Foot-and-mouth disease-associated myocarditis is age dependent in suckling calves.

Myocarditis is considered a fatal form of foot-and-mouth disease (FMD) in suckling calves. In the present study, a total of 17 calves under 4 months of age and suspected clinically for FMD were examined for clinical lesions, respiratory rate, heart rate, and heart rhythm. Lesion samples, saliva, nasal swabs, and whole blood were collected from suspected calves and subjected to Sandwich ELISA and reverse transcription multiplex polymerase chain reaction (RT-mPCR) for detection and serotyping of FMD virus (FMDV). The samples were found to be positive for FMDV serotype "O". Myocarditis was suspected in 6 calves based on tachypnoea, tachycardia, and gallop rhythm. Serum aspartate aminotransferase (AST), creatinine kinase myocardial band (CK-MB) and lactate dehydrogenase (LDH), and cardiac troponins (cTnI) were measured. Mean serum AST, cTn-I and LDH were significantly higher (P < 0.001) in < 2 months old FMD-infected calves showing clinical signs suggestive of myocarditis (264.833 ± 4.16; 11.650 ± 0.34 and 1213.33 ± 29.06) than those without myocarditis (< 2 months old: 110.00 ± 0.00, 0.06 ± 0.00, 1050.00 ± 0.00; > 2 months < 4 months: 83.00 ± 3.00, 0.05 ± 0.02, 1159.00 ± 27.63) and healthy control groups (< 2 months old: 67.50 ± 3.10, 0.047 ± 0.01, 1120.00 ± 31.62; > 2 months < 4 months: 72.83 ± 2.09, 0.47 ± 0.00, 1160.00 ± 18.44). However, mean serum CK-MB did not differ significantly amongst the groups. Four calves under 2 months old died and a necropsy revealed the presence of a pathognomic gross lesion of the myocardial form of FMD known as "tigroid heart". Histopathology confirmed myocarditis. This study also reports the relevance of clinical and histopathological findings and biochemical markers in diagnosing FMD-related myocarditis in suckling calves.

Endemic persistence of a highly contagious pathogen: Foot-and-mouth disease in its wildlife host.

Extremely contagious pathogens are a global biosecurity threat because of their high burden of morbidity and mortality, as well as their capacity for fast-moving epidemics that are difficult to quell. Understanding the mechanisms enabling persistence of highly transmissible pathogens in host populations is thus a central problem in disease ecology. Through a combination of experimental and theoretical approaches, we investigated how highly contagious foot-and-mouth disease viruses persist in the African buffalo, which serves as their wildlife reservoir. We found that viral persistence through transmission among acutely infected hosts alone is unlikely. However, the inclusion of occasional transmission from persistently infected carriers reliably rescues the most infectious viral strain from fade-out. Additional mechanisms such as antigenic shift, loss of immunity, or spillover among host populations may be required for persistence of less transmissible 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.

Epidemiological study on foot-and-mouth disease in small ruminants: Sero-prevalence and risk factor assessment in Kenya.

Foot-and-mouth disease (FMD) is endemic in Kenya affecting cloven-hoofed ruminants. The epidemiology of the disease in small ruminants (SR) in Kenya is not documented. We carried out a cross-sectional study, the first in Kenya, to estimate the sero-prevalence of FMD in SR and the associated risk factors nationally. Selection of animals to be sampled used a multistage cluster sampling approach. Serum samples totaling 7564 were screened for FMD antibodies of non-structural-proteins using ID Screen® NSP Competition ELISA kit. To identify the risk factors, generalized linear mixed effects (GLMM) logistic regression analysis with county and villages as random effect variables was used. The country animal level sero-prevalence was 22.5% (95% CI: 22.3%-24.3%) while herd level sero-prevalence was 77.6% (95% CI: 73.9%-80.9%). The risk factor that was significantly positively associated with FMD sero-positivity in SR was multipurpose production type (OR = 1.307; p = 0.042). The risk factors that were significantly negatively associated with FMD sero-positivity were male sex (OR = 0.796; p = 0.007), young age (OR = 0.470; p = 0.010), and sedentary production zone (OR = 0.324; p<0.001). There were no statistically significant intra class correlations among the random effect variables but interactions between age and sex variables among the studied animals were statistically significant (p = 0.019). This study showed that there may be widespread undetected virus circulation in SR indicated by the near ubiquitous spatial distribution of significant FMD sero-positivity in the country. Strengthening of risk-based FMD surveillance in small ruminants is recommended. Adjustment of husbandry practices to control FMD in SR and in-contact species is suggested. Cross-transmission of FMD and more risk factors need to be researched.

shRNA transgenic swine display resistance to infection with the foot-and-mouth disease virus.

Foot-and-mouth disease virus (FMDV) is one of the most important animal pathogens in the world. FMDV naturally infects swine, cattle, and other cloven-hoofed animals. FMD is not adequately controlled by vaccination. An alternative strategy is to develop swine that are genetically resistant to infection. Here, we generated FMDV-specific shRNA transgenic cells targeting either nonstructural protein 2B or polymerase 3D of FMDV. The shRNA-positive transgenic cells displayed significantly lower viral production than that of the control cells after infection with FMDV (P < 0.05). Twenty-three transgenic cloned swine (TGCS) and nine non-transgenic cloned swine (Non-TGCS) were produced by somatic cell nuclear transfer (SCNT). In the FMDV challenge study, one TGCS was completely protected, no clinical signs, no viremia and no viral RNA in the tissues, no non-structural antibody response, another one TGCS swine recovered after showing clinical signs for two days, whereas all of the normal control swine (NS) and Non-TGCS developed typical clinical signs, viremia and viral RNA was determined in the tissues, the non-structural antibody was determined, and one Non-TGCS swine died. The viral RNA load in the blood and tissues of the TGCS was reduced in both challenge doses. These results indicated that the TGCS displayed resistance to the FMDV infection. Immune cells, including CD3+, CD4+, CD8+, CD21+, and CD172+ cells, and the production of IFN-γ were analyzed, there were no significant differences observed between the TGCS and NS or Non-TGCS, suggesting that the FMDV resistance may be mainly derived from the RNAi-based antiviral pathway. Our work provides a foundation for a breeding approach to preventing infectious disease in swine.

Phylogenetic and evolutionary analysis of foot-and-mouth disease virus A/ASIA/Sea-97 lineage.

Foot-and-mouth disease virus (FMDV) A/ASIA/Sea-97 is a predominant lineage in Southeast Asia and East Asia. However, Sea-97 lineage has not been well studied since its first outbreak in Thailand in 1997. Thus, we conducted phylogenetic and evolutionary analysis of Sea-97 using 224 VP1 sequences of FMDV A/ASIA during 1960 and 2018. Phylogenetic analysis revealed that Sea-97 lineage can be classified into five groups (G1-G5). After the emergence of G2 from G1, the genetic diversity of Sea-97 increased sharply, causing divergence into G3, G4 and G5. During this evolutionary process, Sea-97 lineage, which was initially found only in some countries in Southeast Asia, gradually spread to East Asia. The evolution rate of this lineage was estimated to be 1.2 × 10-2 substitutions/site/year and there were many differences in amino acid residues compared to vaccine strain. Substitutions at antigenically important sites may affect the efficacy of the vaccine, suggesting the need for appropriate vaccine strains. Our results could provide meaningful information to understand comprehensive characteristic of Sea-97 lineage.

Adherent and suspension baby hamster kidney cells have a different cytoskeleton and surface receptor repertoire.

Animal cell culture, with single cells growing in suspension, ideally in a chemically defined environment, is a mainstay of biopharmaceutical production. The synthetic environment lacks exogenous growth factors and usually requires a time-consuming adaptation process to select cell clones that proliferate in suspension to high cell numbers. The molecular mechanisms that facilitate the adaptation and that take place inside the cell are largely unknown. Especially for cell lines that are used for virus antigen production such as baby hamster kidney (BHK) cells, the restriction of virus growth through the evolution of undesired cell characteristics is highly unwanted. The comparison between adherently growing BHK cells and suspension cells with different susceptibility to foot-and-mouth disease virus revealed differences in the expression of cellular receptors such as integrins and heparan sulfates, and in the organization of the actin cytoskeleton. Transcriptome analyses and growth kinetics demonstrated the diversity of BHK cell lines and confirmed the importance of well-characterized parental cell clones and mindful screening to make sure that essential cellular features do not get lost during adaptation.

Pathogenesis of non-epithelial foot-and-mouth disease in neonatal animals.

Foot-and-mouth disease virus (FMDV), which causes a highly contagious viral disease of cloven-hoofed animals, is notable for epithelial cell tropism, resulting in the appearance of vesicles on the feet and in and around the mouth in infected animals, while FMDV infection in neonatal animals is also associated with not only epithelial lesions, but also muscle-associated lesions, which leads to myocarditis, resulting in high-mortality. However, critical knowledge about the non-epithelial tropism of FMDV is still lacking. In this paper, the current progress of the FMDV non-epithelial tropisms is summarized and the possible role of the key viral and cellular components involved is discussed.

New possibilities for egg white lysozyme: heat-denatured lysozyme partially inactivates select foot-and-mouth disease virus strains.

Foot-and-mouth disease (FMD) is one of the most contagious diseases of cloven-hoofed animals. Disinfectants are used to inactivate FMD virus (FMDV) in Japan. Reports that heat-denatured lysozyme inactivates bacteria as well as viruses, such as norovirus and hepatitis A virus, led us to determine its effects on FMDV. We show here that heat-denatured lysozyme partially inhibited the infectivity of FMDV O/JPN/2010-1/14C but of FMDVs A/TAI/46-1/2015 and Asia1/Shamir (ISR/3/89). Further, heat-denatured lysozyme variably reduced RNA loads of FMDVs O/JPN/2010-1/14C, O/MOG/2/Ca/BU/2017, O/Taiwan/1997, Asia1/Shamir (ISR/3/89), Asia1/TUR/49/2011, SAT1/KEN/117/2009, SAT2/SAU/6/2000 and SAT3/ZIM/3/83 but could not those of O/JPN/2000, A/TAI/46-1/2015, A22/IRQ/24/64, A15/TAI/1/60 and C/PHI/7/84. These findings indicate that heat-denatured lysozyme may serve as a new disinfectant against FMDV.

A Virus-Like Particle-Based Solid-Phase Competition Enzyme-Linked Immunosorbent Assay for Antibody Detection of Foot-and-Mouth Disease Virus Serotype O.

Foot-and-mouth disease (FMD) is caused by FMD virus (FMDV) is a highly contagious disease of ruminants, which is primarily controlled by vaccination. The monitoring of antisera after vaccination is currently depending on liquid-phase blocking ELISA (LPBE). Recently, bacterium-original FMD virus-like particle (VLP) showed the potential as vaccine candidates. In this study, to minimize the risk of live virus involvement, the Escherichia coli original VLP of FMDV serotype O were used as the immunogen for monoclonal antibodies (Mabs) production and the capture antigen in the development of a solid-phase competition ELISA (SPCE). The samples with a percentage inhibition of >50% were considered positive in the SPCE assay. The concordance rate of the Mab-based SPCE compared with the LPBE for clinical serum samples test was 93.4%, and with a high agreement (kappa = 0.892) with LPBE in antibody duration monitoring. Results indicated that the VLP-based SPCE had high specificity and sensitivity, which provides an alternative method for postimmunization antibody evaluation of FMDV serotype O.

Polymerase Fidelity Contributes to Foot-and-Mouth Disease Virus Pathogenicity and Transmissibility In Vivo.

The low fidelity of foot-and-mouth disease virus (FMDV) RNA-dependent RNA polymerase allows FMDV to exhibit high genetic diversity. Previously, we showed that the genetic diversity of FMDV plays an important role in virulence in suckling mice. Here, we mutated the amino acid residue Phe257, located in the finger domain of FMDV polymerase and conserved across FMDV serotypes, to a cysteine (F257C) to study the relationship between viral genetic diversity, virulence, and transmissibility in natural hosts. The single amino acid substitution in FMDV polymerase resulted in a high-fidelity virus variant, rF257C, with growth kinetics indistinguishable from those of wild-type (WT) virus in cell culture, but it displayed smaller plaques and impaired fitness in direct competition assays. Furthermore, we found that rF257C was attenuated in vivo in both suckling mice and pigs (one of its natural hosts). Importantly, contact exposure experiments showed that the rF257C virus exhibited reduced transmissibility compared to that of wild-type FMDV in the porcine model. This study provides evidence that FMDV genetic diversity is important for viral virulence and transmissibility in susceptible animals. Given that type O FMDV exhibits the highest genetic diversity among all seven serotypes of FMDV, we propose that the lower polymerase fidelity of the type O FMDV could contribute to its dominance worldwide.IMPORTANCE Among the seven serotypes of FMDV, serotype O FMDV have the broadest distribution worldwide, which could be due to their high virulence and transmissibility induced by high genetic diversity. In this paper, we generated a single amino acid substitution FMDV variant with a high-fidelity polymerase associated with viral fitness, virulence, and transmissibility in a natural host. The results highlight that maintenance of viral population diversity is essential for interhost viral spread. This study provides evidence that higher genetic diversity of type O FMDV could increase both virulence and transmissibility, thus leading to their dominance in the global epidemic.

MiR-4334-5p Facilitates Foot and Mouth Disease Virus Propagation by Suppressing Interferon Pathways via Direct Targeting ID1.

Emerging evidence indicates that the host microRNAs (miRNAs) are important intracellular regulators and play pivotal roles in intricate host-pathogen interaction networks. In our previous studies, ssc-microRNA-4334-5p (miR-4334-5p) was identified as a differentially expressed miRNA in microarray-based miRNAs profiling experiment, but whether miR-4334-5p regulates foot and mouth disease virus (FMDV) propagation is less understood. Here, we demonstrated that miR-4334-5p expression level was up-regulated shortly after FMDV infection, transfection of miR-4334-5p mimics promoted, while inhibitor transfection suppressed FMDV replication correspondingly. Further bioinformatic analysis and experimental study suggested ID1 was the direct target of miR-4334-5p, suppressing FMDV replication by regulating interferon (IFN) pathways. These findings shed light on microRNAs-ID1-interferon axis in regulating FMDV replication.

Sequence Analysis of Egyptian Foot-and-Mouth Disease Virus Field and Vaccine Strains: Intertypic Recombination and Evidence for Accidental Release of Virulent Virus.

In spite of annual mass vaccination programs with polyvalent inactivated vaccines, the incidence and economic impact of foot-and-mouth disease (FMD) in Egypt is high. Viruses of the A, O and SAT 2 serotypes are endemic and repeated incursions of new lineages from other countries lead to an unstable situation that makes the selection of appropriate vaccine antigens very difficult. In this study, whole genome sequencing of a 2016 serotype A isolate from Egypt revealed a recombination event with an African serotype O virus. Based on available vaccine matching data, none of the vaccines currently used in Egypt are expected to sufficiently protect against this virus or other viruses of this lineage (A/AFRICA/G-IV) circulating there since 2012. In addition to the risk of vaccine failure caused by strain mismatch, the production of inactivated FMD vaccines is dangerous if adequate biosafety cannot be maintained. Using a high-throughput sequencing protocol optimized for short nucleic acid fragments, the composition of a local inactivated vaccine was analyzed in depth. The serotype O strain identified in the vaccine was genetically identical to viruses found in recent FMD outbreaks in Egypt.

Optimal surveillance against foot-and-mouth disease: A sample average approximation approach.

Decisions surrounding the presence of infectious diseases are typically made in the face of considerable uncertainty. However, the development of models to guide these decisions has been substantially constrained by computational difficulty. This paper focuses on the case of finding the optimal level of surveillance against a highly infectious animal disease where time, space and randomness are fully considered. We apply the Sample Average Approximation approach to solve our problem, and to control model dimension, we propose the use of an infection tree model, in combination with sensible 'tree-pruning' and parallel processing techniques. Our proposed model and techniques are generally applicable to a number of disease types, but we demonstrate the approach by solving for optimal surveillance levels against foot-and-mouth disease using bulk milk testing as an active surveillance protocol, during an epidemic, among 42,279 farms, fully characterised by their location, livestock type and size, in the state of Victoria, Australia.

A Temperature-Dependent Translation Defect Caused by Internal Ribosome Entry Site Mutation Attenuates Foot-and-Mouth Disease Virus: Implications for Rational Vaccine Design.

Foot-and-mouth disease (FMD), which is caused by FMD virus (FMDV), remains a major plague among cloven-hoofed animals worldwide, and its outbreak often has disastrous socioeconomic consequences. A live-attenuated FMDV vaccine will greatly facilitate the global control and eradication of FMD, but a safe and effective attenuated FMDV vaccine has not yet been successfully developed. Here, we found that the internal ribosome entry site (IRES) element in the viral genome is a critical virulence determinant of FMDV, and a nucleotide substitution of cytosine (C) for guanine (G) at position 351 of the IRES endows FMDV with temperature-sensitive and attenuation (ts&att) phenotypes. Furthermore, we demonstrated that the C351G mutation of IRES causes a temperature-dependent translation defect by impairing its binding to cellular pyrimidine tract-binding protein (PTB), resulting in the ts&att phenotypes of FMDV. Natural hosts inoculated with viruses carrying the IRES C351G mutation showed no clinical signs, viremia, virus excretion, or viral transmission but still produced a potent neutralizing antibody response that provided complete protection. Importantly, the IRES C351G mutation is a universal determinant of the ts&att phenotypes of different FMDV strains, and the C351G mutant was incapable of reversion to virulence during in vitro and in vivo passages. Collectively, our findings suggested that manipulation of the IRES, especially its C351G mutation, may serve as a feasible strategy to develop live-attenuated FMDV vaccines.IMPORTANCE The World Organization for Animal Health has called for global control and eradication of foot-and-mouth disease (FMD), the most economically and socially devastating disease affecting animal husbandry worldwide. Live-attenuated vaccines are considered the most effective strategy for prevention, control, and eradication of infectious diseases due to their capacity to induce potent and long-lasting protective immunity. However, efforts to develop FMD virus (FMDV) live-attenuated vaccines have achieved only limited success. Here, by structure-function study of the FMDV internal ribosome entry site (IRES), we find that the C351 mutation of the IRES confers FMDV with an ideal temperature-sensitive attenuation phenotype by decreasing its interaction with cellular pyrimidine tract-binding protein (PTB) to cause IRES-mediated temperature-dependent translation defects. The temperature-sensitive attenuated strains generated by manipulation of the IRES address the challenges of FMDV attenuation differences among various livestock species and immunogenicity maintenance encountered previously, and this strategy can be applied to other viruses with an IRES to rationally design and develop live-attenuated vaccines.

Cellular Vimentin Interacts with Foot-and-Mouth Disease Virus Nonstructural Protein 3A and Negatively Modulates Viral Replication.

Nonstructural protein 3A of foot-and-mouth disease virus (FMDV) is a partially conserved protein of 153 amino acids that is in most FMDVs examined to date, and it plays important roles in virus replication, virulence, and host range. To better understand the role of 3A during FMDV infection, we used coimmunoprecipitation followed by mass spectrometry to identify host proteins that interact with 3A in FMDV-infected cells. Here, we report that cellular vimentin is a host binding partner for 3A. The 3A-vimentin interaction was further confirmed by coimmunoprecipitation, glutathione S-transferase (GST) pull down, and immunofluorescence assays. Alanine-scanning mutagenesis indicated that amino acid residues 15 to 21 at the N-terminal region of the FMDV 3A are responsible for the interaction between 3A and vimentin. Using reverse genetics, we demonstrate that mutations in 3A that disrupt the interaction between 3A and vimentin are also critical for virus growth. Overexpression of vimentin significantly suppressed the replication of FMDV, whereas knockdown of vimentin significantly enhanced FMDV replication. However, chemical disruption of the vimentin network by acrylamide resulted in a significant decrease in viral yield, suggesting that an intact vimentin network is needed for FMDV replication. These results indicate that vimentin interacts with FMDV 3A and negatively regulates FMDV replication and that the vimentin-3A interaction is essential for FMDV replication. This study provides information that should be helpful for understanding the molecular mechanism of FMDV replication.IMPORTANCE Foot-and-mouth disease virus (FMDV) nonstructural protein 3A plays important roles in virus replication, host range, and virulence. To further understand the role of 3A during FMDV infection, identification of host cell factors that interact with FMDV 3A is needed. Here, we found that vimentin is a direct binding partner of FMDV 3A, and manipulation of vimentin has a negative effect on virus replication. We also demonstrated that amino acid residues 15 to 21 at the N-terminal region of the FMDV 3A are responsible for the interaction between 3A and vimentin and that the 3A-vimentin interaction is critical for viral replication since the full-length cDNA clone harboring mutations in 3A, which were disrupt 3A-vimentin reactivity, could not produce viable virus progeny. This study provides information that not only provides us a better understanding of the mechanism of FMDV replication but also helps in the development of novel antiviral strategies in the future.

Multilevel model for airborne transmission of foot-and-mouth disease applied to Swedish livestock.

The foot-and-mouth disease is an ever-present hazard to the livestock industry due to the huge economic consequences following an outbreak that necessitates culling of possibly infected animals in vast numbers. The disease is highly contagious and previous epizootics have shown that it spreads by many routes. One such route is airborne transmission, which has been investigated in this study by means of a detailed multilevel model that includes all scales of an outbreak. Local spread within an infected farm is described by a stochastic compartment model while the spread between farms is quantified by atmospheric dispersion simulations using a network representation of the set of farms. The model was applied to the Swedish livestock industry and the risk for an epizootic outbreak in Sweden was estimated using the basic reproduction number of each individual livestock-holding farm as the endpoint metric. The study was based on comprehensive official data sets for both the current livestock holdings and regional meteorological conditions. Three species of farm animals are susceptible to the disease and are present in large numbers: cattle, pigs and sheep. These species are all included in this study using their individual responses and consequences to the disease. It was concluded that some parts of southern Sweden are indeed preconditioned to harbor an airborne epizootic, while the sparse farm population of the north renders such events unlikely to occur there. The distribution of the basic reproduction number spans over several orders of magnitudes with low risk of disease spread from the majority of the farms while some farms may act as very strong disease transmitters. The results may serve as basic data in the planning of the national preparedness for this type of events.

Impairment of the DeISGylation Activity of Foot-and-Mouth Disease Virus Lpro Causes Attenuation In Vitro and In Vivo.

Foot-and-mouth disease virus (FMDV) leader proteinase (Lpro) affects several pathways of the host innate immune response. Previous studies in bovine cells demonstrated that deletions (leaderless [LLV]) or point mutations in Lpro result in increased expression of interferon (IFN) and IFN-stimulated genes (ISGs), including, among others, the ubiquitin-like protein modifier ISG15 and the ubiquitin specific peptidase USP18. In addition to its conventional papain-like protease activity, Lpro acts as a deubiquitinase (DUB) and deISGylase. In this study, we identified a conserved residue in Lpro that is involved in its interaction with ISG15. Mutation W105A rendered Escherichia coli-expressed Lpro unable to cleave the synthetic substrate pro-ISG15 while preserving cellular eIF4G cleavage. Interestingly, mutant FMDV W105A was viable. Overexpression of ISG15 and the ISGylation machinery in porcine cells resulted in moderate inhibition of FMDV replication, along with a decrease of the overall state of ISGylation in wild-type (WT)-infected cells. In contrast, reduced deISGylation was observed upon infection with W105A and leaderless virus. Reduction in the levels of deubiquitination was also observed in cells infected with the FMDV LproW105A mutant. Surprisingly, similarly to WT, infection with W105A inhibited IFN/ISG expression despite displaying an attenuated phenotype in vivo in mice. Altogether, our studies indicate that abolishing/reducing the deISGylase/DUB activity of Lpro causes viral attenuation independently of its ability to block the expression of IFN and ISG mRNA. Furthermore, our studies highlight the potential of ISG15 to be developed as a novel biotherapeutic molecule against FMD.IMPORTANCE In this study, we identified an aromatic hydrophobic residue in foot-and-mouth disease virus (FMDV) leader proteinase (Lpro) (W105) that is involved in the interaction with ISG15. Mutation in Lpro W105 (A12-LproW105A) resulted in reduced deISGylation in vitro and in porcine-infected cells. Impaired deISGylase activity correlated with viral attenuation in vitro and in vivo and did not affect the ability of Lpro to block expression of type I interferon (IFN) and other IFN-stimulated genes. Moreover, overexpression of ISG15 resulted in the reduction of FMDV viral titers. Thus, our study highlights the potential use of Lpro mutants with modified deISGylase activity for development of live attenuated vaccine candidates, and ISG15 as a novel biotherapeutic against FMD.

Pathogenesis, biophysical stability and phenotypic variance of SAT2 foot-and-mouth disease virus.

Foot-and-mouth disease (FMD) is a highly contagious vesicular disease of cloven-hoofed animals, which severely decreases livestock productivity. FMD virus (FMDV), the causative agent, initiates infection by interaction with integrin cellular receptors on pharyngeal epithelium cells, causing clinical signs one to four days after transmission to a susceptible host. However, some Southern African Territories (SAT) viruses have been reported to cause mild or subclinical infections that may go undiagnosed in field conditions and are likely to be more common than previously expected. The studies presented here demonstrate that not all SAT2 viruses are equally virulent in cattle. The two SAT2 viruses, ZIM/5/83 and ZIM/7/83, were both highly attenuated in cattle, as evidenced by the mild clinical signs observed after needle challenge, while two incongruent SAT2 viruses showed significantly different clinical signs in challenged cattle. We then explored the ability of the SAT2 viruses to infect different cell types with defined receptors that are utilised by FMDV and found differences in their ability to lyse cells in culture and to compete in a controlled cell culture environment. The population sequence variation between ZIM/5/83 and ZIM/7/83 revealed multiple sites of single nucleotide variants of low frequency between the predominant virus populations, as could be expected from the genome of an RNA virus. An assessment of the biophysical stability of SAT2 virions during acidification indicated that the SAT2 virus EGY/09/12 was more resilient to acidification than the ZIM/5/83 and ZIM/7/83 viruses; however, whether this difference relates to differences in virulence in vivo is unclear. This study is a consolidated view of the key findings of SAT2 viruses studied over a 14-year period involving many different experiments.

A new implication of quasispecies dynamics: Broad virus diversification in absence of external perturbations.

RNA genetic elements include many important animal and plant pathogens. They share high mutability, a trait that has multiple implications for the interactions with their host organisms. Here we review evidence of a new adaptive feature of RNA viruses that we term "broadly diversifying selection". It constitutes a new type of positive selection without participation of any external selective agent, and which is built upon a progressive increase of the number of different genomes that dominate the population. The evidence was provided by analyses of mutant spectrum composition of two important viral pathogens, foot-and-mouth disease virus (FMDV) and hepatitis C virus (HCV) after prolonged replication in their respective cell culture environment. Despite being fueled by mutations that arise randomly and in absence of an external guiding selective force, this type of selection prepares the viral population for a response to selective forces still to occur. Since current evidence suggests that broadly diversifying selection is favored by elevated mutation rates and population sizes, it may constitute a more general behavior, relevant also to the adaptive dynamics of microbial populations and cancer cells.