Estimating the time of infection for African swine fever in pig farms in Korea.

African swine fever (ASF) is a highly contagious and lethal disease with characteristics of hemorrhagic fever. ASF outbreaks in pig farms significantly damage the entire pork industry. Understanding the transmission dynamics of ASF is crucial to effectively respond. Notably, it is important to know when the infection started on the outbreak farm. This study aimed at establishing a procedure for estimating the time of infection on pig farms affected by the ASF outbreak in Korea. The protocol for sampling to detect ASF virus infection, the estimation of the time interval between infection and detection, and the estimation of the infection stage parameters for the simulation model were described. After infection, fattening sheds (9.8 days in median) had the longest detection time compared with pregnant (8.6 days) or farrowing sheds (8.0 days). The intervals were 8.8 days for farrow-to-finisher farms, 7.0 days for farrow-to-weaning farms, and 9.5 days for fattening farms. The findings of this study provide valuable insights into ASF outbreaks in pig farms thus, improving the disease control ability.

Big data-based risk assessment of poultry farms during the 2020/2021 highly pathogenic avian influenza epidemic in Korea.

Outbreaks of H5-type highly pathogenic avian influenza (HPAI) in poultry have been reported in various parts of the world. To respond to these continuous threats, numerous surveillance programs have been applied to poultry raising facilities as well as wild birds. In Korea, a surveillance program was developed aimed at providing a preemptive response to possible outbreaks at poultry farms. The purpose of this study is to comprehensively present the risks of HPAI evaluated by this program in relation to actual outbreak farms during the epidemic of 2020/2021. A deep learning-based risk assessment program was trained based on the pattern of livestock vehicles visiting poultry farms and HPAI outbreaks to calculate the risk of HPAI for farms linked by the movement of livestock vehicles (such farms are termed "epidemiologically linked farms"). A total of 7,984 risk assessments were conducted, and the results were categorized into four groups. The proportion of the highest risk level was greater in duck farms (13.6%) than in chicken farms (8.8%). Among the duck farms, the proportion of the highest risk level was much greater in farms where breeder ducks were raised (accounting for 26.4% of the risk) than in farms where ducks were raised to obtain meat (12.8% of the risk). A higher risk level was also found in cases where the species of the outbreak farm and epidemiologically linked farms were the same (proportion of the highest risk level = 13.2%) compared to that when the species between the two farms were different (7.9%). The overall proportion of farms with HPAI outbreaks among epidemiologically linked farms (attack rate, AR) was 1.7% as HPAI was confirmed on 67 of the 3,883 epidemiologically linked farms. The AR was highest for breeder ducks (15.3%) among duck farms and laying hens (4.8%) among chicken farms. The AR of the pairs where livestock vehicles entered the inner farm area was 1.3 times (95% confidence interval: 1.4-2.9) higher than that of all pairs. With the risk information provided, customized preventive measures can be implemented for each epidemiologically linked farm. The use of this risk assessment program would be a good example of information-based surveillance and support decision-making for controlling animal diseases.

Arthropods as potential vectors of African swine fever virus outbreaks in pig farms in the Republic of Korea.

The seasonality of African swine fever (ASF) in the summers and outbreaks in farms with high biosecurity levels suggest that the ASF virus (ASFV) may be transmitted by arthropod vectors. Arthropods were collected in this study from 14 pig farms with ASF outbreaks in Korea in 2019 to explore the role of arthropods as potential ASFV vectors. A total of 28,729 arthropods, including 28,508 (99.2%) Diptera, were collected using blacklight traps, insect nets and yellow sticky strips. All arthropod samples were negative for ASFV genomic DNA according to laboratory tests using real-time polymerase chain reaction. This result may reflect the effects of immediate control measures following the detection of farms with ASF outbreaks in the early phase of infection in Korea.

Epidemiological Characteristics of Foot-and-Mouth Disease in the Republic of Korea, 2014-2019.

This study describes the epidemiological characteristics of six epidemics of foot-and-mouth disease (FMD) in the Republic of Korea between 2014 and 2019. A total of 223 outbreaks had been confirmed in 40 municipalities across nine provinces. Most farms with FMD (194, 87%) were located in three densely populated livestock areas (Chungcheongnam-do, Gyeonggi-do, and Chungcheongbuk-do). More cases of FMD were found in farms with more than 1,000 pigs or 50 cattle (risk ratios = 1.27 for pigs; 9.46 for Korean native cattle) and fattening pigs. In farms affected by FMD, the proportion of animals with vaccine antibodies was low (5%-50% for Korean native beef cattle farms with FMD in 2017 vs. 97.5% in the surveillance in 2016). Effective control of FMD can be achieved through strict biosecurity measures, proper vaccination, regionalized management, and instilling awareness of FMD prevention in farmers.

Fine-scale tracking of wild waterfowl and their impact on highly pathogenic avian influenza outbreaks in the Republic of Korea, 2014-2015.

Wild migratory waterfowl are considered one of the most important reservoirs and long-distance carriers of highly pathogenic avian influenza (HPAI). Our study aimed to explore the spatial and temporal characteristics of wild migratory waterfowl's wintering habitat in the Republic of Korea (ROK) and to evaluate the impact of these habitats on the risk of HPAI outbreaks in commercial poultry farms. The habitat use of 344 wild migratory waterfowl over four migration cycles was estimated based on tracking records. The association of habitat use with HPAI H5N8 outbreaks in poultry farms was evaluated using a multilevel logistic regression model. We found that a poultry farm within a wild waterfowl habitat had a 3-8 times higher risk of HPAI outbreak than poultry farms located outside of the habitat. The range of wild waterfowl habitats increased during autumn migration, and was associated with the epidemic peak of HPAI outbreaks on domestic poultry farms in the ROK. Our findings provide a better understanding of the dynamics of HPAI infection in the wildlife-domestic poultry interface and may help to establish early detection, and cost-effective preventive measures.

Clinical symptoms of African swine fever in domestic pig farms in the Republic of Korea, 2019.

This study describes the clinical characteristics of the African swine fever (ASF) outbreaks in 14 domestic pig farms in the Republic of Korea. ASF outbreak was identified by farmers' notifications in 11 farms and by active surveillance in the remaining three. At the time of notification, farmers reported sudden death, abortion and anorexia in sows. Death was the primary symptom identified by farmers in fattener pigs. The number of animals exhibiting clinical symptoms did not exceed four heads at notification, and the number of asymptomatic virus positives was four heads per farm on average. As ASF virus was detected only in the same pig house (in a pen for fattener pigs) in each of 14 ASF outbreak farms, there has been no evidence of house-to-house viral spread within any of the ASF outbreak farms. This in turn supports our hypothesis that infection was successfully detected during its initial phase.

Viral Fitness Landscapes in Diverse Host Species Reveal Multiple Evolutionary Lines for the NS1 Gene of Influenza A Viruses.

Influenza A viruses (IAVs) have a remarkable tropism in their ability to circulate in both mammalian and avian species. The IAV NS1 protein is a multifunctional virulence factor that inhibits the type I interferon host response through a myriad of mechanisms. How NS1 has evolved to enable this remarkable property across species and its specific impact in the overall replication, pathogenicity, and host preference remain unknown. Here we analyze the NS1 evolutionary landscape and host tropism using a barcoded library of recombinant IAVs. Results show a surprisingly great variety of NS1 phenotypes according to their ability to replicate in different hosts. The IAV NS1 genes appear to have taken diverse and random evolutionary pathways within their multiple phylogenetic lineages. In summary, the high evolutionary plasticity of this viral protein underscores the ability of IAVs to adapt to multiple hosts and aids in our understanding of its global prevalence.

One-step multiplex real-time RT-PCR for detection and typing of dengue virus.

Previous studies reported that severity of dengue is associated with multiple factors, including secondary infection, age, viral load and infecting serotype and genotype. In addition, other studies have reported that a dengue virus-2 (DENV-2) infection is associated with a prognosis of more severe clinical manifestations than DENV-1 and DENV-4 infections. For these reasons, the ability to identify the DENV serotypes is critical for optimal patient diagnosis and epidemiological studies. In this study, we developed a TaqMan probe-based, one-step real-time reverse transcriptase-polymerase chain reaction (RT-PCR) system for detection and serotyping DENV. Our linear dynamic range (101 to 107 copies/reaction) showed the R2 values of DENV-1, 2, 3 and 4 as 0.998, 0.998, 0.994, and 0.998, respectively. The detection limits of DENV-1, 2, 3, and 4, were 10 copies/reaction, 100 copies/reaction, 10 copies/reaction, and 100 copies/reaction, respectively. Specificity test results indicated that this system is specific for DENV-1, 2, 3, and 4 and does not react with other viruses. Finally, we validated our results with five different real-time PCR instruments. Our results showed that the Ct values of the four serotype templates were similar in five real-time PCR instruments. Thus, this system provides an accurate method for detection and serotyping of DENV, which can be applied in diagnostics, surveillance, and epidemiology. Dengue can be found in many nations with varying socioeconomic and monetary resources. The results of our validation analyses using five different real-time PCR instruments suggest that this method can easily and confidently be used world-wide.

Distinct molecular evolution of influenza H3N2 strains in the 2016/17 season and its implications for vaccine effectiveness.

Influenza virus is a respiratory pathogen that causes seasonal epidemics by resulting in a considerable number of influenza-like illness (ILI) patients. During the 2016/17 season, ILI rates increased unusually earlier and higher than previous seasons in Korea, and most viral isolates were subtyped as H3N2 strains. Notably, the hemagglutinin (HA) of most Korean H3N2 strains retained newly introduced lysine signatures in HA antigenic sites A and D, compared with that of clade 3C.2a vaccine virus, which affected antigenic distances to the standard vaccine antisera in a hemagglutination inhibition assay. The neuraminidase (NA) of Korean H3N2 strains also harbored amino acid mutations. However, neither consistent amino acid mutations nor common phylogenetic clustering patterns were observed. These suggest that Korean H3N2 strains of the 2016/17 season might be distantly related with the vaccine virus both in genotypic and phenotypic classifications, which would adversely affect vaccine effectiveness.

Effects of Lactobacillus plantarum and Leuconostoc mesenteroides Probiotics on Human Seasonal and Avian Influenza Viruses.

Influenza viruses that cause recurrent seasonal epidemics to humans can be controlled with vaccine and antiviral therapy. However, the medical treatments often exhibit limited efficacy in the elderly or immunosuppressed individuals. In these cases, daily uptake of probiotic microbes may be an option to bring in health benefits against influenza. Here, we demonstrate the effects of probiotics Lactobacillus plantarum (Lp) and Leuconostoc mesenteroides (Lm) against seasonal and avian influenza viruses. As assessed by the plaque size reduction of human H1N1 and avian influenza H7N9 viruses, including green fluorescent protein-tagged H1N1 strain in cells, the selected Lp and Lm strains restrained viral replication in mouse lungs with statistical significance. Against lethal viral challenge, the Lp and Lm strains exhibited their beneficial effects by increasing the mean days and rates of survival of infected mice. These results suggest that, despite rather narrow ranges of protective efficacy, the dietary supplement of Lactobacillus and Leuconostoc probiotics may promote health benefits against influenza.

A Single Amino Acid in the Polymerase Acidic Protein Determines the Pathogenicity of Influenza B Viruses.

Influenza B virus (IBV) is one of the human respiratory viruses and one of the targets of seasonal vaccination. However, the bifurcation of two antigenically distinct lineages of IBVs makes it difficult to arrange proper medical countermeasures. Moreover, compared with pathogenicity-related molecular markers known for influenza A virus, little has been known for IBVs. To understand pathogenicity caused by IBVs, we investigated the molecular determinants of IBV pathogenicity in animal models. After serial lung-to-lung passages of Victoria lineage B/Brisbane/60/2008 (Vc_BR60) and Yamagata lineage B/Wisconsin/01/2010 (Ym_WI01) viruses in BALB/c mice, we identified the mouse-adapted Vc_BR60 (maVc_BR60) and Ym_WI01 (maYm_WI01) viruses, respectively. To find a molecular clue(s) to the increased pathogenicity of maVc_BR60 and maYm_WI01, we determined their genetic sequences. Several amino acid mutations were identified in the PB2, PB1, PA, BM2, and/or NS1 protein-coding regions, and one concurrent lysine (K)-to-arginine (R) mutation in PA residue 338 (PA K338R) was found in both maVc_BR60 and maYm_WI01 viruses. When analyzed using viruses rescued through reverse genetics, it was shown that PA K338R alone could increase the pathogenicity of both IBVs in mice and viral replication in the respiratory tracts of ferrets. In a subsequent minireplicon assay, the effect of PA K338R was highlighted by the enhancement of viral polymerase complex activity of both Vc_BR60 and Ym_WI01 viruses. These results suggest that the PA K338R mutation may be a molecular determinant of IBV pathogenicity via modulating the viral polymerase function of IBVs.IMPORTANCE To investigate molecular pathogenic determinants of IBVs, which are one of the targets of seasonal influenza vaccines, we adapted both Victoria and Yamagata lineage IBVs independently in mice. The recovered mouse-adapted viruses exhibited increased virulence, and of the various mutations identified from both mouse-adapted viruses, a concurrent amino acid mutation was found in the PA protein-coding region. When analyzed using viruses rescued through reverse genetics, the PA mutation alone appeared to contribute to viral pathogenicity in mice within the compatible genetic constellation between the IBV lineages and to the replication of IBVs in ferrets. Regarding the potential mechanism of increased viral pathogenicity, it was shown that the PA mutation could upregulate the viral polymerase complex activity of both IBV lineages. These results indicate that the PA mutation could be a newly defined molecular pathogenic determinant of IBVs that substantiates our understanding of the viral pathogenicity and public health risks of IBVs.

Effects of heat-killed Lactobacillus plantarum against influenza viruses in mice.

The potential use of dietary measures to treat influenza can be an important alternative for those who lack access to influenza vaccines or antiviral drugs. Lactobacillus plantarum (Lp) is one of many lactic acid bacteria that grow in 'kimchi', an essential part of Korean meal, and several strains of Lp reportedly show protective effects against influenza. Using heat-killed Lp (nF1) isolated from kimchi, which is known for its immunomodulatory effects, we investigated whether regular oral intake of nF1 could influence the outcome of influenza virus infection in a mouse model. In a lethal challenge with influenza A (H1N1 and H3N2 subtypes) and influenza B (Yamagata lineage) viruses, daily oral administration of nF1 delayed the mean number of days to death of the infected mice and resulted in increased survival rates compared with those of the non-treated mice. Consistent with these observations, nF1 treatment also significantly reduced viral replication in the lungs of the infected mice. Taken together, our results might suggest the remedial potential of heatkilled Lactobacillus probiotics against influenza.

Towards the Application of Human Defensins as Antivirals.

Defensins are antimicrobial peptides that participate in the innate immunity of hosts. Humans constitutively and/or inducibly express α- and ß-defensins, which are known for their antiviral and antibacterial activities. This review describes the application of human defensins. We discuss the extant experimental results, limited though they are, to consider the potential applicability of human defensins as antiviral agents. Given their antiviral effects, we propose that basic research be conducted on human defensins that focuses on RNA viruses, such as human immunodeficiency virus (HIV), influenza A virus (IAV), respiratory syncytial virus (RSV), and dengue virus (DENV), which are considered serious human pathogens but have posed huge challenges for vaccine development for different reasons. Concerning the prophylactic and therapeutic applications of defensins, we then discuss the applicability of human defensins as antivirals that has been demonstrated in reports using animal models. Finally, we discuss the potential adjuvant-like activity of human defensins and propose an exploration of the 'defensin vaccine' concept to prime the body with a controlled supply of human defensins. In sum, we suggest a conceptual framework to achieve the practical application of human defensins to combat viral infections.

Adaptive mutations of neuraminidase stalk truncation and deglycosylation confer enhanced pathogenicity of influenza A viruses.

It has been noticed that neuraminidase (NA) stalk truncation has arisen from evolutionary adaptation of avian influenza A viruses (IAVs) from wild aquatic birds to domestic poultry. We identified this molecular alteration after the adaptation of a 2009 pandemic H1N1 virus (pH1N1) in BALB/c mice. The mouse-adapted pH1N1 lost its eight consecutive amino acids including one potential N-linked glycosite from the NA stalk region. To explore the relationship of NA stalk truncation or deglycosylation with viral pathogenicity changes, we generated NA stalk mutant viruses on the pH1N1 backbone by reverse genetics. Intriguingly, either NA stalk truncation or deglycosylation changed pH1N1 into a lethal virus to mice by resulting in extensive pathologic transformation in the mouse lungs and systemic infection affecting beyond the respiratory organs in mice. The increased pathogenicity of these NA stalk mutants was also reproduced in ferrets. In further investigation using a human-infecting H7N9 avian IAV strain, NA stalk truncation or deglycosylation enhanced the replication property and pathogenicity of H7N9 NA stalk mutant viruses in the same mouse model. Taken together, our results suggest that NA stalk truncation or deglycosylation can be the pathogenic determinants of seasonal influenza viruses associated with the evolutionary adaptation of IAVs.

Evolutionary relationships of the hexon and penton base genes of novel squirrel adenovirus.

Squirrel adenovirus (SqAdV) was reported previously. However, only partial sequences of its hexon and polymerase genes have been revealed. For the first time, we report the full-length genome of SqAdV including the complete hexon and penton base genes. From internal body organs of 59 red squirrels archived in Korea Bank for Pathogenic Viruses, the hexon, penton base, and full-length genome of SqAdV were determined by a PCR method. Of the internal body organs examined, the spleen showed the highest detection rate (25.42%) for SqAdV whereas the kidney and lung exhibited 18.64% and 3.39% rates, respectively. Based on the phylogenetic relationships of the hexon and penton base genes, SqAdV appears to belong to the genus Mastadenovirus, and, at least in our study, the hexon of SqAdV exhibits the closest relationship to that of an alpaca AdV. Compared with the hexon, the penton base of SqAdV appears to be genetically more divergent from that of other mastadenoviruses. It was also revealed that the full-length SqAdV genome retained AT nucleotide content similar level to AT-rich atadenoviruses, which is unusual for mastadenoviruses. Our results emphasize that SqAdV is classified into the genus Mastadenovirus and demonstrate the AT-biased nucleotide constitution of SqAdV.

Phylogenetic relationships of the HA and NA genes between vaccine and seasonal influenza A(H3N2) strains in Korea.

Seasonal influenza is caused by two influenza A subtype (H1N1 and H3N2) and two influenza B lineage (Victoria and Yamagata) viruses. Of these antigenically distinct viruses, the H3N2 virus was consistently detected in substantial proportions in Korea during the 2010/11-2013/14 seasons when compared to the other viruses and appeared responsible for the influenza-like illness rate peak during the first half of the 2011/12 season. To further scrutinize possible causes for this, we investigated the evolutionary and serological relationships between the vaccine and Korean H3N2 strains during the 2011/12 season for the main antigenic determinants of influenza viruses, the hemagglutinin (HA) and neuraminidase (NA) genes. In the 2011/12 season, when the number of H3N2 cases peaked, the majority of the Korean strains did not belong to the HA clade of A/Perth/16/2009 vaccine, and no Korean strains were of this lineage in the NA segment. In a serological assay, post-vaccinated human sera exhibited much reduced hemagglutination inhibition antibody titers against the non-vaccine clade Korean H3N2 strains. Moreover, Korean strains harbored several amino acid differences in the HA antigenic sites and in the NA with respect to vaccine lineages during this season. Of these, the HA antigenic site C residues 45 and 261 and the NA residue 81 appeared to be the signatures of positive selection. In subsequent seasons, when H3N2 cases were lower, the HA and NA genes of vaccine and Korean strains were more phylogenetically related to each other. Combined, our results provide indirect support for using phylogenetic clustering patterns of the HA and possibly also the NA genes in the selection of vaccine viruses and the assessment of vaccine effectiveness.

Single PA mutation as a high yield determinant of avian influenza vaccines.

Human infection with an avian influenza virus persists. To prepare for a potential outbreak of avian influenza, we constructed a candidate vaccine virus (CVV) containing hemagglutinin (HA) and neuraminidase (NA) genes of a H5N1 virus and evaluated its antigenic stability after serial passaging in embryonated chicken eggs. The passaged CVV harbored the four amino acid mutations (R136K in PB2; E31K in PA; A172T in HA; and R80Q in M2) without changing its antigenicity, compared with the parental CVV. Notably, the passaged CVV exhibited much greater replication property both in eggs and in Madin-Darby canine kidney and Vero cells. Of the four mutations, the PA E31K showed the greatest effect on the replication property of reverse genetically-rescued viruses. In a further luciferase reporter, mini-replicon assay, the PA mutation appeared to affect the replication property by increasing viral polymerase activity. When applied to different avian influenza CVVs (H7N9 and H9N2 subtypes), the PA E31K mutation resulted in the increases of viral replication in the Vero cell again. Taken all together, our results suggest the PA E31K mutation as a single, substantial growth determinant of avian influenza CVVs and for the establishment of a high-yield avian influenza vaccine backbone.

Original Antigenic Sin Response to RNA Viruses and Antiviral Immunity.

The human immune system has evolved to fight against foreign pathogens. It plays a central role in the body's defense mechanism. However, the immune memory geared to fight off a previously recognized pathogen, tends to remember an original form of the pathogen when a variant form subsequently invades. This has been termed 'original antigenic sin'. This adverse immunological effect can alter vaccine effectiveness and sometimes cause enhanced pathogenicity or additional inflammatory responses, according to the type of pathogen and the circumstances of infection. Here we aim to give a simplified conceptual understanding of virus infection and original antigenic sin by comparing and contrasting the two examples of recurring infections such as influenza and dengue viruses in humans.

Effects of HA and NA glycosylation pattern changes on the transmission of avian influenza A(H7N9) virus in guinea pigs.

Avian influenza H7N9 virus has posed a concern of potential human-to-human transmission by resulting in seasonal virus-like human infection cases. To address the issue of sustained human infection with the H7N9 virus, here we investigated the effects of hemagglutinin (HA) and neuraminidase (NA) N-linked glycosylation (NLG) patterns on influenza virus transmission in a guinea pig model. Based on the NLG signatures identified in the HA and NA genetic sequences of H7N9 viruses, we generated NLG mutant viruses using either HA or NA gene of a H7N9 virus, A/Anhui/01/2013, by reverse genetics on the 2009 pandemic H1N1 virus backbone. For the H7 HA NLG mutant viruses, NLG pattern changes appeared to reduce viral transmissibility in guinea pigs. Intriguingly, however, the NLG changes in the N9 NA protein, such as a removal from residue 42 or 66 or an addition at residue 266, increased transmissibility of the mutant viruses by more than 33%, 50%, and 16%, respectively, compared with a parental N9 virus. Given the effects of HA-NA NLG changes with regard to viral transmission, we then generated the HA-NA NLG mutant viruses harboring the H7 HA of double NLG addition and the N9 NA of various NLG patterns. As seen in the HA NLG mutants above, the double NLG-added H7 HA decreased viral transmissibility. However, when the NA NLG changes occurred by a removal of residue 66 and an addition at 266 were additionally accompanied, the HA-NA NLG mutant virus recovered the transmissibility of its parental virus. These demonstrate the effects of specific HA-NA NLG changes on the H7N9 virus transmission by highlighting the importance of a HA-NA functional balance.

Reassortment compatibility between PB1, PB2, and HA genes of the two influenza B virus lineages in mammalian cells.

In addition to influenza A subtypes, two distinct lineages of influenza B virus also cause seasonal epidemics to humans. Recently, Dudas et al. have done evolutionary analyses of reassortment patterns of the virus and suggested genetic lineage relationship between PB1, PB2, and HA genes. Using genetic plasmids and reassortant viruses, we here demonstrate that a homologous lineage PB1-PB2 pair exhibits better compatibility than a heterologous one and that the lineage relationship between PB1 and HA is more important for viral replication than that between PB2 and HA. However, co-adaptation of PB1-PB2-HA genes appears to be affected by complete gene constellation.