Isolation and characterization of low pathogenic H7N7 avian influenza virus from a red-crowned crane in a zoo in South Korea.

BACKGROUND: South Korea conducts annual national surveillance programs to detect avian influenza (AI) in domestic poultry, live bird markets, and wild birds. In March 2017, an AIV was isolated from fecal samples in an outdoor aviary flight cage in a zoo in Korea. RESULTS: Nucleotide sequencing identified the isolate as low pathogenic avian influenza virus (LPAIV) H7N7, and DNA barcoding analysis identified the host species as red-crowned crane. This isolate was designated A/red-crowned crane/Korea/H1026/2017 (H7N7). Genetic analysis and gene constellation analysis revealed that A/red-crowned crane/Korea/H1026/2017 (H7N7) showed high similarity with four H7N7 LPAIVs isolated from wild bird habitats in Seoul and Gyeonggi in early 2017. CONCLUSIONS: Considering the genetic similarity and similar collection dates of the viruses, and the fact that zoo bird cages are vulnerable to AIV, it is likely that fecal contamination from wild birds might have introduced LPAIV H7N7 into the red-crowned crane at the zoo. Therefore, our results emphasize that enhanced biosecurity measures should be employed during the wild bird migration season, and that continued surveillance should be undertaken to prevent potential threats to avian species in zoos and to humans.

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.

Pathogenicity and genetic characterisation of a novel reassortant, highly pathogenic avian influenza (HPAI) H5N6 virus isolated in Korea, 2017.

We investigated influenza A(H5N6) viruses from migratory birds in Chungnam and Gyeonggi Provinces, South Korea following a reported die-off of poultry in nearby provinces in November 2017. Genetic analysis and virulence studies in chickens and ducks identified our isolate from December 2017 as a novel highly pathogenic avian influenza virus. It resulted from reassortment between the highly virulent H5N8 strain from Korea with the N6 gene from a low-pathogenic H3N6 virus from the Netherlands.

Experimental infection of cows with newly isolated Akabane virus strain (AKAV-7) causing encephalomyelitis.

Akabane virus (AKAV), an arthropod-transmitted bunyavirus, is a major cause of congenital abnormalities and encephalomyelitis in ruminants. In 2010, there was a major outbreak of encephalomyelitis in Korea and fifteen AKAV strains, including AKAV-7, were isolated from cows. To identify the neuropathogenicity of AKAV-7, we performed experimental infection of cows. Six-month-old female Korean Holstein dairy cattle were inoculated with AKAV-7 by various routes, including intracerebral (IC), intrasubarachnoid space (IS), subcutaneous (SC) and intravenous (IV); a separate group was vaccinated before intravenous infection. Five of the six cows in the IC group and two of the six cows in the IS group showed clinical signs such as locomotor ataxia and paralysis of the hind limbs. Three of six cows died after IC infection 9-12 days post infection (dpi). Histopathologic changes such as nonsuppurative encephalomyelitis were confirmed in various parts of the central nervous system in the IC, IS and SC groups. Early onset of neutralizing antibodies in the serum and lower viral mRNA levels in the peripheral blood mononuclear cells (PBMCs) and various tissues in the vaccinated group was noticeable compared to the unvaccinated group (IV group). We suggest that the AKAV vaccine currently used in Korea may be partially effective for protection against AKAV-7 in cows.

Genetic characterization of porcine epidemic diarrhea virus in Korea from 1998 to 2013.

The number of porcine epidemic diarrhea (PED) cases has increased over the past 20 years in Korea, with a major outbreak in 2013. A total of 27 Korean strains from 1998 to 2013 were analyzed (excluding the noncoding regions) and divided into two groups for comparison of the spike (S), ORF3, envelope (E), membrane (M), and nucleocapsid (N) genes with those of reference strains, vaccine strains, and previously identified strains based on phylogenetic analysis. Analysis of the selection patterns of PEDV isolated in Korea indicated positive selection of nine nonsynonymous sites in the S and N proteins and negative selection at 97 sites for all of the proteins. Interestingly, eight nonsynonymous mutations in S showed no significant pattern change over the 15-year period, and one of eight mutation sites was found only in IC05TK, GN05DJ, and KNU0802 in the epidemic years 2005 and 2008. These eight mutations were also present during the epidemic years in China. Furthermore, of the signs of positive selection in the S protein, the conservative substitutions were more frequent than radical substitutions in PEDVs, suggesting that the evolution of Korean strains has been slow. Serological cross-reactivity was detected between three field PEDVs and two vaccine strains, with different serum neutralization titers. In conclusion, although Korean PEDVs have been evolving slowly, their diverse antigenicity and genetics imply that multilateral efforts to prevent future PED outbreaks are required.

Genetic characteristics of the complete feline kobuvirus genome.

We sequenced the complete genome of a feline kobuvirus and determined relationships with other kobuviruses. This kobuvirus has an 8,269-nucleotide-long RNA genome, excluding the poly(A) tail. The genome contains a 7,311-bp open reading frame (ORF) encoding a putative polyprotein precursor of 2,437 amino acids, a 717-bp 5'-untranslated region (UTR), and a 241-bp 3'-UTR. The L protein sequence was found to be the most variable region in the feline kobuvirus genome. Interestingly, the 5'-UTR B and C stem-loops were conserved as observed with other kobuviruses; however, a secondary structure corresponding to stem-loop A was not found in the full length 5'-UTR sequence. Phylogenetic tree analysis showed that kobuviruses can be divided into 3 main groups. The feline kobuvirus belongs to the Aichivirus A species containing Aichivirus, mouse kobuvirus, and canine kobuvirus.

Analysis of signs and pathology of H5N1-infected ducks from the 2010-2011 Korean highly pathogenic avian influenza outbreak suggests the influence of age and management practices on severity of disease.

We compared the clinical signs, histopathological lesions and distribution of viral antigens among infected young (meat-type) and older (breeder) ducks that were naturally infected with the highly pathogenic avian influenza (HPAI) virus during the 2010-2011 Korean outbreak. The meat-type ducks had a high mortality rate (30%) and showed severe neurological signs such as head tremors and paresis. In contrast, HPAI-infected breeder ducks had minimal clinical signs but a decreased egg production rate. The histopathological characteristics of infected meat-type ducks included necrotic lesions of heart and brain, which may have primarily contributed to the high mortality rate. In contrast, the breeder ducks only presented necrotic splenitis, and viral antigens were only detected in the trachea, lungs and spleen. Younger ducks had a high viral titre in the organs, high levels of viral shedding and a high mortality rate after experimental HPAI virus infection. Compared to the breeder ducks, the meat-type ducks were raised in smaller farms that had poor quarantine and breeding facilities. It is therefore possible that better biosecurity in the breeder farms could have reduced the infection dose and subsequently the severity of the disease. Thus, age and management may be the influencing factors for HPAI susceptibility in ducks.

Canine kobuvirus infections in Korean dogs.

To investigate canine kobuvirus (CaKoV) infection, fecal samples (n = 59) were collected from dogs with or without diarrhea (n = 21 and 38, respectively) in the Republic of Korea (ROK) in 2012. CaKoV infection was detected in four diarrheic samples (19.0 %) and five non-diarrheic samples (13.2 %). All CaKoV-positive dogs with diarrhea were found to be infected in mixed infections with canine distemper virus and canine parvovirus or canine adenovirus. There was no significant difference in the prevalence of CaKoV in dogs with and without diarrhea. By phylogenetic analysis based on partial 3D genes and complete genome sequences, the Korean isolates were found to be closely related to each other regardless of whether they were associated with diarrhea, and to the canine kobuviruses identified in the USA and UK. This study supports the conclusion that CaKoVs from different countries are not restricted geographically and belong to a single lineage.

Evaluation of changes induced by temperature, contact time, and surface in the efficacies of disinfectants against avian influenza virus.

Avian influenza viruses (AIV) are highly susceptible to all disinfectants because they are enveloped viruses. Disinfectants effective against AIV have optimum efficacies at temperatures above 20°C. Very few studies on effective disinfectants at low temperatures have been done. Disinfectants were investigated at 4 different temperatures (25, 4, 0, and -10°C) and 2 contact times (1 and 5 min) with suspension tests. Virucidal activity of the disinfectants was evaluated by carrier tests (wood and stainless steel) at 25 and -10°C. The concentration of each disinfectant for efficient disinfection within a short time (<1 min) at 25 and -10°C was also reestablished. The results from the suspension test indicated that low temperatures inhibited the virucidal efficacy of citric acid (CA) and CA + quaternary ammonium compounds (CA+ QAC) for 1 and 5 min, whereas the remaining disinfectants were effective, regardless of the short contact times and low temperatures. The carrier test results suggested that dried virus on wood was more difficult to inactivate compared with that on stainless steel. However, sodium dichloroisocyanurate and glutaraldehyde could inactivate AIV on both wood and stainless steel at -10°C. Citric acid-based agents could not sufficiently inactivate AIV at -10°C; however, the limitation due to low temperatures was overcome by adjusting disinfectant concentration. For a successful disinfection during winter, the disinfectants that could have short contact times with optimum efficacy against the target organism should be selected.

Serological characteristics of affected cattle during an outbreak of bovine enzootic encephalomyelitis caused by Akabane virus.

During an outbreak of bovine enzootic encephalomyelitis caused by the Akabane virus (AKAV) in 2010, 210 serum samples were collected from the affected cattle, and serological investigations for the AKAV were performed using a serum neutralization test (SNT) and an enzyme-linked immunosorbent assay (ELISA). The seropositive rates for SNT and ELISA were 90.0 and 85.2 %, respectively. The titers of SNT (log2) against the AKAV were higher than 4.0 in the highly affected cattle (80.0 %). This finding indicates that most affected cattle were infected with the AKAV and that strong immune responses against this virus were elicited in affected cattle. The strong immune response to the AKAV in cattle may provide insight into the occurrence of bovine encephalomyelitis caused by the AKAV.

Canine adenovirus type 1 in a fennec fox (Vulpes zerda).

A 10-mo-old female fennec fox (Vulpes zerda) with drooling suddenly died and was examined postmortem. Histologic examination of different tissue samples was performed. Vacuolar degeneration and diffuse fatty change were observed in the liver. Several diagnostic methods were used to screen for canine parvovirus, canine distemper virus, canine influenza virus, canine coronavirus, canine parainfluenza virus, and canine adenovirus (CAdV). Only CAdV type 1 (CAdV-1) was detected in several organs (liver, lung, brain, kidney, spleen, and heart), and other viruses were not found. CAdV-1 was confirmed by virus isolation and nucleotide sequencing.

Complete genome analysis of three live attenuated Rinderpest virus vaccine strains derived through serial passages in different culture systems.

The genomes of three South Korean Rinderpest virus vaccine strains (L72, LA77, and LA96) were analyzed in order to investigate their genetic variability. These three vaccine strains were all derived from the same virus strain origin (Fusan) through repeated passages in different culture systems. The full genome length of the three strains was 15,882 nucleotides, and the sequence similarity between the three South Korean RPV strains at the nucleotide level was 98.1 to 98.9%. The genetic distance between Nakamura III, L72, LA77, LA96, and LATC06 and the Kabete strain was greater than that between the Fusan and Kabete strains for the P, V, and C genes. The difference in pathogenicity among these strains might be due to the V gene, which has a positive (>1) selection ratio based on the analysis of synonymous (dS) and nonsynonymous (dN) substitution rates (dN/dS ratio [ω]).

Isolation of influenza A(H3N2)v virus from pigs and characterization of its biological properties in pigs and mice.

Recently, a novel reassortant virus, influenza A(H3N2)v [A(H3N2)v], was identified as the causative pathogen in 307 human cases of influenza in the United States. A(H3N2)v contains the matrix gene from the 2009 pandemic H1N1 (pH1N1) virus, while its other genes originate from H3N2 viruses with triple-reassorted internal genes. In this study, we isolated three A(H3N2)v viruses from commercial pigs in Korea that showed similarities with published human A(H3N2)v viruses in eight segment sequence alignments. After genetic characterization, the pathogenicity of one of these viruses was assessed in pigs and mice. Infection of pigs with this novel virus resulted in mild interstitial pneumonia with marked oronasal shedding of viral RNA for about 14 days. In mice, the virus replicated efficiently in the lungs; viral RNA was detected up to 9 days post-inoculation. However, the virus did not cause severe disease or death in mice, despite the administration of a high infectious dose (10(5.2) TCID50). This study demonstrates that A(H3N2)v causes a high morbidity rate with low virulence; however, global monitoring of A(H3N2)v outbreaks in mammals will be needed to determine whether this novel subtype will shift to a highly pathogenic virus.

Complete genome sequences of three rabies viruses isolated from rabid raccoon dogs and a cow in Korea.

The complete genomes of three rabies viruses (BD0406CC, BV9901PJ, and 08F40) of two raccoon dogs (Nyctereutes procyonoides koreensis) and a cow were determined. The genomic organization is typical of rabies viruses, and the open reading frames of the N, P, M, G, and L genes are 1,353, 894, 609, 1,575, and 6,384 bases in length, respectively. The full genome length of the three strains was 11,928 nucleotides, and the sequence similarity between the rabies viruses at the nucleotide level was 98.5-99.5%. Sequence comparisons indicated that these rabies viruses belong to the "Arctic and Arctic-like" group, with high homology to the Eurasian cluster. All Korean strains were clustered with the Mongolia strains, which belong to Arctic-like 1 clade. The 08F40 and BD0406CC strains were constructed with rabies virus strains isolated in Gangwon province. The BV9901PJ strain was closely related to strains isolated in Gyeonggi province in Korea. Three strains were more dependent upon geographical distribution and time period than host species. Complete genome sequencing of different host-origin rabies viruses will provide information that should contribute to understanding the transmission cycle and genetic variability of rabies from different hosts.

Detection and genetic characterization of feline kobuviruses.

In order to survey for feline kobuviruses infection, fecal samples (n = 39) of cats with diarrhea were collected during 2011-2012. Six (14.5%) of the fecal samples tested were positive for feline kobuviruses. The partial nucleotide sequences of feline kobuviruses based on the RNA-dependent RNA polymerase gene were compared to those of other species. Feline kobuviruses were most closely related to canine kobuvirus in terms of their amino acid and nucleotide levels. In a phylogenetic tree, feline kobuviruses were also closely clustered with canine kobuvirus, Aichi virus (human), and mouse kobuvirus. This is the first report of the detection and genetic characterization of feline kobuviruses.

First isolation of Clostridium perfringens type E from a goat with diarrhea.

A 2-day-old goat died suddenly after the onset of severe diarrhea. No specific gross lesions were observed except for a remarkably thin intestinal wall and watery intestinal contents. Histopathological analysis revealed large numbers of Gram-positive bacilli layered upon the intestinal epithelia of the small intestine. Heavy growth of only Clostridium perfringens type E, and no detection of the other enteric pathogens in the small intestine, suggests that C. perfringens type E contributed to the death of this kid. To our knowledge, this is the first isolation of C. perfringens type E from a goat with diarrhea.

Chemical stability of active ingredients in diluted veterinary disinfectant solutions under simulated storage conditions.

Introduction: The product labels of veterinary disinfectants specify their expiration dates to prevent the use of outdated products, as these may result in disinfection and biosecurity failures during outbreak situations. However, a clear standard for the storage conditions of diluted disinfectant solutions has not yet been established, and the effects of storage conditions have scarcely been investigated. To fill this research gap, our study examined the stability of the active ingredients of diluted veterinary disinfectants based on their change in concentrations when stored at various temperatures for various time periods. Methods: Twenty veterinary disinfectants effective against either foot-and-mouth disease or avian influenza viruses were selected. The disinfectants were diluted to effective concentrations following the manufacturer's instructions. Using selective analytical techniques, the concentrations of the active ingredients of the samples that had been stored for varying intervals at different temperatures (4, 20, 30, and 45°C) were determined. These samples included soaps and detergents, acids, oxidizing agents, aldehydes, and copper compounds. The active ingredient concentrations of two of the samples were determined following freezing/thawing cycle, to establish their stability when exposed to simulated winter conditions. Results: Our results showed that most of the active ingredients had concentrations of 90% or greater of their initial concentrations, indicating ≥90% stability over a 21-day period under the experimental storage conditions. However, there were some exceptions. Glutaraldehyde, formaldehyde, and malic acid are over 90% stable at ≤ 30°C for 21 days, but their concentrations decreased to below 90% of their initial concentrations at 45°C, indicating a decline in stability when stored at 45°C for 21 days. The concentrations of potassium peroxymonosulfate and peracetic acid rapidly declined with increasing time and temperature to less than 90% of their initial concentrations. Discussion: Based on our findings, we propose that diluted disinfectant solutions should preferably be prepared daily. However, if the daily preparation of a diluted disinfectant solution is not feasible, then our results can be used as a reference, providing basic scientific data on the chemical stability of diluted disinfectant solutions commonly used in the veterinary field, thus indicating suitable storage conditions.

Kobuvirus in South Korean black goats.

Kobuviruses have been detected in humans and several animal species, including cattle, swine, sheep, canines, mice, and probably bats. While investigating the possibility of Kobuviruses infecting additional animal host species, we detected kobuvirus in three fecal samples from domestic Korean black goats. In a maximum parsimony tree and a Bayesian tree, the 08KG680 strain fell within the bovine kobuvirus lineage, but the 09KG172 and 10KG056 strains did not fall within any of the known animal kobuvirus lineages. Comparative analysis of the partial nucleotide sequences of the RNA-dependent RNA polymerase (RdRp) gene of the 08KG680 strain also revealed high amino acid sequence identity and a close genetic relationship with bovine kobuvirus, but the amino acid sequences of the other two strains had low similarity to those of known kobuvirus isolates from any animal species. The similarity of the sequence of the 08KG680 strains with the bovine kobuvirus indicate that the infectious may have originated from cattle, but the possible source for remaining strains could not be classified.

Prevalence of Korean cats with natural feline coronavirus infections.

BACKGROUND: Feline coronavirus is comprised of two pathogenic biotypes consisting of feline infectious peritonitis virus (FIPV) and feline enteric coronavirus (FECV), which are both divided into two serotypes. To examine the prevalence of Korean cats infected with feline coronavirus (FCoV) type I and II, fecal samples were obtained from 212 cats (107 pet and 105 feral) in 2009. RESULTS: Fourteen cats were FCoV-positive, including infections with type I FCoV (n = 8), type II FCoV (n = 4), and types I and II co-infection (n = 2). Low seroprevalences (13.7%, 29/212) of FCoV were identified in chronically ill cats (19.3%, 16/83) and healthy cats (10.1%, 13/129). CONCLUSIONS: Although the prevalence of FCoV infection was not high in comparison to other countries, there was a higher prevalence of type I FCoV in Korean felines. The prevalence of FCoV antigen and antibody in Korean cats are expected to gradually increase due to the rising numbers of stray and companion cats.

Development of a recombinant H9N2 influenza vaccine candidate against the Y280 lineage field virus and its protective efficacy.

Since June 2020, a new H9N2 virus of the Y280 lineage has been epidemic in Korea. Initially, a Korean commercial vaccine against the Y280 and Y439 lineages of H9N2 was evaluated for use in SPF chickens. A single vaccination did not protect chickens against virus of the Y280 lineage, with no significant reduction in virus shedding and a 37.5% inhibition in virus recovery rate in cecal tonsil. rgHS314 was selected as a vaccine candidate, showing immunogenicity in SPF chickens, and was highly productive in eggs. Moreover, rgHS314 protected with high levels of protective immunity and significantly reduced virus shedding, with 100% and 83.3% inhibition of virus recovery in the cecal tonsil against homologous and heterologous challenge viruses, respectively. Taken together, these data suggest that a single vaccination with this recombinant vaccine candidate could elicit cross-reactive immune responses capable of protecting chickens against H9N2 viruses of the Y439 and Y280 lineages.