Simultaneous and Staggered Foot-and-Mouth Disease Virus Coinfection of Cattle.

Foot-and-mouth disease (FMD) field studies have suggested the occurrence of simultaneous infection of individual hosts by multiple virus strains; however, the pathogenesis of foot-and-mouth disease virus (FMDV) coinfections is largely unknown. In the current study, cattle were experimentally exposed to two FMDV strains of different serotypes (O and A). One cohort was simultaneously infected with both viruses, while additional cohorts were initially infected with FMDV A and subsequently superinfected with FMDV O after 21 or 35 days. Coinfections were confirmed during acute infection, with both viruses concurrently detected in blood, lesions, and secretions. Staggered exposures resulted in overlapping infections as convalescent animals with persistent subclinical FMDV infection were superinfected with a heterologous virus. Staggering virus exposure by 21 days conferred clinical protection in six of eight cattle, which were subclinically infected following the heterologous virus exposure. This effect was transient, as all animals superinfected at 35 days post-initial infection developed fulminant FMD. The majority of cattle maintained persistent infection with one of the two viruses while clearing the other. Analysis of viral genomes confirmed interserotypic recombination events within 10 days in the upper respiratory tract of five superinfected animals from which the dominant genomes contained the capsid coding regions of the O virus and nonstructural coding regions of the A virus. In contrast, there were no dominant recombinant genomes detected in samples from simultaneously coinfected cattle. These findings inculpate persistently infected carriers as potential FMDV mixing vessels in which novel strains may rapidly emerge through superinfection and recombination. IMPORTANCE Foot-and-mouth disease (FMD) is a viral infection of livestock of critical socioeconomic importance. Field studies from areas of endemic FMD suggest that animals can be simultaneously infected by more than one distinct variant of FMD virus (FMDV), potentially resulting in emergence of novel viral strains through recombination. However, there has been limited investigation of the mechanisms of in vivo FMDV coinfections under controlled experimental conditions. Our findings confirmed that cattle could be simultaneously infected by two distinct serotypes of FMDV, with different outcomes associated with the timing of exposure to the two different viruses. Additionally, dominant interserotypic recombinant FMDVs were discovered in multiple samples from the upper respiratory tracts of five superinfected animals, emphasizing the potential importance of persistently infected FMDV carriers as sources of novel FMDV strains.

Phylodynamics of foot-and-mouth disease virus O/PanAsia in Vietnam 2010-2014.

Foot-and-mouth disease virus (FMDV) is endemic in Vietnam, a country that plays an important role in livestock trade within Southeast Asia. The large populations of FMDV-susceptible species in Vietnam are important components of food production and of the national livelihood. In this study, we investigated the phylogeny of FMDV O/PanAsia in Vietnam, reconstructing the virus' ancestral host species (pig, cattle or buffalo), clinical stage (subclinical carrier or clinically affected) and geographical location. Phylogenetic divergence time estimation and character state reconstruction analyses suggest that movement of viruses between species differ. While inferred transmissions from cattle to buffalo and pigs and from pigs to cattle are well supported, transmission from buffalo to other species, and from pigs to buffalo may be less frequent. Geographical movements of FMDV O/PanAsia virus appears to occur in all directions within the country, with the South Central Coast and the Northeast regions playing a more important role in FMDV O/PanAsia spread. Genetic selection of variants with changes at specific sites within FMDV VP1 coding region was different depending on host groups analyzed. The overall ratio of non-synonymous to synonymous nucleotide changes was greater in pigs compared to cattle and buffalo, whereas a higher number of individual amino acid sites under positive selection were detected in persistently infected, subclinical animals compared to viruses collected from clinically diseased animals. These results provide novel insights to understand FMDV evolution and its association with viral spread within endemic countries. These findings may support animal health organizations in their endeavor to design animal disease control strategies in response to outbreaks.

Direct RNA Sequencing of Foot-and-mouth Disease Virus Genome Using a Flongle on MinION.

Foot-and-mouth disease (FMD) is a severe and extremely contagious viral disease of cloven-hoofed domestic and wild animals, which leads to serious economic losses to the livestock industry globally. FMD is caused by the FMD virus (FMDV), a positive-strand RNA virus that belongs to the genus Aphthovirus, within the family Picornaviridae. Early detection and characterization of FMDV strains are key factors to control new outbreaks and prevent the spread of the disease. Here, we describe a direct RNA sequencing method using Oxford Nanopore Technology (ONT) Flongle flow cells on MinION Mk1C (or GridION) to characterize FMDV. This is a rapid, low cost, and easily deployed point of care (POC) method for a near real-time characterization of FMDV in endemic areas or outbreak investigation sites. Key features • Saves ~35 min of the original protocol time by omitting the reverse transcription step and lowers the costs of reagents and consumables. • Replaces the GridION flow cell from the original protocol with the Flongle, which saves ~90% on the flow cell cost. • Combines the NGS benchwork with a modified version of our African swine fever virus (ASFV) fast analysis pipeline to achieve FMDV characterization within minutes. Graphical overview Schematic of direct RNA sequencing of foot-and-mouth disease virus (FMDV) process, which takes ~50 min from extracted RNA to final loading, modified from the ONT SQK-RNA002 protocol (Version: DRS_9080_v2_revO_14Aug2019).

Genome Sequences of Foot-and-Mouth Disease Virus SAT1 Strains Purified from Coinfected Cape Buffalo in Kenya.

Nearly complete genomes of 49 novel foot-and-mouth disease virus (FMDV) SAT1 strains acquired from oropharyngeal fluid samples from asymptomatic African Cape buffalo in Kenya in 2016 were determined. Sequences were from primary passage or plaque-purified dually SAT1/SAT2-infected samples. These sequences are important for elucidation of the molecular epidemiology of persistent and subclinical FMDV infections.

Genome Sequences of Foot-and-Mouth Disease Virus SAT2 Strains Purified from Coinfected Cape Buffalo in Kenya.

Foot-and-mouth disease virus (FMDV) SAT2 sequences were acquired from Cape buffalo in Kenya in 2016, from either primary passage (n = 38) or plaque purification of dually SAT1/SAT2-infected samples (n = 61). All samples were derived from asymptomatic animals. These sequences contribute to our understanding of FMDV diversity in reservoirs and during subclinical FMDV infections.

Viral Population Diversity during Co-Infection of Foot-And-Mouth Disease Virus Serotypes SAT1 and SAT2 in African Buffalo in Kenya.

African buffalo are the natural reservoirs of the SAT serotypes of foot-and-mouth disease virus (FMDV) in sub-Saharan Africa. Most buffalo are exposed to multiple FMDV serotypes early in life, and a proportion of them become persistently infected carriers. Understanding the genetic diversity and evolution of FMDV in carrier animals is critical to elucidate how FMDV persists in buffalo populations. In this study, we obtained oropharyngeal (OPF) fluid from naturally infected African buffalo, and characterized the genetic diversity of FMDV. Out of 54 FMDV-positive OPF, 5 were co-infected with SAT1 and SAT2 serotypes. From the five co-infected buffalo, we obtained eighty-nine plaque-purified isolates. Isolates obtained directly from OPF and plaque purification were sequenced using next-generation sequencing (NGS). Phylogenetic analyses of the sequences obtained from recombination-free protein-coding regions revealed a discrepancy in the topology of capsid proteins and non-structural proteins. Despite the high divergence in the capsid phylogeny between SAT1 and SAT2 serotypes, viruses from different serotypes that were collected from the same host had a high genetic similarity in non-structural protein-coding regions P2 and P3, suggesting interserotypic recombination. In two of the SAT1 and SAT2 co-infected buffalo identified at the first passage of viral isolation, the plaque-derived SAT2 genomes were distinctly grouped in two different genotypes. These genotypes were not initially detected with the NGS from the first passage (non-purified) virus isolation sample. In one animal with two SAT2 haplotypes, one plaque-derived chimeric sequence was found. These findings demonstrate within-host evolution through recombination and point mutation contributing to broad viral diversity in the wildlife reservoir. These mechanisms may be critical to FMDV persistence at the individual animal and population levels, and may contribute to the emergence of new viruses that have the ability to spill-over to livestock and other wildlife species.

Multiple Genomes of Foot-and-Mouth Disease Virus Serotype Asia-1 Obtained from Subclinically Infected Asian Buffalo (Bubalus bubalis) in Pakistan.

We report the near-full-genome sequences of 49 isolates of serotype Asia-1 foot-and-mouth disease virus obtained from subclinically infected Asian buffalo in Islamabad Capital Region, Pakistan, in 2011 to 2012. Sequences from subclinically infected animals are exceedingly rare and complement the more commonly available sequences acquired from clinical cases.

Multiple Genome Sequences of Foot-and-Mouth Disease Virus Asia-1 Lineage Sindh-08 from Outbreaks in Pakistan, 2011 to 2012.

We report the near-full-length genome sequences of 22 isolates of foot-and-mouth disease virus (FMDV) serotype Asia-1, lineage Sindh-08, obtained from foot-and-mouth disease outbreaks in Pakistan between 2011 and 2012. The scarcity of full-length FMDV sequences from this region enhances the importance of these new genomes for understanding the regional molecular epidemiology.

Novel Recombinant Foot-and-Mouth Disease Virus Circulating in Vietnam.

We report the genome sequences of 12 recombinant foot-and-mouth disease virus isolates from Vietnam. The recombinant strain has a capsid region from an A/Sea-97 strain and a nonstructural segment from an O/ME-SA/PanAsia strain. The isolates were obtained from two outbreak samples collected in June 2017 and 10 subclinical samples collected between 2017 and 2019.

Into the Deep (Sequence) of the Foot-and-Mouth Disease Virus Gene Pool: Bottlenecks and Adaptation during Infection in Naïve and Vaccinated Cattle.

Foot-and-mouth disease virus (FMDV) infects hosts as a population of closely related viruses referred to as a quasispecies. The behavior of this quasispecies has not been described in detail in natural host species. In this study, virus samples collected from vaccinated and non-vaccinated cattle up to 35 days post-experimental infection with FMDV A24-Cruzeiro were analyzed by deep-sequencing. Vaccination induced significant differences compared to viruses from non-vaccinated cattle in substitution rates, entropy, and evidence for adaptation. Genomic variation detected during early infection reflected the diversity inherited from the source virus (inoculum), whereas by 12 days post infection, dominant viruses were defined by newly acquired mutations. Mutations conferring recognized fitness gain occurred and were associated with selective sweeps. Persistent infections always included multiple FMDV subpopulations, suggesting distinct foci of infection within the nasopharyngeal mucosa. Subclinical infection in vaccinated cattle included very early bottlenecks associated with reduced diversity within virus populations. Viruses from both animal cohorts contained putative antigenic escape mutations. However, these mutations occurred during later stages of infection, at which time transmission is less likely to occur. This study improves upon previously published work by analyzing deep sequences of samples, allowing for detailed characterization of FMDV populations over time within multiple hosts.

Genome Sequences of Seven Foot-and-Mouth Disease Virus Isolates Reveal Diversity in the O/ME-SA/Ind2001 Lineage in India between 1997 and 2009.

We report the genome sequences of seven foot-and-mouth disease (FMD) virus (FMDV) isolates collected in India between 1997 and 2009. The strains represented four sublineages within the O/ME-SA/Ind2001 lineage. These viruses provide insights into FMDV diversity and evolution in India and may influence future control measures, including vaccine selections.

Foot-and-Mouth Disease Virus Serotype O/CATHAY Genome Sequences from Five Outbreaks in Vietnam, 2017 to 2019.

We report the genomes of five foot-and-mouth disease viruses (FMDVs) from distinct provinces in Vietnam. All five viruses were grouped within the O/CATHAY topotype. Sequences contain the full polyprotein coding sequence and partial untranslated regions. These genomes provide critical data on the spread and evolution of FMDVs in the region.

Field evaluation of a multiplex real-time reverse transcription polymerase chain reaction assay for detection of Vesicular stomatitis virus.

Sporadic outbreaks of vesicular stomatitis (VS) in the United States result in significant economic losses for the U.S. livestock industries because VS is a reportable disease that clinically mimics foot-and-mouth disease. Rapid and accurate differentiation of these 2 diseases is critical because their consequences and control strategies differ radically. The objective of the current study was to field validate a 1-tube multiplexed real-time reverse transcription polymerase chain reaction (real-time RT-PCR) assay for the rapid detection of Vesicular stomatitis New Jersey virus and Vesicular stomatitis Indiana virus strains occurring in Mexico and North and Central America. A comprehensive collection of 622 vesicular lesion samples obtained from cattle, horses, and swine from throughout Mexico and Central America was tested by the real-time RT-PCR assay and virus isolation. Overall, clinical sensitivity and specificity of the real-time RT-PCR were 83% and 99%, respectively. Interestingly, VS virus isolates originating from a specific region of Costa Rica were not detected by real-time RT-PCR. Sequence comparisons of these viruses with the real-time RT-PCR probe and primers showed mismatches in the probe and forward and reverse primer regions. Additional lineage-specific primers and a probe corrected the lack of detection of the missing genetic lineage. Thus, this assay reliably identified existing Mexican and Central American VS viruses and proved readily adaptable as new VS viruses were encountered. An important secondary result of this research was the collection of hundreds of new VS virus isolates that provide a foundation from which many additional studies can arise.

Near-Full-Length Genome Sequence of a Foot-and-Mouth Disease Virus of Serotype Southern African Territories 2 Isolated from Nigeria in 2014.

We report a near-full-length genome sequence of a foot-and-mouth disease virus (FMDV) of serotype Southern African Territories 2 (SAT 2), topotype VII, isolated from cattle during an FMDV outbreak in Bauchi State, Nigeria, in October 2014. This provides the first SAT 2 near-full-length genome sequence from West Africa and contributes to our understanding of viral spread and evolution.

First Report of Near-Complete Genome Sequences of Foot-and-Mouth Disease Virus Serotype O Strains from Kenya.

This is the first report of two near-complete genome sequences of foot-and-mouth disease virus (FMDV) serotype O from Kenya. The viruses were isolated from bovine epithelium collected in 2014 and 2016 from local FMD outbreaks. These full-genome sequences are critical for improving the understanding of regional FMDV molecular epidemiology.

The evolution of a super-swarm of foot-and-mouth disease virus in cattle.

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.

Genome Sequences of Foot-and-Mouth Disease Virus SAT1 and SAT2 Strains from Kenya in 2014 to 2016.

Here, we report the near-complete genomes of three Southern African Territories 1 (SAT1) serotype strains and one SAT2 serotype strain of foot-and-mouth disease virus (FMDV) recently isolated from Kenya. Viral isolates were obtained from bovine epithelial tissues collected in 2014 and 2016 following outbreaks of foot-and-mouth disease (FMD). These near-complete genome sequences provide a critical update of Kenyan FMDV molecular epidemiology.

Genome Sequences of Four Foot-and-Mouth Disease Virus SAT 1 Topotype X Isolates from Cameroon.

We report the genomes of four foot-and-mouth disease virus (FMDV) serotype SAT 1 topotype X isolates from Cameroon. The viruses were isolated from bovine epithelium collected during an outbreak in 2016. These novel sequences update knowledge of FMDV diversity in Central Africa and contribute to regional FMDV molecular epidemiology.

Foot-and-Mouth Disease Virus Serotype A Genome Sequence from Kenya in 2016.

We report the genome sequence of a foot-and-mouth disease virus (FMDV) serotype A topotype Africa isolate collected from bovine vesicular epithelium from Kenya in 2016. This novel sequence updates the knowledge of FMDV diversity in eastern Africa and has important implications for FMDV epidemiology and molecular analyses.

Genome Sequences of 18 Foot-and-Mouth Disease Virus Outbreak Strains of Serotype O Sublineage Ind2001d from India, 2013 to 2014.

We report the full polyprotein-coding sequences and partial untranslated regions (UTRs) of 18 foot-and-mouth disease (FMD) viruses from 4 outbreaks in India in 2013 and 2014. All strains grouped within the O/ME-SA/Ind2001d sublineage. These genomes update knowledge of FMD virus (FMDV) diversity in South Asia and may contribute to molecular epidemiology studies and vaccine selections.