Genetic Characteristics of Coronaviruses from Korean Bats in 2016.

Bats have increasingly been recognized as the natural reservoir of severe acute respiratory syndrome (SARS), coronavirus, and other coronaviruses found in mammals. However, little research has been conducted on bat coronaviruses in South Korea. In this study, bat samples (332 oral swabs, 245 fecal samples, 38 urine samples, and 57 bat carcasses) were collected at 33 natural bat habitat sites in South Korea. RT-PCR and sequencing were performed for specific coronavirus genes to identify the bat coronaviruses in different bat samples. Coronaviruses were detected in 2.7% (18/672) of the samples: 13 oral swabs from one species of the family Rhinolophidae, and four fecal samples and one carcass (intestine) from three species of the family Vespertiliodae. To determine the genetic relationships of the 18 sequences obtained in this study and previously known coronaviruses, the nucleotide sequences of a 392-nt region of the RNA-dependent RNA polymerase (RdRp) gene were analyzed phylogenetically. Thirteen sequences belonging to SARS-like betacoronaviruses showed the highest nucleotide identity (97.1-99.7%) with Bat-CoV-JTMC15 reported in China. The other five sequences were most similar to MERS-like betacoronaviruses. Four nucleotide sequences displayed the highest identity (94.1-95.1%) with Bat-CoV-HKU5 from Hong Kong. The one sequence from a carcass showed the highest nucleotide identity (99%) with Bat-CoV-SC2013 from China. These results suggest that careful surveillance of coronaviruses from bats should be continued, because animal and human infections may result from the genetic variants present in bat coronavirus reservoirs.

Complete genome sequence of a novel avian paramyxovirus isolated from wild birds in South Korea.

A novel avian paramyxovirus (APMV), Cheonsu1510, was isolated from wild bird feces in South Korea and serologically and genetically characterized. In hemagglutination inhibition tests, antiserum against Cheonsu1510 showed low reactivity with other APMVs and vice versa. The complete genome of Cheonsu1510 comprised 15,408 nucleotides, contained six open reading frames (3'-N-P-M-F-HN-L-5'), and showed low sequence identity to other APMVs (< 63%) and a unique genomic composition. Phylogenetic analysis revealed that Cheonsu1510 was related to but distinct from APMV-1, -9, and -15. These results suggest that Cheonsu1510 represents a new APMV serotype, APMV-17.

Identification of Two novel reassortant avian influenza a (H5N6) viruses in whooper swans in Korea, 2016.

BACKGROUND: On November 20, 2016 two novel strains of H5N6 highly pathogenic avian influenza virus (HPAIVs) were isolated from three whooper swans (Cygnus cygnus) at Gangjin Bay in South Jeolla province, South Korea. Identification of HPAIVs in wild birds is significant as there is a potential risk of transmission of these viruses to poultry and humans. RESULTS: Phylogenetic analysis revealed that Gangjin H5N6 viruses classified into Asian H5 clade 2.3.4.4 lineage and were distinguishable from H5N8 and H5N1 HPAIVs previously isolated in Korea. With the exception of the polymerase acidic (PA) gene, the viruses were most closely related to A/duck/Guangdong/01.01SZSGXJK005-Y/2016 (H5N6) (98.90 ~ 99.74%). The PA genes of the two novel Gangjin H5N6 viruses were most closely related to AIV isolates previously characterized from Korea, A/hooded crane/Korea/1176/2016 (H1N1) (99.16%) and A/environment/Korea/W133/2006 (H7N7) (98.65%). The lack of more recent viruses to A/environment/Korea/W133/2006 (H7N7) indicates the need for analysis of recent wild bird AIVs isolated in Korea because they might provide further clues as to the origin of these novel reassortant H5N6 viruses. CONCLUSIONS: Although research on the origins and epidemiology of these infections is ongoing, the most likely route of infection for the whooper swans was through direct or indirect contact with reassortant viruses shed by migratory wild birds in Korea. As H5N6 HPAIVs can potentially be transmitted to poultry and humans, continuous monitoring of AIVs among wild birds will help to mitigate this risk.

Novel reassortant clade 2.3.4.4 avian influenza A (H5N8) virus in a grey heron in South Korea in 2017.

We report the identification of a novel reassortant clade 2.3.4.4 H5N8 virus from a dead grey heron in Korea in 2017. Outbreaks of clade 2.3.4.4 H5 HPAIVs have been reported worldwide, and they have evolved into multiple genotypes among wild birds. Phylogenetic analysis indicated that this virus likely originated from Qinghai Lake and Western Siberia and further evolved through reassortment with Eurasian LPAI during the 2016 fall migration of wild birds. Enhanced surveillance and comparative genetic analysis will help to monitor the further evolution and dissemination of clade 2.3.4.4 HPAIVs.

Novel reassortant influenza A(H5N8) viruses among inoculated domestic and wild ducks, South Korea, 2014.

An outbreak of highly pathogenic avian influenza, caused by a novel reassortant influenza A (H5N8) virus, occurred among poultry and wild birds in South Korea in 2014. The aim of this study was to evaluate the pathogenesis in and mode of transmission of this virus among domestic and wild ducks. Three of the viruses had similar pathogenicity among infected domestic ducks: the H5N8 viruses were moderately pathogenic (0%-20% mortality rate); in wild mallard ducks, the H5N8 and H5N1 viruses did not cause severe illness or death; viral replication and shedding were greater in H5N8-infected mallards than in H5N1-infected mallards. Identification of H5N8 viruses in birds exposed to infected domestic ducks and mallards indicated that the viruses could spread by contact. We propose active surveillance to support prevention of the spread of this virus among wild birds and poultry, especially domestic ducks.

Genetic evolution of H5 highly pathogenic avian influenza virus in domestic poultry in Vietnam between 2011 and 2013.

In spite of highly pathogenic avian influenza H5N1 vaccination campaigns for domestic poultry, H5N1 viruses continue to circulate in Vietnam. To estimate the prevalence of avian influenza virus in Vietnam, surveillance was conducted between November 2011 and February 2013. Genetic analysis of 312 highly pathogenic avian influenza H5 viruses isolated from poultry in Vietnam was conducted and possible genetic relationships with strains from neighboring countries were investigated. As previously reported, phylogenetic analysis of the avian influenza virus revealed two H5N1 HPAI clades that were circulating in Vietnam. Clade 1.1, related to Cambodian strains, was predominant in the southern provinces, while clade 2.3.2.1 viruses were predominant in the northern and central provinces. Sequence analysis revealed evidence of active genetic evolution. In the gene constellation of clade 2.3.2.1, genotypes A, B, and B(II) existed during the 2011/2012 winter season. In June 2012, new genotype C emerged by reassortment between genotype A and genotype B(II), and this genotype was predominant in 2013 in the northern and central provinces. Interestingly, enzootic Vietnamese clade 2.3.2.1C H5 virus subsequently reassorted with N2, which originated from wild birds, to generate H5N2 highly pathogenic avian influenza, which was isolated from duck in the northeast region. This investigation indicated that H5N1 outbreaks persist in Vietnam and cause genetic reassortment with circulating viruses. It is necessary to strengthen active influenza surveillance to eradicate highly pathogenic avian influenza viruses and sever the link between highly pathogenic avian influenza and other circulating influenza viruses.

Downregulation of chicken interleukin-17 receptor A during Eimeria infection.

Both interleukin-17A (IL-17A) and IL-17F are proinflammatory cytokines that have an important role in intestinal homeostasis via receptor signaling. These cytokines have been characterized in chickens, but very little is known about their receptors and their functional activity. We provide here the first description of the sequence analysis, bioactivity, and comparative expression analysis of chicken IL-17RA (chIL-17RA) in chickens infected with Salmonella and Eimeria, two major infectious agents of gastrointestinal diseases of poultry of economic importance. A full-length chIL-17RA cDNA with a 2,568-bp coding region was identified from chicken thymus cDNA. chIL-17RA shares ca. 46% identity with mammalian homologues and 29.2 to 31.5% identity with its piscine counterparts. chIL-17RA transcript expression was relatively high in the thymus and in the chicken macrophage cell line HD11. The chIL-17RA-specific small interfering RNA inhibits interleukin-6 (IL-6), IL-8, and IL-1ß mRNA expression in chicken embryo fibroblast cells (but not in DF-1 cells) stimulated with chIL-17A or chIL-17F. Interaction between chIL-17RA and chIL-17A was confirmed by coimmunoprecipitation. Downregulation of chIL-17RA occurred in concanavalin A- or lipopolysaccharide-activated splenic lymphocytes but not in poly(I·C)-activated splenic lymphocytes. In Salmonella- and Eimeria-infected chickens, the expression levels of the chIL-17RA transcript were downregulated in intestinal tissues from chickens infected with two Eimeria species, E. tenella or E. maxima, that preferentially infect the cecum and jejunum, respectively. However, chIL-17RA expression was generally unchanged in Salmonella infection. These results suggest that chIL-17RA has an important role in mucosal immunity to intestinal intracellular parasite infections such as Eimeria infection.

Genetic and pathogenic characteristics of H1 avian and swine influenza A viruses.

This study examined the potential for cross-species transmission of influenza viruses by comparing the genetic and pathogenic characteristics of H1 avian influenza viruses (AIVs) with different host origins in Korea. Antigenic and phylogenetic analyses of H1 AIVs circulating in Korea provided evidence of genetic similarity between viruses that infect domestic ducks and those that infect wild birds, although there was no relationship between avian and swine viruses. However, there were some relationships between swine and human viral genes. The replication and pathogenicity of the H1 viruses was assessed in chickens, domestic ducks and mice. Viral shedding in chickens was relatively high. Virus was recovered from both oropharyngeal and cloacal swabs up to 5-10 days post-inoculation. The titres of domestic duck viruses in chickens were much higher than those of wild-bird viruses. Both domestic duck and wild-bird viruses replicated poorly in domestic ducks. None of the swine viruses replicated in chickens or domestic ducks; however, six viruses showed relatively high titres in mice, regardless of host origin, and induced clinical signs such as ruffled fur, squatting and weight loss. Thus, although the phylogenetic and antigenic analyses showed no evidence of interspecies transmission between birds and swine, the results suggest that Korean H1 viruses have the potential to cause disease in mammals. Therefore, we should intensify continuous monitoring of avian H1 viruses in mammals and seek to prevent interspecies transmission.

Effects of simple and disposable chicken cages for experimental Eimeria infections.

During experimental Eimeria infections in chickens, facilities are often contaminated by fecal oocysts known to be highly resistant to both chemical and enzymatic treatments. Thus, studies using experimental Eimeria infections have been limited due to the difficulty of complete elimination of residual oocysts from both cages and facilities. To overcome this limitation, simple, inexpensive, and disposable cages were constructed from cardboard boxes and tested during experimental Eimeria maxima infections. The cages were used in animal rooms with only a 1.7% evidence of coccidia contamination between adjacent cages. No significant differences in fecal oocyst output and body weight gain were noted between animals housed in disposable cages and animals housed in wire control cages. This cage design is a useful means for preventing oocyst contamination during experimental conditions, suggesting that this disposable cage design could be used for other avian infectious disease studies.

Prevalence and cross-immunity of Eimeria species on Korean chicken farms.

Epidemiology of Eimeria species in poultry flocks is important to increase the effectiveness of vaccinations and prophylactic strategies on chicken farms. In this study, fecal samples from 356 chicken farms were collected randomly and examined for the prevalence of Eimeria species. Through microscopic examination, it was determined that 78.7% of the tested farms were positive in Eimeria-infection. Seven Eimeria species were detected in all the positive farms by PCR amplification of the internal transcribed spacer 1 (ITS-1) region with species-specific primers. E. acervulina and E. tenella were the most prevalent, followed by E. brunetti and E. praecox (87.5, 62.5, 59.3, and 37.5% of the farms, respectively). Each of E. maxima, E. mitis, and E. necatrix was identified in 31.3% of the farms. Individual positive fecal samples contained multiple Eimeria species (mean=3.4). Since E. maxima is known to generate antigenic variants, cross-immunity was investigated for four isolates of E. maxima from the poultry farms in different regions of Korea. The extent of cross-protection varied from 54.3 to 100% against the heterologous isolates. The results obtained from this large-scale survey will be a useful reference for controlling coccidiosis in the poultry industry.

Minimizing an outbreak of avian botulism (Clostridium botulinum type C) in Incheon, South Korea.

An outbreak of botulism occurred over a two-month period beginning July 20, 2016. In all, 697 wild birds were found paralyzed or dead at the Namdong reservoir and 11 Gong-gu. Using a mouse bioassay, type C botulinum toxin was identified in the bird serum, liquid cultures of soil samples, and maggot extracts. To minimize further infection of wild birds, we opened the floodgates of the Namdong reservoir adjacent to the Yellow Sea; this decreased the water temperature and the nutrient load such as nitrogen and phosphorus. The outbreak stopped shortly after taking these actions. It is not known if these efforts decreased the number of dead and diseased wild birds. Our study demonstrates one potential approach to minimize future botulism outbreaks among wild birds and their habitats.

Downregulation of common cytokine receptor γ chain inhibits inflammatory responses in macrophages stimulated with Riemerella anatipestifer.

Th17-cell-mediated inflammation is affected by the soluble form of common cytokine receptor γ chain (γc). We previously suggested that inflammatory cytokines including interleukin (IL)-17A are associated with Riemerella anatipestifer infection, which a harmful bacterial pathogen in ducks. Here, the expression profiles of membrane-associated γc (duγc-a) and soluble γc (duγc-b) in R. anatipestifer-stimulated splenic lymphocytes and macrophages, and in the spleens and livers of R. anatipestifer-infected ducks, were investigated. In vitro and in vivo results indicated that the expression levels of both forms of γc were increased, showing that marked increases were detected in the expression of the duγc-b form rather than the duγc-a form. Treatment with γc-specific siRNA downregulated mRNA expression of Th17-related cytokines, including IL-17A and IL-17F, in duck splenic macrophages stimulated with R. anatipestifer, whereas the expressions of interferon (IFN)-γ and IL-2 were enhanced. The results showed that the upregulation of γc, especially the duγc-b form, was associated with expression of Th17-related cytokines during R. anatipestifer infection.

Identification of duck IL-4 and its inhibitory effect on IL-17A expression in R. anatipestifer-stimulated splenic lymphocytes.

As the dysregulation of IL-17 is implicated in the pathogenesis of various autoimmune and inflammatory diseases, the suppression of IL-17 production by Th2 cytokines could alleviate the development of these diseases. Previously, we confirmed that inflammatory cytokines including IL-17A are strongly associated with R. anatipestifer infection, which is one of the most important bacterial pathogens in the duck industry. Here, we found that IL-4 treatment downregulated the expression of IL-17A and IL-17F transcripts in splenic lymphocytes stimulated with R. anatipestifer. Moreover, duck IL-4 (duIL-4) treatment in R. anatipestifer-stimulated lymphocytes suppressed the expression of IL-23p19 and IL-12p40 transcripts compared to untreated and stimulated lymphocytes. Conversely, duIL-4 increased levels of IFN-γ and IL-10. We identified a full-length duIL-4 cDNA encoding 136 amino acids from ConA-activated splenic lymphocytes that shares 49.3-50% amino acid sequence identity with chicken and quail IL-4 and 21-29.7% with mammalian and piscine homologues. Low or moderate levels of duIL-4 transcript were observed in healthy tissues, including the spleen, bursa, and thymus, whereas duIL-4 expression was higher in the kidney and lung. Levels of duIL-4 were generally upregulated in mitogen-activated splenic lymphocytes but lower in the liver and spleen of R. anatipestifer-infected ducks compared to those of infected chickens. Recombinant duIL-4 promoted nitric oxide synthesis in duck macrophages stimulated by R. anatipestifer compared to untreated and stimulated control macrophages. These results demonstrate that IL-4 is an important Th2 cytokine that inhibits inflammatory responses in splenic lymphocytes stimulated with R. anatipestifer.

Molecular prevalence and genotyping of Chlamydia spp. in wild birds from South Korea.

Wild birds are reservoirs for Chlamydia spp. Of the total 225 samples from wild birds during January to September 2016 in Korea, 4 (1.8%) and 2 (0.9%) showed positive for Chlamydia psittaci and Chlamydia gallinacea, respectively. Phylogenetic analyses and comparisons of sequence identities for outer-membrane protein A (ompA) revealed that Korean C. psittaci fall into three previously known genotypes; genotype E, 1V and 6N, whereas the Korean C. gallinacea were classified as new variants of C. gallinacea. Our study demonstrates that wild birds in South Korea carry at least two Chlamydia species: C. psittaci and C. gallinacea, and provides new information on the epidemiology of avian chlamydiosis in wild birds.

Downregulation of inflammatory cytokines by berberine attenuates Riemerella anatipestifer infection in ducks.

Riemerella anatipestifer, an important infectious bacterium affecting the duck industry, has 5-75% mortality, depending on strain virulence. We previously demonstrated that proinflammatory cytokines are involved in inflammation during, and regulating susceptibility to, R. anatipestifer infection. We investigated the effects of the anti-inflammatory compound berberine in duck splenic lymphocytes stimulated with killed R. anatipestifer, and in R. anatipestifer-infected ducks. IL-17A, IL-17F, and IL-1ß transcripts were downregulated, and IFN-γ and IL-10 transcripts enhanced, in berberine-treated stimulated splenic lymphocytes, compared to stimulated untreated splenic lymphocytes. Similarly, IL-17A, IL-17F, IL-6, and IL-1ß expressions were significantly reduced, and IFN-γ and IL-10 expressions significantly upregulated, in spleens and livers of R. anatipestifer-infected berberine-treated ducks, compared to infected untreated birds. Moreover, infected and treated birds showed increased survival rates and significantly decreased bacterial burdens compared to infected untreated birds, confirming that inflammatory cytokines are strongly associated with R. anatipestifer infection in ducks.

Identification and expression analysis of duck interleukin-17D in Riemerella anatipestifer infection.

Interleukin (IL)-17D is a proinflammatory cytokine with currently largely unknown biological functions. Here we provide the description of the sequence, bioactivity, and mRNA expression profile of duck IL-17D homologue. A full-length duck IL-17D (duIL-17D) cDNA with a 624-bp coding region was identified from the large intestine. duIL-17D shares approximately 94.7% identity with its chicken counterpart, which is also identified in this work. duIL-17D exhibits 62.6-68.4% and 52.1-53.1% identity with mammalian and piscine homologues. Recombinant duIL-17D promoted the expression of proinflammatory cytokines such as IL-6, IL-8, and IL-1ß in duck embryo fibroblast cells. Very low levels of duIL-17D transcript were observed in healthy lymphoid tissues, including bursa, thymus, and spleen, while duIL-17D expression was relatively high in the heart. The duIL-17D expression profiles were examined in mitogen-stimulated splenic lymphocytes, as well as tissues affected by Riemerella anatipestifer infection. The levels of duIL-17D were mostly upregulated in mitogen-activated splenic lymphocytes but downregulated in the liver and spleen of R. anatipestifer-infected ducks. These results provide new insights into the roles of IL-17D in host protective immune responses to Riemerella infection, which can therefore lead to further studies of its biological functions in different disease models of ducks and other avian species.

Upregulation of duck interleukin-17A during Riemerella anatipestifer infection.

Although IL-17 cytokines play critical roles in host defense immunity, dysregulated expression of these cytokines is associated with inflammation and autoimmune diseases. Riemerella anatipestifer is the most important infectious bacterium in the duck industry. Interestingly, not all avian species are equally susceptible to R. anatipestifer infection. This paper reports the first description of mortality rate, bacterial burden, and expression profiles of immune-related genes between ducks and chickens infected with R. anatipestifer. Ducks exhibited increased susceptibility to R. anatipestifer infection compared to chickens, as determined by mortality rate and bacterial burden. Comparative expression analyses of immune-related genes in R. anatipestifer-infected tissues obtained from both species revealed that TLR3, TLR7, IL-2, IL-4, and IFN-γ transcript levels were higher in chickens, whereas TLR4 and IL-17A transcript levels were higher in ducks. Marked increases in expression of IL-17A and IL-6, but not TGF-ß, were associated with Th17 cell differentiation in duck splenic lymphocytes, but not in chicken splenic lymphocytes, stimulated with R. anatipestifer. Moreover, upregulation of IL-1ß, IL-6, and IL-17A mRNA expressions, but not TGF-ß, was confirmed in the liver and spleen of ducks infected with R. anatipestifer, indicating that IL-17A is strongly associated with Riemerella infection in ducks.

Tracking Mallards (Anas platyrhynchos) with GPS Satellite Transmitters Along Their Migration Route Through Northeast Asia.

In this study, Global Positioning System satellite transmitters were attached to three mallards (Anas platyrhynchos) wintering in South Korea to track their migration routes, stopover sites, breeding sites, and migration patterns. We successfully tracked only one mallard (no. 108917) from November 15, 2011, to November 29, 2013, and determined separate migration routes in two cases of spring migration and one case of fall migration. The mallard repeatedly migrated to the same final destination, even though the travel path varied. We identified six stopover sites: Hunhe River, Liaohe River, Yinma River, Yalu River, Songjeon Bay, and Dahuofang Reservoir in China and South Korea. The wintering sites of two migration cases were discovered to be identical (Gokgyo River in Asan, South Korea). The terminal sites, which were presumed to be breeding grounds, were the same in both cases (Hinggan League in Inner Mongolia Autonomous Region, China). On the basis of the migration routes identified in this study, we suggest that future efforts to control highly pathogenic avian influenza (HPAI) should not only include avian influenza surveillance but also implement flyway-based strategies, with regard to all countries affected by potential HPAI outbreaks.

Different strategies for producing naturally soluble form of common cytokine receptor γ chain.

The common cytokine receptor γ chain (γc) plays an essential role in regulating lymphoid homeostasis. In fact, alteration of this gene causes severe immunodeficiency in humans and animals. Although soluble γc (sγc) was identified in the late 1990s, much remains unknown about its production. This study describes various mechanisms underlying the generation of sγc isoforms in different species. Our data demonstrate that mouse γc and the avian ortholog γc-a did not generate sγc. Moreover, two mouse isoforms, CRA-a and mγc-b, encoded by transcripts lacking a transmembrane region by alternative splicing, did not yield sγc. However, in ducks, sγc was produced from a γc-b transcript lacking a transmembrane region by alternative splicing. In chickens, sγc was produced in normal cells and cell lines by proteolytic shedding of the γc-b isoform containing intron 5, which displayed a relatively high probability of proteolytic cleavage of the ectodomain. This shedding was suppressed by leupeptin, serine and cysteine protease inhibitor. Compared to the chicken ortholog γc-a, expression of γc-b mRNA was differentially regulated according to tissue type, developmental stage, and antigen stimulation. These data demonstrate several mechanisms for producing sγc and suggest a potential role for sγc in avian lymphoid homeostatic responses to environmental antigens.