threshold quantity of FMDV detection in donor pigs was necessary in order to achieve transmission by direct contact. The outcomes from this investigation demonstrate that variability of infection dynamics which occurs during the progression of FMD in naturally exposed pigs can be partially attributed to variations in exposure dose. Moreover, these modeling approaches for dose-quantitation may be retrospectively applied to contact-exposure experiments or field scenarios. Hence, robust information could be incorporated into models used to evaluate FMD spread and control.

Foot-and-mouth disease (FMD) is an important transboundary disease with substantial economic impacts. Although between-herd transmission of the disease has been well studied, studies focusing on within-herd transmission using farm-level outbreak data are rare. The aim of this study was to estimate parameters associated with within-herd transmission, host physiological factors and FMD virus (FMDV) persistence using data collected from an outbreak that occurred at a large, organized dairy farm in India. Of 1,836 regularly vaccinated, adult dairy cattle, 222 had clinical signs of FMD over a 39-day period. Assuming homogenous mixing, a frequency-dependent compartmental model of disease transmission was built. The transmission coefficient and basic reproductive number were estimated to be between 16.2-18.4 and 67-88, respectively. Non-pregnant animals were more likely to manifest clinical signs of FMD as compared to pregnant cattle. Based on oropharyngeal fluid (probang) sampling and FMDV-specific RT-PCR, four of 36 longitudinally sampled animals (14%) were persistently infected carriers 10.5 months post-outbreak. There was no statistical difference between subclinical and clinically infected animals in the duration of the carrier state. However, prevalence of NSP-ELISA antibodies differed significantly between subclinical and clinically infected animals 12 months after the outbreak with 83% seroprevalence amongst clinically infected cattle compared to 69% of subclinical animals. This study further elucidates within-herd FMD transmission dynamics during the acute-phase and characterizes duration of FMDV persistence and seroprevalence of FMD under natural conditions in an endemic setting.

We report two full-genome sequences of vesicular stomatitis New Jersey virus (VSNJV) obtained by Illumina next-generation sequencing of RNA isolated from epithelial suspensions of cattle naturally infected in Mexico. These genomes represent the first full-genome sequences of vesicular stomatitis New Jersey viruses circulating in Mexico deposited in the GenBank database.

Several foot-and-mouth disease virus (FMDV) carrier cattle were identified in Vietnam by the recovery of infectious virus from oropharyngeal fluid. This report contains the first near-complete genome sequences of seven viruses from sequential samples from one carrier animal collected over the course of 1 year. The characterization of within-host viral evolution has implications for FMDV control strategies.

We report the laboratory analysis of 125 clinical samples from suspected cases of foot-and-mouth disease (FMD) in cattle and Asian buffalo collected in Pakistan between 2008 and 2012. Of these samples, 89 were found to contain viral RNA by rRT-PCR, of which 88 were also found to contain infectious FMD virus (FMDV) by virus isolation (VI), with strong correlation between these tests (κ = 0.96). Samples that were VI-positive were serotyped by antigen detection ELISA (Ag-ELISA) and VP1 sequence acquisition and analysis. Sequence data identified FMDV serotypes A (n = 13), O (n = 36) and Asia-1 (n = 41), including three samples from which both serotypes Asia-1 and O were detected. Serotype A viruses were classified within three different Iran-05 sublineages: HER-10, FAR-11 and ESF-10. All serotype Asia-1 were within Group VII (Sindh-08 lineage), in a genetic clade that differs from viruses isolated prior to 2010. All serotypes O were classified as PanAsia-2 within two different sublineages: ANT-10 and BAL-09. Using VP1 sequencing as the gold standard for serotype determination, the overall sensitivity of Ag-ELISA to correctly determine serotype was 74%, and serotype-specific sensitivity was 8% for serotype A, 88% for Asia-1 and 89% for O. Serotype-specific specificity was 100% for serotype A, 93% for Asia-1 and 94% for O. Interestingly, 12 of 13 serotype A viruses were not detected by Ag-ELISA. This study confirms earlier accounts of regional genetic diversity of FMDV in Pakistan and highlights the importance of continued validation of diagnostic tests for rapidly evolving pathogens such as FMDV.

Foot-and-mouth disease (FMD) is, arguably, the animal disease with the most devastating global economic impact owing in part, to the severe trade restrictions imposed upon affected countries and regions. South Asia is one of the regions where widespread lineages of the FMDV virus (FMDV) have emerged. Here, we performed an integrative phylogenetic analysis of all FMDV serotypes (A, O and Asia-1) circulating in southern Asia, including viral sequences collected until 2013. Our results describe the occurrence of FMD caused by different serotypes and lineages, focusing in the cycles where a specific lineage predominates within a region for a protracted period and then are rapidly or progressively replaced by an emergent or re-emergent strain that is introduced from an adjacent region. Transmission between the two main regions in southern Asia (the Indian subcontinent and the region comprised by Afghanistan, Iran and Pakistan) has been limited. Results of time divergence estimation of lineages that currently circulate in this region indicate that the most recent common ancestor of endemic lineages are: 1992 [1989-1995] for lineage O/PanAsia; 1997 [1995-1999] for PanAsia2; 2001 [1998-2004] for O/Ind2001; 2001 [2000-2002] for A/Iran-05; 1990 [1988-1991] for A/G-18 (G-VII); 2003 [2000-2006] for Asia-1 Sindh08 and 2002 [1999-2004] for Asia-1 G-VIII. We estimated the mean of the overall substitution rate of the VP1 coding region (substitution/site/year) for serotype O (5.95 × 10-3 ), serotype A (1.19 × 10-2 ) and serotype Asia-1 (3.08 × 10-3 ). The potential factors driving the lineage turnover are discussed. Our results provide insights into the ecological and evolutionary factors driving the emergence of FMDV.

In recent years, foot-and-mouth disease virus (FMDV) serotype O, topotype Middle East- South Asia (ME-SA), lineage Ind-2001d has spread from the Indian subcontinent to the Middle East, North Africa, and Southeast Asia. In the current report, we describe the first detection of this lineage in Vietnam in May, 2015 in Dak Nông province. Three subsequent outbreaks caused by genetically related viruses occurred between May-October, 2015 after which the virus was not detected in clinical outbreaks for at least 15 subsequent months. The observed outbreaks affected (in chronological order): Cattle in Dak Nông province, pigs in Dak Lak province and Dak Nông province, and cattle in Ninh Thuân province. The clinical syndromes associated with these outbreaks were consistent with typical FMD in the affected species. Overall attack rate on affected premises was 0.85 in pigs and 0.93 in cattle over the course of the outbreak. Amongst 378 pigs at risk on affected premises, 85 pigs died during the outbreaks; there were no deaths among cattle. The manner in which FMDV/O/ME-SA/Ind-2001d was introduced into Vietnam remains undetermined; however, movement of live cattle is the suspected route. This incursion has substantial implications for epidemiology and control of FMD in Southeast Asia.

In 2015, foot-and-mouth disease (FMD) virus lineage Ind-2001 was detected for the first time in Southeast Asia. This report contains the first near-complete genome sequence of a viral isolate from this lineage collected from an outbreak in Vietnam. This novel incursion has substantial implications for regional FMD control measures.

Foot-and-mouth disease virus (FMDV) produces a disease in cattle characterized by vesicular lesions and a persistent infection with asymptomatic low-level production of virus in pharyngeal tissues. Here we describe the establishment of a persistently infected primary cell culture derived from bovine pharynx tissue (PBPT) infected with FMDV serotype O1 Manisa, where surviving cells were serially passed until a persistently infected culture was generated. Characterization of the persistent virus demonstrated changes in its plaque size, ability to grow in different cell lines, and change in the use of integrins as receptors, when compared with the parental virus. These results demonstrate the establishment of persistently infected PBPT cell cultures where co-adaptation has taken place between the virus and host cells. This in vitro model for FMDV persistence may help further understanding of the molecular mechanisms of the cattle carrier state.

In order to characterize foot-and-mouth disease virus (FMDV) infection dynamics in pigs, two simulated-natural inoculation systems were developed and evaluated. Intra-oropharyngeal (IOP) and intra-nasopharyngeal (INP) inoculation both enabled precise control of dose and timing of inoculation while simulating field exposure conditions. There were substantial differences between outcomes of infections by the two routes. IOP inoculation resulted in consistent and synchronous infection, whereas INP inoculation at similar doses resulted in delayed, or completely absent infection. All pigs that developed clinical infection had detectable levels of FMDV RNA in their oropharynx directly following inoculation. Furthermore, FMDV antigens were localized to the oropharyngeal tonsils suggesting a role in early infection. The utility of IOP inoculation was further demonstrated in a vaccine-challenge experiment. Thus, the novel system of IOP inoculation described herein, offers a valid alternative to traditionally used systems for FMDV inoculation of pigs, applicable for experimental studies of FMDV pathogenesis and vaccinology.

Early events in the pathogenesis of foot-and-mouth disease virus (FMDV) infection in cattle were investigated through aerosol and intraepithelial lingual (IEL) inoculations of a cDNA-derived FMDV-A24 wild type virus (FMDV-WT) or a mutant derived from the same clone (FMDV-Mut). After aerosolization of FMDV-WT, primary infection sites had significantly greater quantities of FMDV, viral RNA, and type I/III interferon (IFN) activity compared to corresponding tissues from cattle infected with FMDV-Mut. Additionally, FMDV-WT-infected cattle had marked induction of systemic IFN activity in serum. In contrast, FMDV-Mut aerosol-infected cattle did not manifest systemic IFN response nor had viremia. Interestingly, IEL inoculation of FMDV-Mut in cattle restored the virulent phenotype and systemic IFN response. These data indicate that the attenuated phenotype in cattle is associated with decreased replicative efficiency, reflected by decreased innate response. However, attenuation is abrogated by bypassing the common primary infection sites, inducing accelerated viral replication at the inoculation site.

Foot-and-mouth disease virus (FMDV) has a substantial impact on cattle populations in Uganda, causing short- and long-term production losses and hampering local and international trade. Although FMDV has persisted in Uganda for at least 60 years, its epidemiology there and in other endemic settings remains poorly understood. Here, we utilized a large-scale cross-sectional study of cattle to elucidate the dynamics of FMDV spread in Uganda. Sera samples (n = 14,439) from 211 herds were analyzed for non-structural protein reactivity, an indication of past FMDV exposure. Serological results were used to determine spatial patterns, and a Bayesian multivariable logistic regression mixed model was used to identify risk factors for FMDV infection. Spatial clustering of the disease was evident, with higher risk demonstrated near international borders. Additionally, high cattle density, low annual rainfall, and pastoralism were associated with increased likelihood of FMD seropositivity. These results provide insights into the complex epidemiology of FMDV in Uganda and will help inform refined control strategies in Uganda and other FMDV-endemic settings.

Bovine rhinitis B virus (BRBV) shares many motifs and sequence similarities with foot-and-mouth disease virus (FMDV). This study examined if the BRBV leader proteinase (L(pro) ) could functionally replace that of FMDV. A mutant A24LBRV3DYR FMDV engineered with the BRBV L(pro) and an antigenic marker in the 3D polymerase exhibited growth properties and eIF4G cleavage similar to parental A24WT virus. The A24LBRV3DYR type I interferon activity in infected bovine cells resembled that of A24LL virus that lacks L(pro), but this effect was less pronounced for A24LBRV3DYR infected porcine cells. In vivo studies showed that the A24LBRV3DYR virus was attenuated in cattle, and exhibited low virulence in pigs exposed by direct contact. The mutant virus induced protective immunity in cattle against challenge with parental A24WT. These results provide evidence that L(pro) of different Aphthoviruses are not fully functionally interchangeable and have roles that may depend on the nature of the infected host.

Foot-and-mouth disease (FMD) vaccines are routinely used as effective control tools in large regions worldwide and to limit outbreaks during epidemics. Vaccine-induced protection in cattle has been largely correlated with the FMD virus (FMDV)-specific antibodies. Genetic control of cattle immune adaptive responses has been demonstrated only for peptide antigens derived from FMDV structural proteins. Here, we quantify the heterogeneity in the antibody response of cattle primo-vaccinated against FMD and study its association with the genetic background in Holstein and Jersey sires. A total of 377 FMDV-seronegative calves (122 and 255 calves from 16 and 15 Holstein and Jersey sires, respectively) were included in the study. Samples were taken the day prior to primo-vaccination and 45 days post-vaccination (dpv). Animals received commercial tetravalent FMD single emulsion oil vaccines formulated with inactivated FMDV. Total FMDV-specific antibody responses were studied against three viral strains included in the vaccine, and antibody titres were determined by liquid-phase blocking ELISA. Three linear hierarchical mixed regression models, one for each strain, were formulated to assess the heterogeneity in the immune responses to vaccination. The dependent variables were the antibody titres induced against each FMDV strain at 45 dpv, whereas sire's 'breed' was included as a fixed effect, 'sire' was included as a random effect, and 'farm' was considered as a hierarchical factor to account for lack of independence of within herd measurements. A significant association was found between anti-FMDV antibody responses and sire's breed, with lower immune responses found in the Jersey sires' offspring compared with those from Holstein sires. No significant intrabreed variation was detected. In addition, farm management practices were similar in this study, and results of the serological assays were shown to be repeatable. It therefore seems plausible that differences in the immune response may be expected in the event of a mass vaccination campaigns.

Recombinant adenovirus-5 vectored foot-and-mouth disease constructs (Ad5- FMD) were made for three Indian vaccine virus serotypes O, A and Asia 1. Constructs co-expressing foot-and- mouth disease virus (FMDV) capsid and viral 3C protease sequences, were evaluated for their ability to induce a neutralizing antibody response in indigenous cattle (Bos indicus). Purified Ad5-FMD viruses were inoculated in cattle as monovalent (5×109 pfu/animal) or trivalent (5×109 pfu/animal per serotype) vaccines. Animals vaccinated with monovalent Ad5-FMD vaccines were boosted 63days later with the same dose. After primary immunization, virus neutralization tests (VNT) showed seroconversion in 83, 67 and 33% of animals vaccinated with Ad5-FMD O, A and Asia 1, respectively. Booster immunization elicited seroconversion in all of the animals (100%) in the monovalent groups. When used in a trivalent form, the Ad5-FMD vaccine induced neutralizing antibodies in only 33, 50 and 16% of animals against serotypes O, A and Asia 1, respectively on primo-vaccination, and titers were significantly lower than when the same vectors were used in monovalent form. Neutralizing antibody titers differed by serotype for both Ad5-FMD monovalent and trivalent vaccines, with Asia 1 serotype inducing the lowest titers. Antibody response to Ad5 vector in immunized cattle was also assessed by VNT. It appeared that the vector immunity did not impact the recall responses to expressed FMDV antigens on booster immunization. In summary, the study suggested that the recombinant Ad5-FMD vaccine has a potential use in monovalent form, while its application in multivalent form is not currently encouraging.

The epidemiological situation of foot-and-mouth disease virus (FMDV) is uncertain in Nigeria, where the disease is endemic, and the majority of outbreaks are unreported. Control measures for FMD in Nigeria are not being implemented due to the absence of locally produced vaccines and an official ban on vaccine importation. This study summarizes the findings of a 3-year study aimed at quantifying the seroprevalence of FMD, its distribution in susceptible species and the genetic diversity of FMDV isolated from the Plateau State of Nigeria. A 29% FMD prevalence was estimated using 3ABC enzyme-linked immunosorbent assay (3ABC ELISA). Farms with suspected FMD nearby, with contact with wildlife, that used drugs or FMD vaccines or with100 animals, and animals of large ruminant species and in pastures other than nomadic grazing were significantly (P 0.05) associated with FMD. Antibodies against five FMDV serotypes, (A, O, SAT1, SAT2 and SAT3) were detected by the virus neutralization test (VNT) at various titres (100-800) from all tested sera from most parts of the region. This is probably the first report of the presence of FMDV SAT3 in Nigeria. Further studies to investigate the potential probable presence and prevalence of SAT 3 virus in Nigeria are required. Tissue samples collected from clinical animals were positive for FMDV. Virus isolates were sequenced and confirmed as serotype A. All of the isolates showed marked genetic homogeneity with99% genetic identity in the VP1 region and were most closely related to a previously described virus collected from Cameroon in 2000. This study provides knowledge on the epidemiological situation of FMD in Plateau State, Nigeria, and will probably help to develop effective control and preventive strategies for the disease in Nigeria and other countries in the West African subregion.

In order to investigate the mechanisms of persistent foot-and-mouth disease virus (FMDV) infection in cattle, transcriptome alterations associated with the FMDV carrier state were characterized using a bovine whole-transcriptome microarray. Eighteen cattle (8 vaccinated with a recombinant FMDV A vaccine, 10 non-vaccinated) were challenged with FMDV A24 Cruzeiro, and the gene expression profiles of nasopharyngeal tissues collected between 21 and 35 days after challenge were compared between 11 persistently infected carriers and 7 non-carriers. Carriers and non-carriers were further compared to 2 naive animals that had been neither vaccinated nor challenged. At a controlled false-discovery rate of 10% and a minimum difference in expression of 50%, 648 genes were differentially expressed between FMDV carriers and non-carriers, and most (467) had higher expression in carriers. Among these, genes associated with cellular proliferation and the immune response-such as chemokines, cytokines and genes regulating T and B cells-were significantly overrepresented. Differential gene expression was significantly correlated between non-vaccinated and vaccinated animals (biological correlation +0.97), indicating a similar transcriptome profile across these groups. Genes related to prostaglandin E2 production and the induction of regulatory T cells were overexpressed in carriers. In contrast, tissues from non-carrier animals expressed higher levels of complement regulators and pro-apoptotic genes that could promote virus clearance. Based on these findings, we propose a working hypothesis for FMDV persistence in nasopharyngeal tissues of cattle, in which the virus may be maintained by an impairment of apoptosis and the local suppression of cell-mediated antiviral immunity by inducible regulatory T cells.

Background In order to investigate host factors associated with the establishment of persistent foot-and-mouth disease virus (FMDV) infection, the systemic response to vaccination and challenge was studied in 47 steers. Eighteen steers that had received a recombinant FMDV A vaccine 2 weeks earlier and 29 non-vaccinated steers were challenged by intra-nasopharyngeal deposition of FMDV A24. For up to 35 days after challenge, host factors including complete blood counts with T lymphocyte subsets, type I/III interferon (IFN) activity, neutralizing and total FMDV-specific antibody titers in serum, as well as antibody-secreting cells (in 6 non-vaccinated animals) were characterized in the context of viral infection dynamics. Results Vaccination generally induced a strong antibody response. There was a transient peak of FMDV-specific serum IgM in non-vaccinated animals after challenge, while IgM levels in vaccinated animals did not increase further. Both groups had a lasting increase of specific IgG and neutralizing antibody after challenge. Substantial systemic IFN activity in non-vaccinated animals coincided with viremia, and no IFN or viremia was detected in vaccinated animals. After challenge, circulating lymphocytes decreased in non-vaccinated animals, coincident with viremia, IFN activity, and clinical disease, whereas lymphocyte and monocyte counts in vaccinated animals were unaffected by vaccination but transiently increased after challenge. The CD4+/CD8+ T cell ratio in non-vaccinated animals increased during acute infection, driven by an absolute decrease of CD8+ cells. Conclusions The incidence of FMDV persistence was 61.5 % in non-vaccinated and 54.5 % in vaccinated animals. Overall, the systemic factors examined were not associated with the FMDV carrier/non-carrier divergence; however, significant differences were identified between responses of non-vaccinated and vaccinated cattle. Electronic supplementary material The online version of this article (doi:10.1186/s12917-016-0838-x) contains supplementary material, which is available to authorized users.

Foot-and-mouth disease (FMD) is a highly contagious and economically important viral disease of cloven-hoofed animals, including domestic and wild host species. During recent FMD outbreaks in India, spontaneous abortions were reported amongst FMD-affected and asymptomatic cows. The current study was an opportunistic investigation of these naturally occurring bovine abortions to assess causality of abortion and vertical transmission of FMDV from infected cows to fetuses. For this purpose, fetal tissue samples of eight abortuses (heart, liver, kidney, spleen, palatine tonsil, umbilical cord, soft palate, tongue, lungs, and submandibular lymph node) were collected and screened by various detection methods, including viral genome detection, virus isolation, and immunomicroscopy. Amongst these cases, gross pathological changes were observed in 3 abortuses. Gross pathological findings included blood-tinged peritoneal and pleural effusions and myocarditis. Hearts of infected calves had mild to moderate degeneration and necrosis of the myocardium with moderate infiltration by mixed inflammatory cells. Localization of FMDV antigen was demonstrated in lungs and soft palate by immunomicroscopy. FMDV serotype O viral genome was recovered from 7 of 8 cases. Infectious FMDV serotype O was rescued by chemical transfection of the total RNA extracted from three soft palate samples and was sequenced to confirm 100% identity of the VP1 (capsid) coding region with isolates collected from infected cattle during the acute phase of infection. Based upon these findings, it may be concluded that FMDV-associated abortion occurred among the infected pregnant cows included within this study and FMDV was subsequently transmitted vertically to fetuses. This is the first documentation of FMDV-associated abortions in cattle.

Foot-and-mouth disease (FMD) is a highly transmissible disease of hooved livestock. Although FMD has been eradicated from many countries, economic and social consequences of FMD reintroductions are devastating. After achieving disease eradication, Argentina was affected by a major epidemic in 2000-2002, and within few months, FMD virus spread throughout most of the country and affected >2500 herds. Available records and viral strains allowed us to assess the origins, spread and progression of this FMD epidemic, which remained uncertain. We used whole genome viral sequences and a continuous phylogeographic diffusion approach, which revealed that the viruses that caused the outbreaks spread fast in different directions from a central area in Argentina. The analysis also suggests that the virus that caused the outbreaks in the year 2000 was different from those found during the 2001 epidemic. To estimate if the approximate overall genetic diversity of the virus was related to disease transmission, we reconstructed the viral demographic variation in time using Bayesian Skygrid approach and compared it with the epidemic curve and the within-herd transmission rate and showed that the genetic temporal diversity of the virus was associated with the increasing number of outbreaks in the exponential phase of the epidemic. Results here provide new evidence of how the disease entered and spread throughout the country. We further demonstrate that genetic data collected during a FMD epidemic can be informative indicators of the progression of an ongoing epidemic. Fil: Brito Rodriguez, Barbara Patricia. United States Department of Agriculture. Agricultural Research Service; Argentina. Universidad de Chile; Chile Fil: König, Guido Alberto. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Museo Argentino de Ciencias Naturales "Bernardino Rivadavia"; Argentina. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Biotecnología; Argentina Fil: Cabanne, Gustavo Sebastián. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Museo Argentino de Ciencias Naturales "Bernardino Rivadavia"; Argentina. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Biotecnología; Argentina Fil: Pérez Beascoechea, Claudia. Ministerio de Agricultura, Ganadería, Pesca y Alimento. Servicio Nacional de Sanidad y Calidad Agroalimentaria; Argentina Fil: Rodriguez, Luis Carlos. United States Department of Agriculture. Agricultural Research Service; Argentina Fil: Perez, Andres. University of Minnesota; Estados Unidos

Arboviruses (arthropod borne viruses) have life cycles that include both vertebrate and invertebrate hosts with substantial differences in vector and host specificity between different viruses. Most arboviruses utilize RNA for their genetic material and are completely dependent on host tRNAs for their translation, suggesting that virus codon usage could be a target for selection. In the current study we analyzed the relative synonymous codon usage (RSCU) patterns of 26 arboviruses together with 25 vectors and hosts, including 8 vertebrates and 17 invertebrates. We used hierarchical cluster analysis (HCA) and principal component analysis (PCA) to identify trends in codon usage. HCA demonstrated that the RSCU of arboviruses reflects that of their natural hosts, but not that of dead-end hosts. Of the two major components identified by PCA, the first accounted for 62.1% of the total variance, and among the 59 codons analyzed in this study, the leucine codon CTG had the highest correlation with the first principal component, however isoleucine had the highest correlation during amino acid analysis. Nucleotide and dinucleotide composition were the variables that explained most of the total codon usage variance. The results suggest that the main factors driving the evolution of codon usage in arboviruses is based on the nucleotide and dinucleotide composition present in the host. Comparing codon usage of arboviruses and potential vector hosts can help identifying potential vectors for emerging arboviruses.

This report establishes and validates a standard operating procedure (SOP) for select agents (SAs) and other biosafety level 3 and/or 4 (BSL-3/4) RNA viruses to rapidly generate noninfectious, barcoded cDNA amenable for next-generation sequencing (NGS). This eliminates the burden of testing all processed samples derived from high-consequence pathogens prior to transfer from high-containment laboratories to lower-containment facilities for sequencing. Our established protocol can be scaled up for high-throughput sequencing of hundreds of samples simultaneously, which can dramatically reduce the cost and effort required for NGS library construction. NGS data from this SOP can provide complete genome coverage from viral stocks and can also detect virus-specific reads from limited starting material. Our data suggest that the procedure can be implemented and easily validated by institutional biosafety committees across research laboratories., Several biosafety level 3 and/or 4 (BSL-3/4) pathogens are high-consequence, single-stranded RNA viruses, and their genomes, when introduced into permissive cells, are infectious. Moreover, many of these viruses are select agents (SAs), and their genomes are also considered SAs. For this reason, cDNAs and/or their derivatives must be tested to ensure the absence of infectious virus and/or viral RNA before transfer out of the BSL-3/4 and/or SA laboratory. This tremendously limits the capacity to conduct viral genomic research, particularly the application of next-generation sequencing (NGS). Here, we present a sequence-independent method to rapidly amplify viral genomic RNA while simultaneously abolishing both viral and genomic RNA infectivity across multiple single-stranded positive-sense RNA (ssRNA+) virus families. The process generates barcoded DNA amplicons that range in length from 300 to 1,000 bp, which cannot be used to rescue a virus and are stable to transport at room temperature. Our barcoding approach allows for up to 288 barcoded samples to be pooled into a single library and run across various NGS platforms without potential reconstitution of the viral genome. Our data demonstrate that this approach provides full-length genomic sequence information not only from high-titer virion preparations but it can also recover specific viral sequence from samples with limited starting material in the background of cellular RNA, and it can be used to identify pathogens from unknown samples. In summary, we describe a rapid, universal standard operating procedure that generates high-quality NGS libraries free of infectious virus and infectious viral RNA. IMPORTANCE This report establishes and validates a standard operating procedure (SOP) for select agents (SAs) and other biosafety level 3 and/or 4 (BSL-3/4) RNA viruses to rapidly generate noninfectious, barcoded cDNA amenable for next-generation sequencing (NGS). This eliminates the burden of testing all processed samples derived from high-consequence pathogens prior to transfer from high-containment laboratories to lower-containment facilities for sequencing. Our established protocol can be scaled up for high-throughput sequencing of hundreds of samples simultaneously, which can dramatically reduce the cost and effort required for NGS library construction. NGS data from this SOP can provide complete genome coverage from viral stocks and can also detect virus-specific reads from limited starting material. Our data suggest that the procedure can be implemented and easily validated by institutional biosafety committees across research laboratories.