This study evaluated the virucidal efficacy of a chlorine dioxide (ClO2) gas-generating fumigation disinfectant consisting of sodium chlorate solution (25% sodium chlorate) and reaction solution against avian influenza virus (AIV). After AIV suspensions had been deposited on stainless steel carriers, the 9 dried carriers were exposed to the fumigant (sodium chlorate solution: 8.5, 17, 34, 50, and 100 mL) in a 25-m³ test room for 2, 3, and 4 h, respectively. Thereafter, all carriers were submerged in a neutralizing solution (20% fetal bovine serum) to scrape off the surviving viruses, and the respective suspensions were diluted. Each diluent was inoculated into the allantoic membrane of five 10-day-old embryos. After incubation for 5 days at 37℃, AIV viability in the collected allantoic fluids was examined, and the egg infectious dose 50 (EID50) was calculated. When the carrier was exposed to ClO2 gas generated from reacting 34 mL of the fumigant for 3 h, the AIV titer reduced by more than 104.0 EID50/carrier compared to the control, which was exposed to the fumigant without inoculation of AIV suspension. In addition, the control was non-toxic to the embryos.

Pigs are considered a “mixing vessel” that can produce new influenza strains through genetic reassortments, which threaten public health and cause economic losses worldwide. We performed surveillance of swine influenza virus (SIV) using 114,140 nasal swabs from 11,414 Korean farms from 2009 to 2022, and characterized their genetic evolution at each farm level. A total of 169 farms out of the 11,414 (1.48%) were SIV-positive. The positivity rate for the H1N2 subtype, which is most dominant in the pig population, was 37.87% (64/169). Through hemagglutinin (HA) gene analysis, 64 H1N2-positive farms were classified into Eurasian avian-like (46/64, 71.88%), triple-reassortant (14/64, 21.88%), pdm09 (3/64, 4.69%), and classical swine (1/64, 1.56%) groups. The estimated evolutionary rate of HA in H1N2 from 2009 to 2022 in Korea is 1.5309 × 10-3/site/year (95% HPD intervals from 1.0003 × 10-3 to 2.1735 × 10-3) with an estimated mean growth rate of 0.0114. Estimates of the relative genetic diversity of clades over time suggested that the HA of H1N2 exhibited an increase in population size. The results of this study showed that the Eurasian avian-like-HA of the H1N2 subtype was dominant in the pig population. The continued evolution of the HA of H1N2, which is critical for cell entry, might lead to genetic diversity and the loss of vaccine cross-protection. These results indicate that continus surveillance is imperative for monitoring the evolution of the swine influenza virus.

Wild birds, especially aquatic birds, are the natural reservoir of avian influenza virus (AIV), and many kinds of water body can be contaminated with feces of these birds. Seasonally, AIVs can be dissolved in the environmental water from the feces of the infected birds, and this water can be a target for viral detection and identification. In this study, we employed and tested three different filters for concentrating AIV, and it was shown that high concentration factor in terms of viral density could be achieved with viral samples diluted with natural water. Wild bird fecal samples containing low pathogenicity H5 AIVs were successfully concentrated with the adsorption and elution method using mixed cellulose esters membrane; the recovery rate of virus was 35.5 % and the concentration factor was about 50 on average. For the larger volume of water sample, we proved that an inline disposable filter with high surface area, 300 cm2, has a comparable concentration factor to the adsorption and elution method and the filter could be used in the field conveniently by being plugged into peristaltic pump. These validated methods for water sampling may be used as a supplementary for virological surveillance on wild migratory birds or during the epidemiological investigation on the environment near affected premises by AIV.

Influenza A viruses (IAVs) are members of the family Orthomyxoviridae and genus Orthomyxovirus. Avian and mammalian species are the host of IAVs, which includes humans and dogs. Canine influenza virus (CIV) is an emerging pathogen that causes severe and acute respiratory diseases in dogs. This study monitored the antigen and antibody against CIV in dogs in the Republic of Korea (ROK) from 2016 to 2021. One thousand and seventy-two nasal swabs and 1,545 blood samples were collected from animal hospitals and animal shelters. Five nasal swabs in 2017 and seven in 2018 from stray dogs were positive for CIV according to RT-PCR. The prevalence of H3N2 CIV ranged from 9.5% to 24.8%, according to the hemagglutination inhibition (HI) assay. On the other hand, none of the serum samples from 2018 to 2021 showed seropositivity against the avian H5, H7, and H9 viruses. The HI titers for H3N2 ranged from 16 to 512. The distribution of HI titer 16–32 was 57.6% in seropositive samples. The pet dogs were vaccinated against CIV, but the stray and military dogs were unvaccinated. In 2017 and 2018, the seroprevalence of CIV in stray dogs was higher than in the other years, and viral RNA was detected in nasal swabs. It may mean previous exposure of stray dogs to CIV. With the increasing number of pet dogs and the close contact between humans and dogs, canines could serve as an intermediate host for transmitting IAVs to humans. Therefore, continuous surveillance of CIV is needed for public health and the potential emergence of novel zoonotic viruses.

In this study, the near-complete genome sequence of the novel reassortant H1N2 influenza A virus strain A/swine/Korea/KS60/2016 is reported. Sequences of the hemagglutinin (HA), neuraminidase (NA), and polymerase basic 2 (PB2) genes were analyzed, revealing that the isolates contain segments from previous Korean swine H1N2 strains. Additionally, the remaining genes of this strain originated from human H1N1 strains in 2009.

Influenza A virus (IAV) causes respiratory disease in birds and mammals, including pigs and humans. Infection by IAV in pigs increases not only economic losses in the swine industry but also the emergence of novel IAV variants via gene reassortment, which is important due to the susceptibility of both birds and humans to IAV. This study provides serological data obtained during a study to detect IAV infections in pigs in the Republic of Korea during 2018 and 2019. A total of 1,559 samples were collected from 74 domestic pig farms. Hemagglutination inhibition (HI) assays were performed using the A/Swine/Korea/25-13(H1N1), A/Swine/Korea/E102 (H1N2), and A/Swine/Korea/Cy10/2007 (H3N2) viruses as antigens. The HI assay results showed that 266 of the 1,559 samples were seropositive (17.0%). Among these, H1N1, H1N2, and H3N2 comprised 7.3% (114), 6.0% (93), and 8.8% (137) of the 1,559 samples, respectively. Co-infections of H1N1/H1N2, H1N1/H3N2, H1N2/H3N2 and H1N1/H1N2/H3N2 were observed in 2.1% (31), 1.5% (23), 1.5% (24), and 0.8% (13) of the 1,559 samples, respectively. Interestingly, IAV infections were detected in all nine provinces of the country.

In this study, antibody responses after vaccination against equine influenza were investigated among 1,591 horses in the Republic of Korea using the hemagglutination inhibition (HI) test. Equine influenza has not occurred since 2011 and a commercial vaccine against H3N8 has been used. The equine influenza virus, A/equine/South Africa/4/03 (H3N8), was used as the antigen in the HI assay. The mean seropositive rate was 90.5% in 2019. Except for stallion whose seropositive rate was 78.5%, all seropositive rates of other horse types were over 90%. Regionally, except for Gangwon-do and Jeju-do whose seropositive rates were 89.0% and 87.1%, all seropositive rates in other provinces were over 90%. In the future, more through vaccination against equine influenza needs to be done based on this investigation result.

Salmonella Enteritidis (SE) infection, one of a major foodborne disease remains considerable public health concerns globally. In this study, we have constructed that the genetically inactivated SE ghost vaccine candidate, JOL2220 (Δlon ΔcpxR Δasd), which express the immunogenic hemagglutinin portion (HA1) of H1N1 virus on the Salmonella surface. The lysis gene cassette, holin-endolysin system originated from double DNA bacteriophage λ was cloned into the ghost vector plasmid pJHL464, which activated under the control of the convergent promoter system. Scanning electronic microscopic examination revealed that transmembrane tunnels of JOL2220 was formed following the 48 hr of the lysis mediated by the holin-endolysin system. In vitro expression of HA1 antigenic protein was also determined by Western blot analysis. The Chickens immunized with JOL2220 ghost via intramuscular and oral routes showed significant increase in IgG level against both SE and HA1 antigen. Further, we observed markedly enhancement of lymphocyte proliferation and T-cell differentiation in the primed PBMC of the immunized chicken, compared to those in the control group. Collectively, the results indicated that the SE ghosts expressing HA1 antigen may have immunostimulatory properties against Salmonella and influenza infection where the chicken is the predominant reservoir.

To attenuate and control the spread of infectious disease, a body of research has been conducted to generate safe vaccines and to continue national-level surveillance. However, understanding on viability and persistence of avian influenza virus (AIV) in infected carcasses, and effective disposal approaches are still limited up to date. Here, using HA test and RT-PCR, we assessed active status of AIV and degradation of viral RNA in collected specimens at different sites and time points. First, AIV infectivity was recovered until day 2, and viral nucleic acids persisted to day 14 and 21 in inorganic and organic samples, respectively, in sealed vials incubated at room temperature. Second, AIV was totally inactivated in all examined specimens, and viral RNA was not detectable at all time points tested at least one month post-infection in AIV-inoculated carcasses buried directly in soil or fiber reinforced plastic (FRP) bin. Lastly, among different burial sites in South Korea, 6 out of 17 sampling sites in Jeonbuk province showed the presence of viral genetic materials, while the rest of the field samples displayed neither the presence of infective AIV nor detectable viral RNA. This study showed a linear relation between time and degradation degree of viral RNA in buried samples suggesting that burial disposal method is effective for the control or at least attenuation of spread of AI infection in infected animals although consistent monitoring is required to verify safety of disposal.

Highly pathogenic avian influenza virus (HPAIV) damages vital organs and tissues, frequently leading to death in birds, and causes serious economic losses in the poultry industry. In addition, HPAIV can infect humans and other mammals, often with fatal outcomes. In this study, the virucidal efficacy of Clean-Zone®, which contains citric acid, malic acid and phosphoric acid, against avian influenza virus (AIV, H9N2) was investigated. Virucidal efficacy was determined by examining the viability of AIV after contact with the disinfectant in the allantoic membrane of chicken embryos. The disinfectant and AIV were reacted under hard water (HW) and organic matter suspension (OM) condition. AIV was inactivated with 200- and 50-fold dilutions of the disinfectant under HW and OM conditions, respectively. As the disinfectant, Clean-Zone®, has a virucidal efficacy against AIV, it can be used to prevent the spread of animal viral diseases.

In this study, the virucidal efficacy of a fumigant (35% paraformaldehyde) against avian influenza virus (AIV) was examined. After AIV suspensions had been deposited on stainless steel carriers, the dried carriers were exposed to the fumigant in a 300-m3 test room for 3 h. Thereafter, all carriers were submerged in a neutralizing solution to scrape off the surviving viruses, and the respective suspensions were diluted. Each dilution factor was respectively inoculated into the allantoic membrane of five 10-day-old embryos. After incubation, AIV viability in the collected allantoic fluids was examined and the EID50 was calculated. The fumigant inactivated ≥5.7log10EID50 of AIV and was nontoxic to the embryos.

The efficacy of chemical disinfectants is reduced owing to the inactivation of active ingredients after dilution. This study investigated the effect of time on the efficacy of six different disinfectants, after dilution, against avian influenza virus. When used at the recommended concentration, most disinfectants showed efficacy at a high concentrations in the presence of organic materials immediately after dilution, while sodium dichloroisocyanurate-based products, after dilution, showed reduced efficacy over time at low concentrations in the absence of organic materials. Most disinfectants were neutralized by organic materials; however, this could be compensated for by increasing the product dosage. For successful decontamination in farms, disinfectants should be used at high concentrations in accordance with the manufacturers’ recommendations. Furthermore, the presence of organic materials must be taken into consideration, and diluted disinfectant solution should be prepared no more than a day before use.

In the past four years, outbreaks of acute respiratory diseases associated with canine influenza H3N2 viruses in dogs and cats have been reported in South Korea and China. For prevention of disease from spread of the disease and for administration of timely medical treatments, including countermeasures for quarantine, use of a rapid and highly sensitive detection method are important to detection of the causative viruses. This study was conducted in order to develop a real time RT-PCR for the H3N2 subtype. It was based on primers targeting the highly homologous sequences of matrix, hemagglutinin, and neuraminidase genes. The detection limit of real time RT-PCR was 10 copies/ul with matrix and hemagglutinin genes, and 1 copy with neuraminidase genes, respectively. This real time RT-PCR was as sensitive as virus isolation in 52 clinical samples. The detection system developed in this study might provide more rapid and highly sensitive results than commercial rapid kits based on immunochromatographic assay.

The influenza virus is an important respiratory risk affecting humans, and effective treatments are needed. Some oriental medicines are currently applied for treatment of common colds as well as influenza infection. Previous studies have reported that the therapeutic properties of MA-128 are effective for treatment of psoriasis antiasthmatic and atopic dermatitis. In this study, we investigated the therapeutic properties of the novel herbal medicine, MA-128, for treatment of influenza virus infection by oral administration. MA-128 is an active natural biological compound from herbal-marine origin. The results showed that oral administration of MA-128 in mice could confer a survival benefit against Type A influenza virus infection. Daily oral administration of MA-128 resulted in delayed death in infected mice for three days against mouse adapted H3N2 (A/Philippines/2/82). However, it protected more than 60% of mice from lethal infection of 2009 pandemic H1N1 (A/Korea/CJ01/2009) influenza virus. In addition, lung viral titers were significantly reduced at seven days post infection (~100 times) compared with mock-treated mice and viruses were cleared at 9 dpi only in the MA-128 treated groups. This study demonstrated the potential of the novel herbal medicine, MA-128, as an herbal remedy against influenza A viruses.

Highly pathogenic avian influenza virus (HPAIV) is already panzootic in poultry and caused a considerable economic loss in poultry industry. In addition, HPAIV continues to cross species barriers to infect humans and other mammals, often with fatal outcomes. In this study, the virucidal efficacy of Citra-Kill® composed to quaternary ammonium chloride and citric acid was investigated against avian influenza H9N2 virus (AIV). A virucidal efficacy was determined with the viability of AIV contacted with the disinfectant in the allantoic membrane of chicken embryos. Citra-Kill® and AIV was reacted on the distilled water (DW), hard water (HW) or organic matter suspension (OM) condition. On DW condition, AIV was inactivated with 2,000 fold dilutions of Citra-Kill®. When the antiviral effect on HW condition was evaluated, the antiviral activity of the disinfectant showed on 1,500 fold dilutions against AIV. With the investigation of the antiviral effect of the disinfectant on OM condition, AIV was inactivated on 500 fold dilutions of Citra-Kill®. As Citra-Kill® possesses virucidal efficacy against AIV, the disinfectant solution can be used to limit the spread of animal viral diseases.

Avian influenza (AI) and Newcastle disease (ND) distress a variety of avian species, especially domestic poultry. Severe syndromes are caused by highly virulent specific virus strains termed highly pathogenic AI and velogenic ND viruses, which are potential agrobioterrorism agents. This outbreak emphasizes the need for continuing cooperation between public health and veterinary medical communities in controlling AI and ND when it has a zoonotic potential. Up to date, the stamping out and burying system were applied for controlling methods against these highly infectious diseases in the ordinary way, however these methods had many environmental problems, including leachat and effluvium. Thus, we assessed that sterilization effect of AI and ND virus dependent on several treatment conditions, such as autoclaving time and cutting types of chicken. As a result, we found that the cutting type of chicken meat revealed a reduced HA titer (20) against both of AI and ND virus after 10 min of autoclaving, while whole chicken showed same titer after 30 and 60 min. Therefore, we propose that the conditions of treatment on infected chicken should be developed for convenient, affordable, and effective prevention methods against for AI and ND.

Korea had experienced second epidemic highly pathogenic avian influenza (HPAI), and there were the seven affected farms, including two breeder duck farms, from 22nd November 2006 to 6th March 2007. Here, we reported the clinico-pathological characteristics of domestic breeder ducks farms naturally infected with HPAI virus (H5N1). Clinically, the most ducks showed various signs from depression, decreased egg production and feed consumption to even, death. The most commonly gross changes were hepatomegaly, splenomegaly, petechial and ecchymotic hemorrhage on the liver surface, a white stripe on the cardiac muscle, and severely hemorrhagic and deformed eggs. The most significant histopathological changes were necrosis of various cells such as neuron, lymphocytes, cardiac myocytes, hepatocytes, blood vessels and pancreatic acinar epithelium. The viral antigen was mainly detected in the endothelium of blood vessels of various organs and tissues, peripheral nerves and neuronal cells. Based on the above results, we identified that HPAI H5N1 induced systemic infection in the adult breeder ducks.

Influenza virus infection is a zoonosis that results in high mortality in animals and humans. Several recent avian influenza outbreaks have posed a significant public health threat to humans because there have been many cases of direct interspecies transmission from birds to humans. Influenza virus infection causes acute respiratory failure, which is the main cause of death, while chronic influenza virus infection in the central nervous system results in neural dysfunction. Of particular interest, according to one report, a group of patients who recovered from influenza virus infection during pandemics showed neuropsychiatric disorders, including depression, schizophrenia, and Parkinsonism. Thus, study of the etiology of neuropsychiatric disorders caused by influenza virus infection is needed. In order to conduct necessary experiments, it is essential to reproduce neurological phenomena that are manifested by human patients who have recovered from influenza virus infection using laboratory animals. In this review, we will discuss some of the facts that should be considered when establishing an animal model for study of central nervous system responses to influenza virus infection using mice.