First detection and prevalence of Apis mellifera filamentous virus in Apis mellifera and Varroa destructor in the Republic of Korea.

Apis mellifera filamentous virus (AmFV) is a double-stranded DNA virus that infects Apis mellifera bees. To our knowledge, this is the first comprehensive study aiming to detect and analyse the genetic diversity and prevalence of AmFV in Korean honeybee colonies. Phylogenetic analysis based on baculovirus repeat open reading frame-N gene (Bro-N) sequences revealed that AmFV isolates from the Republic of Korea (ROK) fell into two distinct lineages, with genetic origins in Switzerland and China, with nucleotide similarities of 98.3% and 98.2%, respectively. Our prevalence analysis demonstrated a noteworthy infection rate of AmFV in 545 honeybee colonies, reaching 33.09% in 2022 and increasing to 44.90% by 2023. Intriguingly, we also detected AmFV in Varroa destructor mites, highlighting their potential role as vectors and carriers of AmFV. The presence of AmFV was correlated with an increased infection rate of sacbrood virus, deformed wing virus, Lake Sinai virus 2, black queen cell virus, and Nosema ceranae in honeybee colonies. These findings provide valuable insight into the prevalence and potential transmission mechanisms of AmFV in honeybee colonies in the ROK. The results of this study may be instrumental in the effective management of viral infections in honeybee apiaries.

Molecular Identification and Prevalence of the Mite Carpoglyphus lactis (Acarina: Carpoglyphidae) in Apis mellifera in the Republic of Korea.

Apis mellifera, especially weak ones, are highly vulnerable to Carpoglyphus lactis mites, which can rapidly infest and consume stored pollen, leading to weakened colonies and potential colony collapse. This study aimed to ascertain and investigate the prevalence of this mite in honeybee colonies across nine provinces in the Republic of Korea (ROK). A total of 615 honeybee colony samples were collected from 66 apiaries during the spring and 58 apiaries during the summer of 2023. A 1242 bp segment of the Cytochrome c oxidase subunit 1 (COI) gene was amplified using the polymerase chain reaction method. The detection levels of C. lactis in the honeybees were compared between winter and summer. Based on the COI sequence analysis, the nucleotide sequence similarity of C. lactis mites isolated in the ROK with those from China (NC048990.1) was found to be 99.5%, and with those from the United Kingdom (KY922482.1) was 99.3%. This study is the first report of C. lactis in Korean apiaries. The findings of this study demonstrate a significantly higher detection rate in winter, which is 4.1 times greater than that in summer (p < 0.001). Furthermore, the results underscore the usefulness of molecular diagnostic techniques for detecting C. lactis mites.

Selection and Evaluation of Porcine circovirus (PCV) 2d Vaccine Strains to Protect against Currently Prevalent PCV2.

Porcine circovirus (PCV) 2d is a common genotype in South Korea, and the cross-protective ability of PCV2a-based vaccines has been reported recently. In this study, a PCV2d vaccine candidate was selected, and its protective efficacy against the PCV2d isolate was evaluated. From 2016 to 2020, 234 PCV2d isolates were phylogenetically analyzed using open reading frame 2 (ORF2) sequences and classified into four subgroups: PCV2d-1, PCV2d-2, PCV2d-3, and PCV2d-4. Except for PCV2d-4, which consisted of ungrouped isolates, the three subgroups showed distinct differences at amino acid positions 53 and 169 in the ORF2. The detection rates of PCV2d-1, PCV2d-2, and PCV2d-3 were 36.5, 37.4, and 3.7%, respectively, and representative isolates were selected from each subgroup (QIA244, QIA126, and QIA169, respectively). In the neutralization assay, QIA244 showed the lowest neutralization efficiency among the three PCV2a-based vaccines, whereas the virus-like particles of QIA244 (rQIA244) provided broader protection against the three genotypes than did those of QIA126 and rQIA169. To further evaluate rQIA244 in pigs, the experimental groups were divided into rQIA244-vaccine (2dVac), commercial PCV2a-vaccine (2aVac), and no-vaccination (noVac) groups. The 2dVac effectively reduced the copy number of PCV2d in blood and tissues, as well as in tissue lesions, compared to the effect of 2aVac. Collectively, 2dVac provided by QIA244 ORF2 successfully demonstrated protective efficacy against the currently prevalent PCV2d in vitro neutralization and in vivo assays.

A Field Efficacy Trial of Recombinant Porcine Circovirus Type 2d Vaccine in Three Herds.

This study aimed to evaluate the efficacy of a virus-like particle (VLP) vaccine containing the open reading frame 2 of porcine circovirus type 2d (PCV2d) in a farm environment where natural infections associated with porcine circovirus-associated disease are endemic. The vaccine trial was conducted on three farms (H, M, and Y) with a history of infections including porcine reproductive and respiratory syndrome virus (PRRSV), PCV, Mycoplasma, and E. coli. Farm H, as well as farms M and Y, experienced natural PCV2 infection between 4 and 8 weeks post-vaccination (wpv), and 8 and 12 wpv, respectively. Viremia levels of all farms were significantly (p < 0.05) lower in vaccinated piglets than the control group after natural infection. In all farms, serum immunoglobulin G levels peaked at 8 wpv in the vaccinated groups, surpassing those in the control groups. Furthermore, neutralizing antibody titers were significantly (p < 0.05) higher in the vaccinated groups than the control groups in farms H and Y (0-8 wpv). However, there were no significant differences between the vaccinated and control group in neutralizing antibody titers of farm M (0-20 wpv). In terms of body weight, vaccinated piglets from all three farms showed significantly increased average weights at 12 wpv compared to the control groups. In conclusion, our study revealed noteworthy differences in viremia and body weight gain between vaccinated and control animals on three farms. As a result, this field trial of PCV2d VLP vaccine was successful in protecting piglets from natural PCV2 infection.

A novel chimeric vaccine candidate for porcine reproductive and respiratory syndrome virus (PRRSV) I and II elicits neutralizing antibodies against both types.

Porcine reproductive and respiratory syndrome virus (PRRSV) is an economically important virus within the swine industry. The virus causes respiratory disease and reproductive failure. Two species of PRRSV-I and II are co-dominant, yet no effective vaccination strategy has been developed to protect against these two types. With an aim to develop a chimeric vaccine strain to protect against both types, in this study, a chimeric porcine reproductive and respiratory syndrome virus (PRRSV) type I and II was rescued using reverse genetics for the first time. Four chimeric infectious clones were designed based on the genomic arrangement of the structural proteins. However, only the clone carrying the transcriptional regulatory sequence (TRS) and ORF6 of a PRRSV-I and ORF6 of a PRRSV-II generated a viable recombinant virus, suggesting that concurrent expression of ORF6 from both parental viruses is essential for the recovery of type I and II chimeric PRRSV. The chimeric virus showed significantly lower replication ability than its parental strains in vitro, which was improved by serial passaging. In vivo, groups of pigs were inoculated with either the chimeric virus, one of the parental strains, or PBS. The chimeric virus replicated in pig tissue and was detected in serum 7 days post-inoculation. Serum neutralization tests indicated that pigs inoculated with the chimeric virus elicited neutralizing antibodies that inhibited infection with strains of both species and with greater coverage than the parental viruses. In conclusion, the application of this technique to construct a chimeric PRRSV holds promise for the development of a highly effective modified live vaccine candidate. This is particularly significant since there are currently no approved commercial divalent vaccines available to combat PRRSV-I and II co-infections.

Molecular Characterization of Porcine Reproductive and Respiratory Syndrome Virus in Korea from 2018 to 2022.

Porcine reproductive and respiratory syndrome (PRRS) is an endemic disease in the Republic of Korea. Surveillance of PRRS virus (PRRSV) types is critical to tailor control measures. This study collected 5062 serum and tissue samples between 2018 and 2022. Open reading frame 5 (ORF5) sequences suggest that subgroup A (42%) was predominant, followed by lineage 1 (21%), lineage 5 (14%), lineage Korea C (LKC) (9%), lineage Korea B (LKB) (6%), and subtype 1C (5%). Highly virulent lineages 1 (NADC30/34/MN184) and 8 were also detected. These viruses typically mutate or recombine with other viruses. ORF5 and non-structural protein 2 (NSP2) deletion patterns were less variable in the PRRSV-1. Several strains belonging to PRRSV-2 showed differences in NSP2 deletion and ORF5 sequences. Similar vaccine-like isolates to the PRRSV-1 subtype 1C and PRRSV-2 lineage 5 were also found. The virus is evolving independently in the field and has eluded vaccine protection. The current vaccine that is used in Korea offers only modest or limited heterologous protection. Ongoing surveillance to identify the current virus strain in circulation is necessary to design a vaccine. A systemic immunization program with region-specific vaccinations and stringent biosecurity measures is required to reduce PRRSV infections in the Republic of Korea.

Evaluation and Determination of a Suitable Passage Number of Codon Pair Deoptimized PRRSV-1 Vaccine Candidate in Pigs.

Porcine reproductive and respiratory syndrome virus (PRRSV) is major economic problem given its effects on swine health and productivity. Therefore, we evaluated the genetic stability of a codon pair de-optimized (CPD) PRRSV, E38-ORF7 CPD, as well as the master seed passage threshold that elicited an effective immune response in pigs against heterologous virus challenge. The genetic stability and immune response of every 10th passage (out of 40) of E38-ORF7 CPD was analyzed through whole genome sequencing and inoculation in 3-week-old pigs. E38-ORF7 CPD passages were limited to 20 based on the full-length mutation analysis and animal test results. After 20 passages, the virus could not induce antibodies to provide effective immunity and mutations accumulated in the gene, which differed from the CPD gene, presenting a reason for low infectivity. Conclusively, the optimal passage number of E38-ORF7 CPD is 20. As a vaccine, this may help overcome the highly diverse PRRSV infection with substantially enhanced genetic stability.

Codon Pair Deoptimization (CPD)-Attenuated PRRSV-1 Vaccination Exhibit Immunity to Virulent PRRSV Challenge in Pigs.

Commercially used porcine respiratory and reproductive syndrome (PRRS) modified live virus (MLV) vaccines provide limited protection with heterologous viruses, can revert back to a virulent form and they tend to recombine with circulating wild-type strains. Codon pair deoptimization (CPD) is an advanced method to attenuate a virus that overcomes the disadvantages of MLV vaccines and is effective in various virus vaccine models. The CPD vaccine against PRRSV-2 was successfully tested in our previous study. The co-existence of PRRSV-1 and -2 in the same herd demands protective immunity against both viruses. In this study, live attenuated PRRSV-1 was constructed by recoding 22 base pairs in the ORF7 gene of the E38 strain. The efficacy and safety of the CPD live attenuated vaccine E38-ORF7 CPD to protect against virulent PRRSV-1 were evaluated. Viral load, and respiratory and lung lesion scores were significantly reduced in animals vaccinated with E38-ORF7 CPD. Vaccinated animals were seropositive by 14 days post-vaccination with an increased level of interferon-γ secreting cells. In conclusion, the codon-pair-deoptimized vaccine was easily attenuated and displayed protective immunity against virulent heterologous PRRSV-1.

Porcine Circovirus (PCV) Genotype 2d-Based Virus-like Particles (VLPs) Induced Broad Cross-Neutralizing Antibodies against Diverse Genotypes and Provided Protection in Dual-Challenge Infection of a PCV2d Virus and a Type 1 Porcine Reproductive and Respiratory Syndrome Virus (PRRSV).

As PCV2d infection has been continuously reported in swine farms in which pigs were vaccinated with PCV2a- or 2d-based vaccines, we attempted to develop a novel vaccine using a PCV2d-based capsid to enhance its protective efficacy. In this study, recombinant virus-like particles (VLPs) of rPCV2a, rPCV2b and rPCV2d were synthesized from the capsid proteins of PCV2a, PCV2b and PCV2d field isolates, respectively. A cross-neutralization assay between the VLPs induced antisera and the field isolates demonstrated the broad cross-neutralizing activities of the rPCV2d-induced antisera. Then, the protective efficacy of rPCV2d as a vaccine candidate was investigated in commercial pigs by rPCV2d vaccination and a single- or dual-challenge infection using a PCV2d strain and a type 1 PRRSV strain. High levels of anti-PCV2d IgG and neutralizing antibodies were induced 3 weeks after vaccination. After the challenge infection, the average ADWG values of the vaccinated group were higher than those of the unvaccinated group. None or a significantly low amount of (p < 0.05) reduced PCV2 genomic DNA was found in the blood, saliva and tissues of the vaccinated pigs, when compared to the unvaccinated group. Moreover, macroscopic and microscopic lesions in the tissues were significantly (p < 0.05) reduced in the vaccinated groups. This study therefore suggests that rPCV2d may be highly useful for the control of diverse field genotypes.

Recombinant vesicular stomatitis virus glycoprotein carrying a foot-and-mouth disease virus epitope as a vaccine candidate.

Foot-and-mouth disease (FMD) is one of the most highly contagious animal diseases. In an effort to overcome the drawbacks of the currently used inactivated foot-and-mouth disease virus vaccine, a novel recombinant protein carrying foot-and-mouth disease virus VP1 GH loop epitope linked to vesicular stomatitis virus glycoprotein was expressed in a baculovirus system. Its antigenicity was confirmed with ELISA using monoclonal antibody against foot-and-mouth disease virus. Twice immunizations one month apart in field pigs resulted in a significant antibody increase compared to the glutathione S-transferase carrier containing the same epitope and the commercial vaccine. To my knowledge, this is the first report that the recombinant protein vaccine was superior to the current vaccine. Although further studies are required to examine their immunogenicity in a large number of animals, this study sheds light on the development of a novel recombinant protein vaccine that could be easily produced in a general laboratory as an alternative to the current FMD vaccine, which requires a biosafety level 3 containment facility for vaccine production.

Evaluation of Quality Control Methods for Foot-And-Mouth Disease Vaccines by High-Performance Liquid Chromatography.

Foot-and-mouth disease (FMD) is considered one of the highly contagious viral infections affecting livestock. In Korea, an FMD vaccination policy has been implemented nationwide since 2010 for the prevention and control of FMD. Since the vaccines are imported from various countries, standardized quality control measures are critical. In this study, we aimed to validate a high-performance liquid chromatography (HPLC) device in the Animal and Plant Quarantine Agency lab and identify an appropriate FMD vaccine pretreatment method for HPLC-a simple, reliable, and practical method to measure antigen content. Based on the analyses of specificity, linearity, accuracy, repeatability, intermediate precision, limits of detection, and limits of quantification using FMD standard samples, we validated the method using a standard material. Overall, we confirmed that the HPLC technique is effective for the quantitative assessment of the FMD virus 146S antigen in Korea. Using commercial FMD vaccines, we evaluated three separation methods and identified the method using n-pentanol and trichloroethylene as optimal for HPLC analysis. Our HPLC method was effective for the analytical detection of the antigen content in FMD vaccine, and it may be useful as a reference method for national lot-release testing.

The interference effect of maternally-derived antibodies on the serological performance of pigs immunized with a foot-and-mouth disease oil emulsion vaccine.

To control foot-and-mouth disease (FMD) outbreaks that originated in Jincheon County in South Korea between 2014 and 2015, several commercial vaccines were studied for their efficacy and serological performance in the field. In this study, the efficacy of the O SKR 7/10 vaccine was evaluated by challenge with the FMD virus (FMDV) O/Jincheon/SKR/2014 (O Jincheon), which has the same O/SEA/Mya-98 lineage as the O/SKR/7/10 strain that was isolated in 2010 in South Korea, in FMD-seronegative pigs. Full protection against the O Jincheon virus was demonstrated as early as 14 days postvaccination, which was explained by the strong serological relationship (r1 value: ≥ 0.92) between the O Jincheon and O SKR 2010 viruses. However, in the field trial, no satisfactory serological elevations against FMDV were observed, even in the double-vaccinated groups. Therefore, it can be concluded that the O SKR 7/10 vaccine may need to be improved to overcome the interference effects from the high levels of maternally-derived antibodies generated due to the mandatory nationwide vaccination of sows in South Korea.

Cross-protective efficacy of the O1 Manisa + O 3039 bivalent vaccine and the O 3039 monovalent vaccine against heterologous challenge with FMDV O/Jincheon/SKR/2014 in pig.

After massive foot-and-mouth disease (FMD) outbreaks originated from Jincheon County from Dec. 2014 to Apr. 2015, the effectiveness of the previous FMD vaccine containing only the O1 Manisa as the O antigen, O1 Manisa + A Malaysia 97 + Asia 1 Sharmir trivalent vaccine, was questioned in South Korea, and a change in the O antigen in FMD vaccines was demanded to control the FMD caused by FMDV O/Jincheon/SKR/2014, the O Jincheon strain. Therefore, the efficacies of O1 Manisa + O 3039 bivalent vaccine and O 3039 monovalent vaccine were studied for cross-protection against heterologous challenge with the O Jincheon strain. In this study, the efficacy of the O1 Manisa + O 3039 bivalent vaccine was better than that of the O 3039 monovalent vaccine, even though the serological relationship (r1 value) between O Jincheon and O 3039 was matched according to the OIE Terrestrial Manual. According to serological test results from vaccinated specific pathogen free pigs, virus neutralization test titers against Jincheon were good estimates for predicting protection against challenge. A field trial of the O1 Manisa + O 3039 bivalent vaccine was performed to estimate the possibility of field application in conventional pig farms, especially due to concerns about the effect of maternally derived antibodies (MDA) in field application of the FMD vaccine. According to the result of the field trial, the O1 Manisa + O 3039 bivalent vaccine was considered to overcome MDA. The results of the efficacy and field trials indicated that the O1 Manisa + O3039 vaccine could be suitable to replace previous FMD vaccines to control the FMD field situation caused by O Jincheon FMDV.

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.

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.

Antiviral activity of ovine interferon tau 4 against foot-and-mouth disease virus.

Foot-and-mouth disease (FMD) is an economically important disease in most parts of the world and new therapeutic agents are needed to protect the animals before vaccination can trigger the host immune response. Although several interferons have been used for their antiviral activities against Foot-and-mouth disease virus (FMDV), ovine interferon tau 4 (OvIFN-τ4), with a broad-spectrum of action, cross-species antiviral activity, and lower incidence of toxicity in comparison to other type І interferons, has not yet been evaluated for this indication. This is the first study to evaluate the antiviral activity of OvIFN-τ4 against various strains of FMDV. The effective anti-cytopathic concentration of OvIFN-τ4 and its effectiveness pre- and post-infection with FMDV were tested in vitro in LFBK cells. In vivo activity of OvIFN-τ4 was then confirmed in a mouse model of infection. OvIFN-τ4 at a concentration of 500 ng, protected mice until 5days post-FMDV challenge and provided 90% protection for 10 days following FMDV challenge. These results suggest that OvIFN-τ4 could be used as an alternative to other interferons or antiviral agents at the time of FMD outbreak.

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.

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.

Evaluation of Infectivity, Virulence and Transmission of FDMV Field Strains of Serotypes O and A Isolated In 2010 from Outbreaks in the Republic of Korea.

Since the early 2000s outbreaks of foot-and-mouth disease (FMD) have been described in several previously FMD-free Asian nations, including the Republic of Korea (South Korea). One outbreak with FMD virus (FDMV) serotype A and two with serotype O occurred in South Korea in 2010/2011. The causative viruses belonged to lineages that had been spreading in South East Asia, far East and East Asia since 2009 and presented a great threat to the countries in that region. Most FMDV strains infect ruminants and pigs, as it happened during the outbreaks of FMDV serotype O in South Korea. Contrastingly, the strain of serotype A affected only ruminants. Based upon these findings, the intention of the work described in the current report was to characterize and compare the infectivity, virulence and transmission of both strains under laboratory conditions in cattle and pigs, by direct inoculation and contact exposure. As expected, FMDV serotype O was highly virulent in both cattle and swine by contact exposure and direct inoculation. Surprisingly, FMDV serotype A was highly virulent in swine, but was less infectious in cattle by contact exposure to infected swine or cattle. Interestingly, similar quantities of aerosolized FMDV RNA were detected during experiments with viruses of serotypes O and A. Specific virus-host interaction of A/SKR/2010 could affect the transmission of this strain to cattle, and this may explain in part the limited spread of the serotype A epizootic.

Inactivation of foot-and-mouth disease virus by citric acid and sodium carbonate with deicers.

Three out of five outbreaks of foot-and-mouth disease (FMD) since 2010 in the Republic of Korea have occurred in the winter. At the freezing temperatures, it was impossible to spray disinfectant on the surfaces of vehicles, roads, and farm premises because the disinfectant would be frozen shortly after discharge and the surfaces of the roads or machines would become slippery in cold weather. In this study, we added chemical deicers (ethylene glycol, propylene glycol, sodium chloride, calcium chloride, ethyl alcohol, and commercial windshield washer fluid) to keep disinfectants (0.2% citric acid and 4% sodium carbonate) from freezing, and we tested their virucidal efficacies under simulated cold temperatures in a tube. The 0.2% citric acid could reduce the virus titer 4 logs at -20°C with all the deicers. On the other hand, 4% sodium carbonate showed little virucidal activity at -20°C within 30 min, although it resisted being frozen with the function of the deicers. In conclusion, for the winter season, we may recommend the use of citric acid (>0.2%) diluted in 30% ethyl alcohol or 25% sodium chloride solvent, depending on its purpose.