Foot-and-mouth disease virus VP1 target the MAVS to inhibit type-I interferon signaling and VP1 E83K mutation results in virus attenuation.

VP1, a pivotal capsid protein encoded by the foot-and-mouth disease virus (FMDV), plays an important role in receptor-mediated attachment and humoral immune responses. Previous studies show that amino acid changes in the VP1 protein of cell culture-adapted strains of FMDV alter the properties of the virus. In addition, FMDV VP1 modulates host IFN signal transduction. Here, we examined the ability of cell culture-adapted FMDV VP1(83K) and wild-type FMDV VP1(83E) to evade host immunity by blocking mitochondrial antiviral signaling protein (MAVS)/TNF Receptor Associated Factor 3 (TRAF3) mediated cellular innate responses. Wild-type FMDV VP1(83E) interacted specifically with C-terminal TRAF3-binding site within MAVS and this interaction inhibited binding of TRAF3 to MAVS, thereby suppressing interferon-mediated responses. This was not observed for cell culture-adapted FMDV VP1(83K). Finally, chimeric FMDV harboring VP1(83K) showed very low pathogenicity in pigs. Collectively, these data highlight a critical role of VP1 with respect to suppression of type-I IFN pathway and attenuation of FMDV by the E83K mutation in VP1.

Rapid Engineering of Foot-and-Mouth Disease Vaccine and Challenge Viruses.

There are seven antigenically distinct serotypes of foot-and-mouth disease virus (FMDV), each of which has intratypic variants. In the present study, we have developed methods to efficiently generate promising vaccines against seven serotypes or subtypes. The capsid-encoding gene (P1) of the vaccine strain O1/Manisa/Turkey/69 was replaced with the amplified or synthetic genes from the O, A, Asia1, C, SAT1, SAT2, and SAT3 serotypes. Viruses of the seven serotype were rescued successfully. Each chimeric FMDV with a replacement of P1 showed serotype-specific antigenicity and varied in terms of pathogenesis in pigs and mice. Vaccination of pigs with an experimental trivalent vaccine containing the inactivated recombinants based on the main serotypes O, A, and Asia1 effectively protected them from virus challenge. This technology could be a potential strategy for a customized vaccine with challenge tools to protect against epizootic disease caused by specific serotypes or subtypes of FMDV.IMPORTANCE Foot-and-mouth disease (FMD) virus (FMDV) causes significant economic losses. For vaccine preparation, the selection of vaccine strains was complicated by high antigenic variation. In the present study, we suggested an effective strategy to rapidly prepare and evaluate mass-produced customized vaccines against epidemic strains. The P1 gene encoding the structural proteins of the well-known vaccine virus was replaced by the synthetic or amplified genes of viruses of seven representative serotypes. These chimeric viruses generally replicated readily in cell culture and had a particle size similar to that of the original vaccine strain. Their antigenicity mirrored that of the original serotype from which their P1 gene was derived. Animal infection experiments revealed that the recombinants varied in terms of pathogenicity. This strategy will be a useful tool for rapidly generating customized FMD vaccines or challenge viruses for all serotypes, especially for FMD-free countries, which have prohibited the import of FMDVs.

Insomnia during coronavirus disease 2019 pandemic in Korea: a National sleep survey.

Coronavirus disease 2019 (COVID-19) pandemic has caused widespread increase in stress and affected sleep quality and quantity, with up to 30% prevalence of sleep disorders being reported after the declaration of the pandemic. This study aimed to assess perceived changes due to the pandemic in the prevalence of insomnia and excessive daytime sleepiness (EDS) in Korea, and identify the associated factors. An online survey was conducted among 4000 participants (2035 men and 1965 women) aged 20-69 years enrolled using stratified multistage random sampling according to age, sex, and residential area, between January, 2021 and February, 2022. The questionnaire included various items, such as socio-demographics, Insomnia Severity Index, and Epworth Sleepiness Scale (ESS). Insomnia was defined as difficulty falling asleep and difficulty maintaining sleep more than twice a week. EDS was classified as an ESS score ≥ 11. Insomnia was reported by 32.9% (n = 1316) of the participants (37.3% among women and 28.6% among men). Multivariate logistic regression revealed that insomnia was associated with female sex [odds ratio (OR) = 1.526, 95% confidence interval (CI) = 1.297-1.796], night workers (OR 1.561, 95% CI 1.160-2.101), and being unmarried (OR 1.256, 95% CI 1.007-1.566). EDS was reported by 12.8% (n = 510) of the participants (14.7% among men and 10.7% among women). EDS was associated with male sex (OR 1.333, 95% CI 1.062-1.674), and being employed (OR 1.292, 95% CI 1.017-1.641). During the COVID-19 pandemic, the prevalence of insomnia increased in Korea, while there was no significant change in EDS compared with pre-pandemic evidence.

Production of Foot-and-Mouth Disease Type O and A Vaccine Antigens on a Pilot Scale and Determination of Optimal Amount of Antigen for Monovalent Vaccines.

Foot-and-mouth disease (FMD) is a highly infectious disease affecting cloven-hoofed animals and causes significant economic losses to the livestock industry. The Type O PanAsia-2 (O PA-2) vaccine strain is protective against a wide range of serotype O FMD virus (FMDV) strains in East Asia, and A22 Iraq/24/64 (A22 IRQ) is the most widely used vaccine strain in FMD vaccine antigen banks. The aim of this study was to produce antigens from O PA-2 and A22 IRQ viruses using a 100 L bioreactor and evaluate the protective efficacy of varying antigen concentrations in pigs. More than 2 µg/mL of the antigen was recovered from the O PA-2 and A22 IRQ virus-infected supernatants. Further, inactivation of O PA-2 and A22 IRQ by binary ethyleneimine revealed that the viral titers decreased below 10-7 TCID50/mL within 13 h and 9 h, respectively. The O PA-2 and A22 IRQ vaccines, containing 10 µg and 5 µg of antigen, respectively, provided protection against homologous viruses in pigs. This is the first report demonstrating that the antigens obtained from the pilot-scale production of O PA-2 and A22 IRQ are viable candidate vaccines. These results will pave the way for industrial-scale FMD vaccine production in South Korea.

Scale-Up Production of Type O and A Foot-and-Mouth Disease Bivalent Vaccine and Its Protective Efficacy in Pigs.

South Korea has experienced FMD outbreaks almost every year since 2014. Therefore, a novel local vaccine that can cover various topotypes of viruses is required. Two virus strains, O/Boeun/SKR/2017 and A/Yeoncheon/SKR/2017, were cultured up to the pilot scale based on the optimized conditions set up on the flask scale. FMDV particles (146S) of 2 µg/mL or more were obtained from the virus culture supernatant using a 100 L bioreactor. The viruses were fully inactivated using binary ethylenimine within 16 h through two inactivation cycles and mixed with an adjuvant into a bivalent vaccine (types O and A) consisting of 15 µg viruses per strain. The experimental bivalent vaccine showed a broad spectrum of high neutralizing antibody titers against heterologous viruses, including type O Cathay strain and type A Asia topotypes, except for GVII. The 50% protective dose was determined as 12.5 for O/Boeun/SKR/2017 and 15.6 for A/Yeoncheon/SKR/2017. Collectively, we expect that the bivalent vaccine could protect against FMDV types O and A circulating in South Korea and neighboring countries. To our knowledge, this is the first report demonstrating that the vaccine strains could be successfully scaled-up to a 100 L bioreactor, with the determination of its protective efficacy in pigs.

Evidence of recombination in a new isolate of foot-and-mouth disease virus serotype Asia 1.

Phylogenetic analysis of the nucleotide sequence of VP1 revealed that a new isolate of foot-and-mouth disease virus (FMDV) serotype Asia 1 identified in Mongolia in 2005 was related to Chinese and Russian strains isolated during the same year. In this study, these strains were defined as East Asian strains having a common geographical origin, and the complete genomic sequence of the Mongolian strain (As1/MOG/05) was determined and compared to other strains of serotype Asia 1. As1/MOG/05 showed 100% identity with an East Asian strain from China (As1/Qinghai/CHA/05) in terms of its VP1 nucleotide sequence. However, the Mongolian strain has a four-amino acid extension in 3D that is missing from all other strains of serotype Asia 1, and which is not due to an insertion. A full genomic scan revealed that the Mongolian strain is closer to the East Asian strain As1/JS/CHA/05 than to all other strains of serotype Asia 1 in nearly all genomic regions. Within the narrow region of low similarity between the two sequences, As1/JS/CHA/05 was found to have a mosaic structure with a partial 2C fragment supposedly transferred from Hong Kong strain As1/HNK/CHA/05. The genomic mosaicism and extension detected in non-structural protein-coding regions in this study may be used to trace the origins and evolution of problematic strains of serotype Asia 1 that have arisen in East Asia since 2005.

Control of type O foot-and-mouth disease by vaccination in Korea, 2014-2015.

On December 3, 2014, a type O foot-and-mouth disease (FMD) outbreak began in Korea. Although vaccinations were administered, FMD cases increased steadily for five months, and reached 185 cases by April 2015. Most of the affected animals were pigs, which are vulnerable to vaccination. The FMD virus belonged to the South-East Asia (SEA) topotype that had been observed three times in Korea between April 2010 and July 2014. However, the FMD virus isolated in December 2014 had a unique feature; that is, partial deletion of the 5´ non-coding region, a deletion not seen in previous SEA topotype isolates identified in Korea. We conclude that this outbreak included the introduction of a new FMD strain to Korea, and that Korea was now affected by genetically similar FMD virus strains that are related to those from neighboring countries.

Immune responses in pigs and cattle vaccinated with half-volume foot-and-mouth disease vaccine.

With the current commercial foot-and-mouth disease vaccine, inoculating twice increases the formation of denatured meat due to granuloma or residual adjuvant at the injection site in pigs, resulting in economic loss. Therefore, we investigated protective antibody levels after reducing the amount of adjuvant in the vaccine. Field applicability of the experimental vaccine, made with a new adjuvant ISA 201, was tested by vaccinating farm animals with half-volume doses (1 mL/animal) of commercial vaccine and monitoring their immunogenicity. Among pigs, the group that received a half-volume dose showed similar or higher titers of structural protein antibody and neutralizing antibody than those receiving the standard dose (2 mL). In pigs, the durable effects of antibody titer of the reduced vaccine volume did not diminish up to the time of slaughter. Among cattle, boosting with a second 1 mL vaccine increased virus neutralizing antibody for the protective effects. The boosting effects were more marked in cattle than in pigs. The immune responses differed between species with the effect of the half-volume vaccination being lower in cattle than in pigs. In conclusion, the immune response to the half-volume vaccine was similar to that from the standard volume vaccine in pigs, but not in cattle.

Complete Genome Sequence of Foot-and-Mouth Disease Virus Serotype SAT3 Zimbabwe/4/81.

The complete genome sequence of a foot-and-mouth disease (FMD) serotype SAT3 virus ZIM/4/81, which belongs to a topotype 1 (SEZ), is reported here.

Novel foot-and-mouth disease virus in Korea, July-August 2014.

Despite nation-wide immunization with O, A, and Asia 1 type vaccines in Republic of Korea, foot-and-mouth disease type O occurred again in July 2014 after three years and three months. This virus was a Mya-98 strain of the Southeast Asian topotype and was most similar to the identified type that circulated in East Asia in 2014. This was new virus with the deletion of 23 amino acids in 3A/3B1 region and low pathogenic property.

Application of mouse model for effective evaluation of foot-and-mouth disease vaccine.

Efficacy evaluation of foot-and-mouth disease (FMD) vaccines has been conducted in target animals such as cows and pigs. In particular, handling FMD virus requires a high level of biosafety management and facilities to contain the virulent viruses. The lack of a laboratory animal model has resulted in inconvenience when it comes to using target animals for vaccine evaluation, bringing about increased cost, time and labor for the experiments. The FMD mouse model has been studied, but most FMD virus (FMDV) strains are not known to cause disease in adult mice. In the present study, we created a series of challenge viruses that are lethal to adult C57BL/6 mice. FMDV types O, A, and Asia1, which are related to frequent FMD outbreaks, were adapted for mice and the pathogenesis of each virus was evaluated in the mouse model. Challenge experiments after vaccination using in-house and commercial vaccines demonstrated vaccine-mediated protection in a dose-dependent manner. In conclusion, we propose that FMD vaccine evaluation should be carried out using mouse-adapted challenge viruses as a swift, effective efficacy test of experimental or commercial vaccines.

Construction of stabilized and tagged foot-and-mouth disease virus.

Foot-and-mouth disease (FMD) is a highly contagious and economically devastating disease that affects cloven-hoofed animals worldwide. Construction and purification of stable antigen for vaccine are necessary but technically difficult and laborious. Here, we have tried to investigate an alternative method by inserting a hexa-histidine tag (6xHIS) in the VP1 C-terminal for easy purification and replacing two amino acids of VP1/VP2 to enhance the stability of the capsid of the FMD virus (FMDV) Asia1/MOG/05. In addition, infectious 6xHIS-tagged stable (S/T) FMDVs were maintained under acidic conditions (pH 6.0) and were readily purified from small-scale cultures using a commercial metal-affinity column. The groups vaccinated with the S/T FMDV antigen showed complete protection comparing to low survival rate in the group vaccinated with non-S/T FMDV against lethal challenge with Asia1 Shamir in mice. Therefore, the present findings indicate that the stabilized and tagged antigen offers an alternative to using the current methods for antigen purification and enhancement of stability and has potential for the development of a new FMD vaccine.

Genetic and immunologic relationships between vaccine and field strains for vaccine selection of type A foot-and-mouth disease virus circulating in East Asia.

Of the seven known serotypes of foot-and-mouth disease virus (FMDV), type A has the most diverse variations. Genetic variations also occur frequently at VP1, VP2, VP3, and VP4 because these proteins constitute the viral capsid. The structural proteins of FMDV, which are closely related to immunologic correlations, are the most easily analyzed because they have highly accessible information. In this study we analyzed the type A vaccine viruses by alignment of available sequences in order to find appropriate vaccine strains. The matching rate of ASIA topotype-specific sites (20 amino acids) located on the viral surface, which are mainly VP1 and VP2, was highly related to immunologic reactivity. Among the available vaccines analyzed in this study, we suggest that A Malaysia 97 could be used as a vaccine virus as it has the highest genetic similarity and immunologic aspects to field strains originating in East Asia.

Antigenic properties and virulence of foot-and-mouth disease virus rescued from full-length cDNA clone of serotype O, typical vaccine strain.

We cloned the full-length cDNA of O Manisa, the virus for vaccinating against foot-and-mouth disease. The antigenic properties of the virus recovered from the cDNA were similar to those of the parental virus. Pathogenesis did not appear in the pigs, dairy goats or suckling mice, but neutralizing antibodies were raised 5-6 days after the virus challenge. The utilization of O Manisa as a safe vaccine strain will increase if recombinant viruses can be manipulated by inserting or removing a marker gene for differential serology or replacing the protective gene from another serotype.

Enhanced immune responses of foot-and-mouth disease vaccine using new oil/gel adjuvant mixtures in pigs and goats.

The immunity and protective capability produced by vaccines can vary remarkably according to the kinds of adjuvants being used. In the case of foot-and-mouth disease (FMD) vaccines in pigs, only oil-adjuvant vaccines have been used, and these tend to show lower immunity in pigs than in cattle. New adjuvants for these vaccines are therefore needed. We made different experimental FMD vaccines using new adjuvants (ISA 201, Carbigen, Emulsigen-D) and well-known adjuvants (ISA 206, aluminum hydroxide gel) and then conducted tests to compare the enhancement in pig immunity. More effective immune responses and protection against challenge were observed with the new adjuvants Emulsigen-D and ISA 201 compared to existing adjuvants. In the case of dairy goats, a mixture of Emulsigen-D and aluminum hydroxide gel produced rapid neutralizing antibody responses that were similar to results from tests conducted with pigs.

Protection to homologous and heterologous challenge in pigs immunized with vaccine against foot-and-mouth disease type O caused an epidemic in East Asia during 2010/2011.

Foot-and-mouth disease (FMD) is a highly contagious infectious disease, and the use of vaccines is known to be effective for its prevention. In 2010/2011, there was an epidemic of the South East Asia (SEA) topotype in East Asian countries. We adapted the SEA topotype virus isolated in November 2010 in Korea in cells to analyze the characteristics of the virus and evaluate its possibility as a vaccine. After cell culture adaptation, the FMD virus particle 146S was purified to develop an inactivated oil vaccine for SEA or other topotypes. To measure its immunogenicity, pigs were inoculated with the experimental vaccine at different concentrations of the antigen. The results indicated that the groups immunized with at least 7.5 µg antigen were protected from homologous challenge. The immunized pigs were also protected against heterologous virus (ME-SA topotype) challenge. The genetic variations between the two field isolates and the adapted vaccine strains were identified in six amino acids by complete genome sequencing.

Antiviral activity of the plant extracts from Thuja orientalis, Aster spathulifolius, and Pinus thunbergii against influenza virus A/PR/8/34.

Influenza viruses cause significant morbidity and mortality in humans through epidemics or pandemics. Currently, two classes of anti-influenza virus drugs, M2 ion-channel inhibitors (amantadin and rimantadine) and neuraminidase inhibitors (oseltamivir and zanamivir), have been used for the treatment of the influenza virus infection. Since the resistance to these drugs has been reported, the development of a new antiviral agent is necessary. In this study, we examined the antiviral efficacy of the plant extracts against the influenza A/PR/8/34 infection. In vitro, the antiviral activities of the plant extracts were investigated using the cell-based screening. Three plant extracts, Thuja orientalis, Aster spathulifolius, and Pinus thunbergii, were shown to induce a high cell viability rate after the infection with the influenza A/PR/8/34 virus. The antiviral activity of the plant extracts also increased as a function of the concentration of the extracts and these extracts significantly reduced the visible cytopathic effect caused by virus infections. Furthermore, the treatment with T. orientalis was shown to have a stronger inhibitory effect than that with A. spathulifolius or P. thunbergii. These results may suggest that T. orientalis has anti-influenza A/PR/8/34 activity.

Direct typing and molecular evolutionary analysis of field samples of foot-and-mouth disease virus collected in Viet Nam between 2006 and 2007.

In this study, we used universal or duplex serotype-specific (O and Asia 1) RT-PCR to analyze clinical field samples of foot-and-mouth disease virus (FMDV) or virus isolates collected in Viet Nam between 2006 and 2007. We found viral serotypes O and Asia 1 circulating concurrently during this period. Direct sequencing of type-specific RT-PCR products revealed the existence of three different topotypes of serotype O: Southeast Asia (SEA), Middle East-South Asia (ME-SA), and Cathay. Of these, SEA was most prevalent during the period. All samples of serotype Asia 1 belonged to genetic group V. Based on the rooted maximum likelihood phylogenetic trees inferred from the VP1 region, new lineages in topotype SEA were originating from Viet Nam, and group V strains of Asia 1 have undergone fewer passages from the common ancestor, compared with other genetic groups. The co-circulation of different types of FMDV may complicate the individual or population genomic structures of FMDV and make conventional multiplex diagnostic methods and phylogenetic analyses with relevant evolutionary models essential in Viet Nam.