Phylogeography as a Proxy for Population Connectivity for Spatial Modeling of Foot-and-Mouth Disease Outbreaks in Vietnam.

Bayesian space-time regression models are helpful tools to describe and predict the distribution of infectious disease outbreaks and to delineate high-risk areas for disease control. In these models, structured and unstructured spatial and temporal effects account for various forms of non-independence amongst case counts across spatial units. Structured spatial effects capture correlations in case counts amongst neighboring provinces arising from shared risk factors or population connectivity. For highly mobile populations, spatial adjacency is an imperfect measure of connectivity due to long-distance movement, but we often lack data on host movements. Phylogeographic models inferring routes of viral dissemination across a region could serve as a proxy for patterns of population connectivity. The objective of this study was to investigate whether the effects of population connectivity in space-time regressions of case counts were better captured by spatial adjacency or by inferences from phylogeographic analyses. To compare these two approaches, we used foot-and-mouth disease virus (FMDV) outbreak data from across Vietnam as an example. We identified that accounting for virus movement through phylogeographic analysis serves as a better proxy for population connectivity than spatial adjacency in spatial-temporal risk models. This approach may contribute to design surveillance activities in countries lacking movement data.

Epidemiologic and economic considerations regarding persistently infected cattle during vaccinate-to-live strategies for control of foot-and-mouth disease in FMD-free regions.

Development of a foot-and-mouth disease (FMD) carrier state following FMD virus (FMDV) infection is a well-established phenomenon in cattle. However, the proportion of cattle likely to become carriers and the duration of the carrier state at a herd or population-level are incompletely understood. The objective of this study was to examine the epidemiologic and economic impacts of vaccination-to-live strategy in a disease-free region or country. We developed and simulated scenarios of FMD spread and control in the US livestock population, which included depopulation for a limited period, followed by a vaccinate-to-live strategy with strong biosecurity and movement restrictions. Six scenarios of FMD spread and control were simulated in the InterSpread Plus (ISP) modeling tool. Data on the number of infected and depopulated cattle (by operation types) from ISP model runs were used to estimate the monthly number of infected but not depopulated (potential carrier) cattle after the infection. Using available literature data on the FMD carrier state, we estimated the monthly proportion of carrier cattle (from infected but not depopulated cattle) over time following infection. Among the simulated scenarios, the median (25th, 75th percentile) number of infected cattle ranged from 43,217 (42,819, 55,274) head to 148,907 (75,819, 205,350) head, and the epidemic duration ranged from 20 (11, 30) to 76 (38, 136) days. In general, larger outbreaks occurred when depopulation was carried out through longer periods, and the onset of the vaccination was late (p > 0.05). The estimated proportion of surviving cattle, which were infected and not depopulated and had the potential to become persistently infected ranged from 14 to 35% of total infected cattle. Production losses in beef and dairy sectors were higher when outbreaks started in multiple states simultaneously, but production losses were small compared to trade losses and consumer avoidance losses. These results can be used to inform the consideration of a vaccinate-to-live strategy for FMD outbreaks and the development of appropriate post-outbreak management strategies. Furthermore, this output will enable a more detailed examination of the epidemiologic and economic implications of allowing convalescent cattle to survive and remain in production chains after FMD outbreaks in FMD-free regions.

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.

Foot-and-Mouth Disease Virus Interserotypic Recombination in Superinfected Carrier Cattle.

Viral recombination contributes to the emergence of novel strains with the potential for altered host range, transmissibility, virulence, and immune evasion. For foot-and-mouth disease virus (FMDV), cell culture experiments and phylogenetic analyses of field samples have demonstrated the occurrence of recombination. However, the frequency of recombination and associated virus-host interactions within an infected host have not been determined. We have previously reported the detection of interserotypic recombinant FMDVs in oropharyngeal fluid (OPF) samples of 42% (5/12) of heterologously superinfected FMDV carrier cattle. The present investigation consists of a detailed analysis of the virus populations in these samples including identification and characterization of additional interserotypic minority recombinants. In every animal in which recombination was detected, recombinant viruses were identified in the OPF at the earliest sampling point after superinfection. Some recombinants remained dominant until the end of the experiment, whereas others were outcompeted by parental strains. Genomic analysis of detected recombinants suggests host immune pressure as a major driver of recombinant emergence as all recombinants had capsid-coding regions derived from the superinfecting virus to which the animals did not have detectable antibodies at the time of infection. In vitro analysis of a plaque-purified recombinant virus demonstrated a growth rate comparable to its parental precursors, and measurement of its specific infectivity suggested that the recombinant virus incurred no penalty in packaging its new chimeric genome. These findings have important implications for the potential role of persistently infected carriers in FMDV ecology and the emergence of novel strains.

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.

Genome of Bovine Viral Diarrhea Virus (BVDV) Contaminating a Continuous LFBK-αVß6 Cell Line.

Here, we report the genome of bovine viral diarrhea virus 1 (BVDV-1) contaminating a continuous fetal bovine kidney cell line. The cell line (LFBK-αVß6) is used for the rapid isolation and serotyping of foot-and-mouth disease virus (FMDV). The sequence contains the full polyprotein-coding sequence and partial untranslated regions (UTRs).

The risk and mitigation of foot-and-mouth disease virus infection of pigs through consumption of contaminated feed.

Transboundary movement of animal feed and feed ingredients has been identified as a route for pathogen incursions. While imports of animals and animal-derived products are highly regulated for the purpose of infectious disease prevention, there has been less consideration of the viability of infectious agents in inanimate products, such as feed. This study investigated the ability of foot-and-mouth disease virus (FMDV) to remain infectious as a contaminant of commercial whole pig feed and select pig feed ingredients, and to establish the minimum infectious dose (MIDF ) required to cause foot-and-mouth disease (FMD) in pigs that consumed contaminated feed. FMDV viability in vitro varied depending on virus strain, feed product, and storage temperature, with increased duration of infectivity in soybean meal compared to pelleted whole feed. Specifically, both strains of FMDV evaluated remained viable through to the end of the 37 day observation period in experimentally contaminated soybean meal stored at 4 or 20°C . The MIDF for pigs consuming contaminated feed varied across virus strains and exposure duration in the range of 106.2 to 107 TCID50 . The ability of FMDV to cause infection in exposed pigs was mitigated by pre-treatment of feed with two commercially available feed additives, based on either formaldehyde (SalCURB®) or lactic acid (Guardian™). Our findings demonstrate that FMDV may remain infectious in pig feed ingredients for durations compatible with transoceanic transport. Although the observed MIDF was relatively high, variations in feeding conditions and biophysical characteristics of different virus strains may alter the probability of infection. These findings may be used to parameterize modelling of the risk of FMDV incursions and to regulate feed importation to minimize the risk of inadvertent importation.

Use of Slaughterhouses as Sentinel Points for Genomic Surveillance of Foot-and-Mouth Disease Virus in Southern Vietnam.

The genetic diversity of foot-and-mouth disease virus (FMDV) poses a challenge to the successful control of the disease, and it is important to identify the emergence of different strains in endemic settings. The objective of this study was to evaluate the sampling of clinically healthy livestock at slaughterhouses as a strategy for genomic FMDV surveillance. Serum samples (n = 11,875) and oropharyngeal fluid (OPF) samples (n = 5045) were collected from clinically healthy cattle and buffalo on farms in eight provinces in southern and northern Vietnam (2015-2019) to characterize viral diversity. Outbreak sequences were collected between 2009 and 2019. In two slaughterhouses in southern Vietnam, 1200 serum and OPF samples were collected from clinically healthy cattle and buffalo (2017 to 2019) as a pilot study on the use of slaughterhouses as sentinel points in surveillance. FMDV VP1 sequences were analyzed using discriminant principal component analysis and time-scaled phylodynamic trees. Six of seven serotype-O and -A clusters circulating in southern Vietnam between 2017-2019 were detected at least once in slaughterhouses, sometimes pre-dating outbreak sequences associated with the same cluster by 4-6 months. Routine sampling at slaughterhouses may provide a timely and efficient strategy for genomic surveillance to identify circulating and emerging FMDV strains.

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.

Identical Viral Genetic Sequence Found in Black Flies (Simulium bivittatum) and the Equine Index Case of the 2006 U.S. Vesicular Stomatitis Outbreak.

In 2006, vesicular stomatitis New Jersey virus (VSNJV) caused outbreaks in Wyoming (WY) horses and cattle after overwintering in 2004 and 2005. Within two weeks of the outbreak onset, 12,203 biting flies and 194 grasshoppers were collected near three equine-positive premises in Natrona County, WY. Insects were identified to the species level and tested by RT-qPCR for VSNJV polymerase (L) and phosphoprotein (P) gene RNA. Collected dipterans known to be competent for VSV transmission included Simulium black flies and Culicoides biting midges. VSNJV L and P RNA was detected in two pools of female Simulium bivittatum and subjected to partial genome sequencing. Phylogenetic analysis based on the hypervariable region of the P gene from black flies showed 100% identity to the isolate obtained from the index horse case on the same premises. This is the first report of VSNJV in S. bivittatum in WY and the first field evidence of possible VSV maintenance in black fly populations during an outbreak.

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.

Extinction Dynamics of the Foot-and-Mouth Disease Virus Carrier State Under Natural Conditions.

Foot-and-mouth disease (FMD) is one of the most economically important livestock diseases worldwide. Following the clinical phase of FMD, a large proportion of ruminants remain persistently infected for extended periods. Although extinction of this carrier state occurs continuously at the animal and population levels, studies vary widely in their estimates of the duration of persistent infection. There is a need for robust statistical models to capture the dynamics of persistent infection for the sake of guiding FMD control and trade policies. The goal of the current study was to develop and assess statistical models to describe the extinction of FMD virus (FMDV) persistent infection using data from primary longitudinal studies of naturally infected cattle and Asian buffalo in Vietnam and India. Specifically, accelerated failure time (AFT) models and generalized linear mixed models (GLMM) were developed to predict the probability of persistent infection in seropositive animals and identified carriers at the individual animal level at sequential time points after outbreaks. The primary studies were analyzed by country and combined using an individual-participant data meta-analysis approach. The models estimated similar trends in the duration of persistent infection for the study/species groups included in the analyses, however the significance of the trends differed between the models. The overall probabilities of persistent infection were similar as predicted by the AFT and GLMM models: 6 months: 99% (AFT) /80% (GLMM), 12 months: 51% (AFT) /32% (GLMM), 18 months: 6% (AFT) /5% (GLMM), 24 months: 0.8% (AFT) /0.6% (GLMM). These models utilizing diverse and robust data sets predict higher probabilities of persistence than previously published, suggesting greater endurance of carriers subsequent to an outbreak. This study demonstrates the utility of statistical models to investigate the dynamics of persistent infection and the importance of large datasets, which can be achieved by combining data from several smaller studies in meta-analyses. Results of this study enhance current knowledge of the FMDV carrier state and may inform policy decisions regarding FMDV persistent infection.

Duration of Contagion of Foot-And-Mouth Disease Virus in Infected Live Pigs and Carcasses.

Data-driven modeling of incursions of high-consequence, transboundary pathogens of animals is a critical component of veterinary preparedness. However, simplifying assumptions and excessive use of proxy measures to compensate for gaps in available data may compromise modeled outcomes. The current investigation was prospectively designed to address two major gaps in current knowledge of foot-and-mouth disease virus (FMDV) pathogenesis in pigs: the end (duration) of the infectious period and the viability of FMDV in decaying carcasses. By serial exposure of sentinel groups of pigs to the same group of donor pigs infected by FMDV A24 Cruzeiro, it was demonstrated that infected pigs transmitted disease at 10 days post infection (dpi), but not at 15 dpi. Assuming a latent period of 1 day, this would result in a conservative estimate of an infectious duration of 9 days, which is considerably longer than suggested by a previous report from an experiment performed in cattle. Airborne contagion was diminished within two days of removal of infected pigs from isolation rooms. FMDV in muscle was inactivated within 7 days in carcasses stored at 4oC. By contrast, FMDV infectivity in vesicle epithelium harvested from intact carcasses stored under similar conditions remained remarkably high until the study termination at 11 weeks post mortem. The output from this study consists of experimentally determined data on contagion associated with FMDV-infected pigs. This information may be utilized to update parameterization of models used for foot-and-mouth disease outbreak simulations involving areas of substantial pig production.

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.

Characterization of transboundary foot-and-mouth disease viruses in Nigeria and Cameroon during 2016.

Continuous surveillance for foot-and-mouth disease (FMD) in endemic settings such as West Africa is imperative to support improved local and regional control plans, with the long-term goal of regional eradication. This paper describes the genetic characterization of FMD viruses (FMDV) obtained from outbreaks in Nigeria (n = 45) and Cameroon (n = 15) during 2016 and from archival samples (n = 3) retrieved from a 2014 outbreak in Nigeria. These viruses were analysed in the context of previously published FMDV sequences from the region. Four FMDV serotypes: O, A, SAT1 and SAT2, were detected. Phylogenetic analyses of the VP1 coding sequences indicate the continuity of FMDV serotype O East Africa-3 (O/EA-3), serotype A AFRICA genotype G-IV (A/AFRICA/G-IV) and serotype South African Territories (SAT) 2 lineage VII (SAT2/VII). The FMDV SAT1 topotype X (SAT1/X), which emerged in Nigeria in 2015, continued to be associated with outbreaks in the region during 2016, and SAT1 is reported for the first time from Cameroon. Additionally, a re-emergence or re-introduction of the serotype O West Africa (O/WA) topotype in Nigeria is described herein. Our findings indicate a consistent, pan-serotypic relationship between FMDV strains detected in Cameroon and Nigeria. Additionally, FMDV strains from West Africa obtained in this study were genetically related to those occurring in East and North Africa. These phylogenetic relationships suggest that animal movements (pastoralism and/or trade) are important factors for virus spread across the African continent. These data provide critical baselines which are a necessary component of Stages 0 and 1 of the Progressive Control Pathway of FMD (PCP-FMD). Specifically, characterizing the existing virus strains (risk) provides the basis for the comprehensive risk-based control plan which is the requisite criteria for Nigeria's transition to Stage 2 of PCP-FMD, and for coordinated regional control of FMD.

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.