Novel pan-lineage VP1 specific degenerate primers for precise genetic characterization of serotype O foot and mouth disease virus circulating in India.

Analysis of the VP1 gene sequence of the foot and mouth disease virus (FMDV) is critical to understanding viral evolution and disease epidemiology. A standard set of primers have been used for the detection and sequence analysis of the VP1 gene of FMDV directly from suspected clinical samples with limited success. The study validated VP1-specific degenerate primer-based reverse transcription polymerase chain reaction (RT-PCR) for the qualitative detection and sequencing of serotype O FMDV lineages circulating in India. The novel degenerate primer-based RT-PCR amplifying the VP1 gene can circumvent the genetic heterogeneity observed in viruses after cell culture adaptation and facilitate precise viral gene sequence analysis from clinical samples.

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.

Genotyping of foot-and-mouth disease viruses collected in Sudan between 2009 and 2018.

Foot-and-mouth disease (FMD) is widely distributed in Sudan where outbreaks occur on an annual basis especially during the winter months (December-February). This study aimed to increase our understanding of the epidemiological patterns of FMD in Sudan and connections to neighbouring countries by characterizing the genetic sequences of FMD viruses (FMDV) collected from samples collected in 10 Sudanese states over a 10-year period (between 2009 and 2018). FMDV was detected in 91 of the 265 samples using an antigen-detection ELISA. Three serotypes were detected: O (46.2%), A (34.0%), and SAT 2 (19.8%). Fifty-two of these samples were submitted for sequence analyses, generating sequences that were characterized as belonging to O/EA-3 (n = 17), A/AFRICA/G-IV (n = 23) and SAT 2/VII/Alx-12 (n = 12) viral lineages. Phylogenetic analyses provided evidence that FMDV lineages were maintained within Sudan, and also highlighted epidemiological connections to FMD outbreaks reported in neighbouring countries in East and North Africa (such as Ethiopia and Egypt). This study motivates continued FMD surveillance in Sudan to monitor the circulating viral lineages and broader initiatives to improve our understanding of the epidemiological risks in the region.

Foot-and-mouth disease viruses of the O/ME-SA/Ind-2001e sublineage in Pakistan.

The presence of foot-and-mouth disease virus (FMDV) of the O/ME-SA/Ind-2001e sublineage within Pakistan was initially detected in two samples collected during 2019. Analysis of further serotype O FMDVs responsible for disease outbreaks in 2019-2020 in the country has now identified the spread of this sublineage to 10 districts within two separate provinces in North-Eastern and North-Western Pakistan. Phylogenetic analysis indicates that these viruses are closely related to those circulating in Bhutan, Nepal and India. The VP1 coding sequences of these viruses from Pakistan belong to three distinct clusters, which may indicate multiple introductions of this virus sublineage, although the routes of introduction are unknown. Vaccine matching studies against O1 Manisa, O 3039 and O TUR/5/2009 support the suitability of existing vaccine strains to control current field outbreaks, but further studies are warranted to monitor the spread and evolution of the O/ME-SA/Ind-2001e sublineage in the region. (145 words).

Adverse effects of foot-and-mouth disease vaccine in dairy cattle / Efeitos adversos da vacina contra febre aftosa em bovinos de leite

Foot-and-mouth disease represents an important barrier to the international commerce of animal products, potentially associated with significant economic losses. The systematic vaccination of bovines and buffaloes was fundamental for the eradication of this disease; however, the use of vaccines can lead to reactions at the application site. The objectives of this study were to evaluate the effects of the vaccination protocol to the production of dairy cows and to observe the occurrence of vaccinal reactions in the animals. At one property located in the municipality of Salvador do Sul, Rio Grande do Sul, 270 dairy cows were vaccinated against foot-and-mouth disease in May 2019. The vaccine was administered via a subcutaneous application using disposable syringes and needles for each animal. Inspection of the animals was performed before and 20 days after the vaccination to verify the presence of reactions to the vaccine. The study's sample was set by convenience, including 203 lactating animals with or without bovine somatotropin (BST) administration during the data collection period, which was limited to 20 days before and 20 days after the vaccination. Milk production data was obtained through SmartDairy® HerdMetrix™ software, tabulated in electronic spreadsheets using Microsoft Excel® and processed using the program SAS®, considering a 5% significance level for mixed model statistical analysis. A total of 160 animals (78.82%) presented local lesions at the application site, even when the recommended vaccination practices were followed, suggesting that the high reaction power was provoked by the vaccinal components. In regards to milk production, a statistically significant (p<0.05) decrease of 0.30kg of milk per animal/day was observed in the average daily production in the 20 days post-vaccination. These results demonstrate the local and systemic effects caused by the foot-and-mouth disease vaccine, evidenced by reduced levels of milk production and the occurrence of vaccine reactions, implying significant economic losses.(AU)

A febre aftosa representa uma importante barreira no comércio internacional de produtos de origem animal, podendo acarretar em significativas perdas econômicas. A vacinação sistemática de bovinos e bubalinos foi fundamental para a erradicação da doença. No entanto, a utilização de vacinas pode causar reações no local da aplicação. O objetivo deste trabalho foi avaliar os efeitos da vacina em bovinos leiteiros e observar a incidência de reações vacinais no local de aplicação. O estudo foi realizado numa propriedade leiteira do município de Salvador do Sul, Rio Grande do Sul, onde foram vacinados 270 bovinos contra febre aftosa no mês de maio de 2019. A vacina foi administrada por via subcutânea, com seringas e agulhas descartáveis para cada animal. Foi realizada inspeção dos animais antes da vacinação e 21 dias após a vacinação, para verificar a presença de reações vacinais. A amostra foi definida por conveniência, incluindo 203 vacas em lactação com ou sem administração de somatotropina bovina (BST) durante o período de coleta de dados, que l foi de 20 dias antes e 20 dias após a vacinação. Estes dados de produção de leite foram obtidos através do software SmartDairy® HerdMetrix™, tabulados em planilhas eletrônicas do Microsoft Excel® e processados usando o programa SAS®, considerando 5% de nível de significância para uma análise estatística modelo misto. Foi observado que 160 (78,82%) vacas apresentaram lesões no local de aplicação, mesmo quando a aplicação era realizada de acordo com as boas práticas de vacinação, o que indica o alto poder de reação provocada pelos componentes da vacina. Em relação à produção de leite, observou-se uma redução significativa (p<0,05) na produção média diária de 0,30kg de leite por animal/dia nos 21 dias após a vacinação. Esses resultados demonstram os efeitos locais e sistêmicos provocados pela vacina da febre aftosa, evidenciados pela redução na produção de leite e pela incidência de reações vacinais, o que implica em significativas perdas econômicas.(AU)

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.

Foot-and-Mouth Disease Virus Leader Protease Cleaves G3BP1 and G3BP2 and Inhibits Stress Granule Formation.

Like other viruses, the picornavirus foot-and-mouth disease virus (FMDV; genus Aphthovirus), one of the most notorious pathogens in the global livestock industry, needs to navigate antiviral host responses to establish an infection. There is substantial insight into how FMDV suppresses the type I interferon (IFN) response, but it is largely unknown whether and how FMDV modulates the integrated stress response. Here, we show that the stress response is suppressed during FMDV infection. Using a chimeric recombinant encephalomyocarditis virus (EMCV), in which we functionally replaced the endogenous stress response antagonist by FMDV leader protease (Lpro) or 3Cpro, we demonstrate an essential role for Lpro in suppressing stress granule (SG) formation. Consistently, infection with a recombinant FMDV lacking Lpro resulted in SG formation. Additionally, we show that Lpro cleaves the known SG scaffold proteins G3BP1 and G3BP2 but not TIA-1. We demonstrate that the closely related equine rhinitis A virus (ERAV) Lpro also cleaves G3BP1 and G3BP2 and also suppresses SG formation, indicating that these abilities are conserved among aphthoviruses. Neither FMDV nor ERAV Lpro interfered with phosphorylation of RNA-dependent protein kinase (PKR) or eIF2α, indicating that Lpro does not affect SG formation by inhibiting the PKR-triggered signaling cascade. Taken together, our data suggest that aphthoviruses actively target scaffolding proteins G3BP1 and G3BP2 and antagonize SG formation to modulate the integrated stress response.IMPORTANCE The picornavirus foot-and-mouth disease virus (FMDV) is a notorious animal pathogen that puts a major economic burden on the global livestock industry. Outbreaks have significant consequences for animal health and product safety. Like many other viruses, FMDV must manipulate antiviral host responses to establish infection. Upon infection, viral double-stranded RNA (dsRNA) is detected, which results in the activation of the RNA-dependent protein kinase (PKR)-mediated stress response, leading to a stop in cellular and viral translation and the formation of stress granules (SG), which are thought to have antiviral properties. Here, we show that FMDV can suppress SG formation via its leader protease (Lpro). Simultaneously, we observed that Lpro can cleave the SG scaffolding proteins G3BP1 and G3BP2. Understanding the molecular mechanisms of the antiviral host response evasion strategies of FMDV may help to develop countermeasures to control FMDV infections in the future.

Development of a droplet digital RT-PCR for the quantification of foot-and-mouth virus RNA.

Foot-and-mouth-disease (FMD) is a highly contagious disease of domestic animals which can result in substantial economic losses, caused by the FMD virus (FMDV). The aim of this study was to develop and standardize a novel reverse transcriptase droplet digital PCR (RT-ddPCR) assay for the quantification of FMDV RNA. This assay was based upon an OIE-recognized real-time RT-PCR that detects the 3D-encoding region of FMDV. The limit of detection at 101.4 TCID50/mL and 26.5 copies was determined using FMDV-A24-Cruzeiro-virus and a plasmid containing the 3D-FMDV sequences, respectively. FMDV O, A and C serotypes and 11 species of non-FMDV were used to confirm the sensitivity and specificity of the assay. The RT-ddPCR was standardized using 60 bovine samples (representing negative and positive samples of epithelium and/or oesophageal-pharyngeal [OP] fluid) from animals suspected of vesicular diseases and previously tested by RT-qPCR. The RT-ddPCR showed robustness, sensitivity, specificity and accuracy, with similar results to the RT-qPCR. Moreover, the new RT-ddPCR diagnostic tool allowed the absolute quantification of FMDV RNA from epithelium and OP-fluid samples, as well as having the advantages of direct quantification by endpoint, eliminating the need for a calibration standard curve required in quantitative real-time RT-PCR.

Foot-and-Mouth Disease in the Middle East Caused by an A/ASIA/G-VII Virus Lineage, 2015-2016.

Phylogenetic analyses of foot-and-mouth disease type A viruses in the Middle East during 2015-2016 identified viruses belonging to the A/ASIA/G-VII lineage, which originated in the Indian subcontinent. Changes in a critical antigenic site within capsid viral protein 1 suggest possible evolutionary pressure caused by an intensive vaccination program.

The VP3 structural protein of foot-and-mouth disease virus inhibits the IFN-ß signaling pathway.

Foot-and-mouth disease is a frequently occurring disease of cloven-hoofed animals that is caused by infection with the foot-and-mouth virus (FMDV). FMDV circumvents the type-I IFN response by expressing proteins that antagonize cellular innate immunity, such as leader protease and 3C protease. We identified the FMDV structural protein VP3 as a negative regulator of the virus-triggered IFN-ß signaling pathway. Expression of FMDV VP3 inhibited the Sendai virus-triggered activation of IFN regulatory factor-3 and the expression of retinoic acid-inducible gene-I/melanoma differentiation-associated protein-5. Transient transfection and coimmunoprecipitation confirmed that the structural protein VP3 interacts with virus-induced signaling adapter (VISA), which is dependent on the C-terminal aa 111-220 of VP3. In addition, we found that FMDV VP3 inhibits the expression of VISA by disrupting its mRNA. Taken together, our findings reveal a novel strategy used by the structural VP3 protein of FMDV to evade host innate immunity.-Li, D., Yang, W., Yang, F., Liu, H., Zhu, Z., Lian, K., Lei, C., Li, S., Liu, X., Zheng, H., Shu, H. The VP3 structural protein of foot-and-mouth disease virus inhibits the IFN-ß signaling pathway.

Outbreaks of Foot-and-Mouth Disease in Libya and Saudi Arabia During 2013 Due to an Exotic O/ME-SA/Ind-2001 Lineage Virus.

Foot-and-mouth disease viruses are often restricted to specific geographical regions and spread to new areas may lead to significant epidemics. Phylogenetic analysis of sequences of the VP1 genome region of recent outbreak viruses from Libya and Saudi Arabia has revealed a lineage, O-Ind-2001, normally found in the Indian subcontinent. This paper describes the characterization of field viruses collected from these cases and provides information about a new real-time RT-PCR assay that can be used to detect viruses from this lineage and discriminate them from other endemic FMD viruses that are co-circulating in North Africa and western Eurasia.

In vitro surrogate models to aid in the development of antivirals for the containment of foot-and-mouth disease outbreaks.

Foot-and-mouth disease virus (FMDV) is a highly pathogenic member of the genus Aphthovirus (family Picornaviridae) that is only to be manipulated in high-containment facilities, thus complicating research on and discovery of antiviral strategies against the virus. Bovine rhinitis B virus (BRBV) and equine rhinitis A virus (ERAV), phylogenetically most closely related to FMDV, were explored as surrogates for FMDV in antiviral studies. Although no efficient cell culture system has been reported so far for BRBV, we demonstrate that infection of primary bovine kidney cells resulted in an extensive but rather poorly-reproducible induction of cytopathic effect (CPE). Madin-Darby bovine kidney cells on the other hand supported viral replication in the absence of CPE. Antiviral tests were developed for ERAV in Vero A cells employing a viral RNA-reduction assay and CPE-reduction assay; the latter having a Z' factor of 0.83±0.07. The BRBV and ERAV models were next used to assess the anti-aphthovirus activity of two broad-spectrum antiviral agents 2'-C-methylcytidine (2CMC) and ribavirin, as well as of the enterovirus-specific inhibitor enviroxime. The effects of the three compounds in the CPE-reduction (ERAV) and viral RNA-reduction assays (BRBV and ERAV) were comparable. Akin to 2CMC, compound A, a recently-discovered non-nucleoside pan-serotype FMDV inhibitor, also inhibited the replication of both BRBV and ERAV, whereas enviroxime was devoid of activity. The BRBV and ERAV surrogate models reported here can be manipulated in BSL-2 laboratories and may facilitate studies to unravel the mechanism of action of novel FMDV inhibitors.

Equine picornaviruses: well known but poorly understood.

Of the many members that comprise the family Picornaviridae, only two species are known to infect horses: equine rhinitis A virus (ERAV) and equine rhinitis B virus (ERBV). Each species now occupies a distinct phylogenetic branch within the family, with the single serotype of ERAV grouping with the aphthoviruses, such as foot-and-mouth disease virus (FMDV), and the three serotypes of ERBV as the sole members of the genus Erbovirus. The high seroprevalence of equine picornaviruses in horse populations worldwide contrasts with the relatively few reports of detection of these viruses and poor understanding of their contribution to disease. This review examines the current knowledge regarding the distribution and pathogenesis of these viruses and discusses recent advances in diagnostic methods that may lead to a better understanding of the role of these viruses as contributors to equine respiratory disease.

Control of foot-and-mouth disease during 2010-2011 epidemic, South Korea.

An outbreak of foot-and-mouth disease caused by serotype O virus occurred in cattle and pigs in South Korea during November 2010-April 2011. The highest rates of case and virus detection were observed 44 days after the first case was detected. Detection rates declined rapidly after culling and completion of a national vaccination program.

Homology modeling and analysis of structure predictions of the bovine rhinitis B virus RNA dependent RNA polymerase (RdRp).

Bovine Rhinitis B Virus (BRBV) is a picornavirus responsible for mild respiratory infection of cattle. It is probably the least characterized among the aphthoviruses. BRBV is the closest relative known to Foot and Mouth Disease virus (FMDV) with a ~43% identical polyprotein sequence and as much as 67% identical sequence for the RNA dependent RNA polymerase (RdRp), which is also known as 3D polymerase (3D(pol)). In the present study we carried out phylogenetic analysis, structure based sequence alignment and prediction of three-dimensional structure of BRBV 3D(pol) using a combination of different computational tools. Model structures of BRBV 3D(pol) were verified for their stereochemical quality and accuracy. The BRBV 3D(pol) structure predicted by SWISS-MODEL exhibited highest scores in terms of stereochemical quality and accuracy, which were in the range of 2Å resolution crystal structures. The active site, nucleic acid binding site and overall structure were observed to be in agreement with the crystal structure of unliganded as well as template/primer (T/P), nucleotide tri-phosphate (NTP) and pyrophosphate (PPi) bound FMDV 3D(pol) (PDB, 1U09 and 2E9Z). The closest proximity of BRBV and FMDV 3D(pol) as compared to human rhinovirus type 16 (HRV-16) and rabbit hemorrhagic disease virus (RHDV) 3D(pols) is also substantiated by phylogeny analysis and root-mean square deviation (RMSD) between C-α traces of the polymerase structures. The absence of positively charged α-helix at C terminal, significant differences in non-covalent interactions especially salt bridges and CH-pi interactions around T/P channel of BRBV 3D(pol) compared to FMDV 3D(pol), indicate that despite a very high homology to FMDV 3D(pol), BRBV 3D(pol) may adopt a different mechanism for handling its substrates and adapting to physiological requirements. Our findings will be valuable in the design of structure-function interventions and identification of molecular targets for drug design applicable to Aphthovirus RdRps.