Immunological responses against vancomycin-resistant Enterococcus faecium and Enterococcus faecalis by mice.

Methicillin-resistant Staphylococcus aureus (MRSA) infections in humans can currently only be treated with vancomycin. Consequently, vancomycin-resistant Enterococcus spp. pose a serious public health hazard because MRSA can acquire their vancomycin resistance. While the microbiological and genetic characteristics of vancomycin-resistant enterococci (VRE) have been extensively studied, serological diagnostic tools for these organisms are lacking. The VanA and VanB classes of VRE show marked resistance. Here, we identified the VanA and VanB proteins that are immunogenic in mice. To do so, mice were orally infected with a VanA strain of E. faecium or a VanB strain of E. faecalis and the serologically immunogenic proteins were identified by SDS-PAGE and Western blot analysis. The mice reacted to the 27 and 65 kDa cell envelope (CE) proteins of VanA at 1 week post-infection (wpi) and then reacted to the 100 kDa cytoplasmic protein (CP) at 2-4 wpi. With regard to VanB, the mice responded at 1-4 wpi, 3-4 wpi, and 4 wpi to the 70 kDa, 25 and 35 kDa, and 79 kDa CE proteins, respectively, and at 3 wpi to the 39 kDa CP. The identification of these immunogenic proteins may be useful for diagnosing and for producing immunotherapeutic VRE antibodies.

Surveillance of avian influenza viruses in South Korea between 2012 and 2014.

BACKGROUND: National surveillance of avian influenza virus (AIV) in South Korea has been annually conducted for the early detection of AIV and responses to the introduction of highly pathogenic avian influenza (HPAI) virus. In this study, we report on a nationwide surveillance study of AIV in domestic poultry and wild birds in South Korea between 2012 and 2014. METHODS: During the surveillance programs between 2012 and 2014, 141,560 samples were collected. Of these, 102,199 were from poultry farms, 8215 were from LBMs, and 31,146 were from wild bird habitats. The virus isolation was performed by inoculation of embryonated chicken eggs and AIV isolates were detected using hemagglutination assay. For subtying of AIV, the hemagglutinin and neuraminidase genes were confirmed by sequencing. Phylogenetic analysis of the H5 subtypes was performed using 28 H5 AIV isolates. RESULTS: Between 2012 and 2014, a total of 819 AIV were isolated from 141,560 samples. Virus isolation rates for AIV were 0.6, 0.4, 0.1, and 2.7% in wild birds (n = 202), domestic ducks (n = 387), minor poultry (n = 11), and the live bird market (LBM) (n = 219), respectively. In wild birds, various subtypes were found including H1-H7 and H9-H13. The major subtypes were H5 (n = 48, 23.9%: N3 (n = 4) and N8 (n = 44)), H4 (n = 39, 19.4%), and H1 (n = 29, 14.4%). In domestic poultry, mainly ducks, the H5N8 (n = 275, 59.3%), H3 (n = 30, 17.2%), and H6 (n = 53, 11.4%) subtypes were predominantly found. The most frequently detected subtypes in LBM, primarily Korean native chicken, were H9 (n = 169, 77.2%). H3 (n = 10, 4%) and H6 (n = 30, 13.7%) were also isolated in LBM. Overall, the prevalence of AIV was found to be higher between winter and spring and in western parts of South Korea. The unusual high prevalence of the H5 subtype of AIV was due to the large scale outbreak of H5N8 HPAI in wild birds and domestic poultry in 2014. CONCLUSIONS: Enhanced surveillance and application of effective control measures in wild birds and domestic poultry, including LBM, should be implemented to control AI and eradicate HPAI.

Prevalence of Campylobacter species in wild birds of South Korea.

Campylobacter species cause human gastrointestinal infections worldwide. They commonly inhabit intestines of avian species including wild birds. They might play a role in the spread of infections to humans and other bird species. The prevalence of Campylobacter species in 2164 faecal samples of wild birds (representing 71 species and 28 families) captured across the Korean peninsula was evaluated in this study. The overall prevalence was 15.3% (332/2164). Bird species belonging to the family Charadriidae had the highest isolation rate (30.0%), followed by those belonging to the families Ardeidae (26.4%), Turdidae (21.9%), and Anatidae (15.3%). The prevalence of Campylobacter spp. differed significantly according to migratory habit. Stopover birds were the most commonly infected (19.0%), followed by winter migratory (16.7%) and summer migratory birds (12.3%). However, indigenous birds showed very low prevalence (2.7%). Antimicrobial susceptibility tests were performed for 213 isolates. Results showed that Campylobacter jejuni isolates (n = 169) exhibited resistance to nalidixic acid (5.3%), ciprofloxacin (3.0%), and tetracycline (1.8%), while Campylobacter lari (n = 1) displayed resistance to nalidixic acid and ciprofloxacin. However, all Campylobacter coli isolates (n = 20) were susceptible to all antimicrobials tested. This is the first report on the prevalence of Campylobacter species in wild birds that seasonally or indigenously inhabit the Korean peninsula. Our results indicate that the overall prevalence of Campylobacter in wild birds is moderate. Therefore, birds might serve as significant reservoirs for Campylobacter pathogens.

Antimicrobial susceptibility and characterization of extended-spectrum ß-lactamases in Escherichia coli isolated from bovine mastitic milk in South Korea from 2012 to 2015.

In this study, we aimed to assess trends in antimicrobial resistance and to investigate the characteristics of extended-spectrum ß-lactamase (ESBL)-producing isolates from bovine mastitic milk from 2012 to 2015. A total of 374 Escherichia coli isolates were analyzed (154 in 2012, 113 in 2013, 76 in 2014, and 31 in 2015). No consistent trends in antimicrobial resistance of E. coli isolates occurred during the 4-yr period. The most frequently observed resistance was tetracycline (23.3%), followed by streptomycin (17.1%), ampicillin (16.6%), neomycin (11.8%), and trimethoprim/sulfamethoxazole (11.2%). Multidrug resistance was observed in 15.5% of isolates. Among these isolates, 15 (4.0%) carried one or more blaCTX-M and AmpC ESBL genes from 11 different farms, including blaCTX-M-15 at 4 farms, blaCTX-M-3 at 2 farms, blaCTX-M-1 at 3 farms, and blaCMY-2 at 3 farms. This study is the first report of blaCTX-M-3-producing E. coli in dairy milk. Transfer of ESBL was observed in 3 blaCTX-M-3-producing isolates, 1 blaCTX-M-1-producing isolate, and all 3 blaCMY-2-producing isolates. Almost all blaCTX-M-15 and blaCTX-M-1 genes possessed an insertion sequence, ISECP1, upstream of the blaCTX-M gene. Identical pulsed-field gel electrophoresis profiles were also observed in blaCTX-M-producing E. coli from the same farm. These results suggested that ESBL might spread by both clonal and horizontal spread in dairy farms in South Korea. Although no significant changes occurred in the antimicrobial resistance of E. coli during the 4-yr study period, the resistance rates and presence of ESBL were high compared with those in other countries. Thus, these findings suggest the importance of control measures for E. coli, particularly ESBL-producing bacteria, on dairy farms to reduce treatment failure and transmission to humans.

Homology differences between complete Sacbrood virus genomes from infected Apis mellifera and Apis cerana honeybees in Korea.

Sacbrood virus (SBV) represents a serious threat to the health of managed honeybees. We determined four complete SBV genomic sequences (AmSBV-Kor1, AmSBV-Kor2, AcSBV-Kor3, and AcSBV-Kor4) isolated from Apis mellifera and Apis cerana in various regions of South Korea. A phylogenetic tree was constructed from the complete genomic sequences of these Korean SBVs (KSBVs) and 21 previously reported SBV sequences from other countries. Three KSBVs (not AmSBV-Kor1) clustered with previously reported Korean genomes, but separately from SBV genomes from other countries. The KSBVs shared 90-98 % identity, and 89-97 % identity with the genomes from other countries. AmSBV-Kor1 was least similar (~90 % identity) to the other KSBVs, and was most similar to previously reported strains AmSBV-Kor21 (97 %) and AmSBV-UK (93 %). Phylogenetic analysis of the partial VP1 region sequences indicated that SBVs clustered by host species and country of origin. The KSBVs were aligned with nine previously reported complete SBV genomes and compared. The KSBVs were most different from the other genomes at the end of the 5' untranslated region and in the entire open reading frame. A SimPlot graph of the VP1 region confirmed its high variability, especially between the SBVs infecting A. mellifera and A. cerana. In this genomic region, SBVs from A. mellifera species contain an extra continuous 51-nucleotide sequence relative to the SBVs from A. cerana. This genomic diversity may reflect the adaptation of SBV to specific hosts, viral cross-infections, and the spatial distances separating the KSBVs from other SBVs.

Pathologic Changes in Wild Birds Infected with Highly Pathogenic Avian Influenza A(H5N8) Viruses, South Korea, 2014.

In January 2014, an outbreak of infection with highly pathogenic avian influenza (HPAI) A(H5N8) virus began on a duck farm in South Korea and spread to other poultry farms nearby. During this outbreak, many sick or dead wild birds were found around habitats frequented by migratory birds. To determine the causes of death, we examined 771 wild bird carcasses and identified HPAI A(H5N8) virus in 167. Gross and histologic lesions were observed in pancreas, lung, brain, and kidney of Baikal teals, bean geese, and whooper swans but not mallard ducks. Such lesions are consistent with lethal HPAI A(H5N8) virus infection. However, some HPAI-positive birds had died of gunshot wounds, peritonitis, or agrochemical poisoning rather than virus infection. These findings suggest that susceptibility to HPAI A(H5N8) virus varies among species of migratory birds and that asymptomatic migratory birds could be carriers of this virus.

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.

Identification of a picornavirus from chickens with transmissible viral proventriculitis using metagenomic analysis.

Transmissible viral proventriculitis (TVP), an infectious disease in chickens, is responsible for economic losses in the commercial poultry industry. The major etiologic agent, however, is unknown. Using metagenomics, we compared the diversity of viruses present in proventriculus samples from flocks diagnosed with TVP to those of healthy flocks in South Korea between 2003 and 2012. Each sample had a mean of 21,538,726 sequence reads generated by high-throughput sequencing, with a mean length of 160 nt. Enrichment in viral sequences suggested that at least three viruses were present in each TVP sample. Although we could not determine a pathogen of TVP that matched the known morphology, picornavirus sequences were present in all five disease samples, suggesting an association with TVP. The five samples yielded 1,045-1,720 bp contigs with 81-84 % nt sequence identity to turkey hepatitis virus (accession number: HM751199). Whole-genome analysis indicated that the QIA01 strain of the novel picornavirus was similar to turkey hepatitis virus in the P2 and P3 regions (82.7 % nt and 95.5 % aa sequence identity), but different in the structural region and partial 2A peptides (56.2 % nt and 23.9 % aa sequence identity). In addition, the QIA01 virus was similar (87.0 % nt and 95.6 % aa sequence identity) to chicken megrivirus, recently detected in chickens with malabsorption syndrome in Hungary. Our results are useful for understanding the genetic diversity of avian picornaviruses and for classifying chicken megrivirus as a pathogen affecting the digestive tract of chickens.

Single-tube nested PCR assay for the detection of avian botulism in cecal contents of chickens.

This paper describes a novel diagnostic method for the detection of avian botulism caused by Clostridium botulinum type C and C/D, using single-tube nested PCR assay. This assay was developed to overcome the disadvantages of bioassays used in experiments with mice. Three primer pairs including an antisense primer were designed to target the N-terminal of the toxin gene from C. botulinum types C and C/D. The specificity of the PCR assay was confirmed by using 33 bacterial strains and chicken cecal contents from farms that experienced botulism outbreaks. The detection limit for purified DNA was 1.1 fg/µl, and for bacterial spores was 4.3 spores/200 mg of cecal contents. While checking for specificity of the PCR assay, the reactions with the templates form C. botulinum type C and C/D which were tested became positive, but the rest of the reactions turned negative. However, the results for all clinical samples (n = 8) were positive. The PCR assay results for cecal samples obtained from 300 healthy chickens (150 Korean native chickens and 150 broilers) were all negative. This assay is rapid and straightforward and evades ethical issues associated with mouse bioassay. Moreover, it is more economical than real-time PCR.

Characterization of Clostridium perfringens isolates obtained from 2010 to 2012 from chickens with necrotic enteritis in Korea.

Clostridium perfringens produces diverse virulent toxins that cause necrotic enteritis in poultry, resulting in a great negative impact on the poultry industry. To study the characteristics of C. perfringens in chickens, we isolated 88 strains from chickens (1 strain per flock) with necrotic enteritis. The isolated bacterial strains were screened for toxin type and antimicrobial susceptibility. Necropsy of 17 chickens that died from necrotic enteritis revealed that their intestines were dilated with inflammatory exudates and characterized by mucosal necrosis. All the isolated strains were identified as toxin type A using multiplex PCR for toxin typing. We found that the rate of netB-positive strains isolated from dead chickens was significantly higher (8 of 17) than the rate among healthy chickens (2 of 50). We performed antimicrobial susceptibility test with 20 selected antimicrobial agents using the disk diffusion test and found that 30 tested strains were completely resistant to 5 antibiotics and partially resistant to 6 antibiotics whereas all the strains were susceptible to 9 antimicrobial agents. Using pulsed-field gel electrophoresis analysis, the 17 strains were divided into 13 genetic clusters showing high genetic diversity. In conclusion, C. perfringens strains isolated from Korean poultry showed a high resistance to antimicrobial drugs and high genetic diversity, suggesting that continuous monitoring is essential to prevent outbreaks of necrotic enteritis in chickens.

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.

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.

Occurrence of avian botulism in Korea during the period from June to September 2012.

Botulism is a paralytic disease caused by the botulinum neurotoxin produced by Clostridium botulinum. In the summer season in Korea, intensive outbreaks of avian botulism were reported in both poultry and wild birds, including five Korean native chicken farms (HanHyup NO.3), one pheasant (Phasianus colchicus karpowi) farm, and one community of spot-billed ducks (Anas poecilorhyncha). The affected domestic birds showed 24.5% to 58.3% mortality, with specific clinical signs including ataxia, limber neck, and diarrhea. To confirm the botulinum toxin, neutralization tests were performed on sera (four Korean native chicken farms and one pheasant farm) or culture supernatant (spot-billed ducks). Additionally, the contents of the cecum and liver from poultry presenting signs suggestive of botulism were inoculated to isolate the pathogen. The toxin genes were then detected by polymerase chain reaction (PCR). Through the neutralization tests, it was possible to diagnose the botulism and, except in the case of one Korean native chicken farm, to identify the type of pathogen. Using detection by PCR, except in two cases of the Korean native chicken farms, the botulinum toxin gene was found. Additionally, in four cases, it was possible to identify the C/D mosaic type using PCR. This paper reports the first occurrence of avian botulism in domestic birds and the first detection of botulism caused by this mosaic type in Korea.

Molecular epidemiology of Newcastle disease viruses in Vietnam.

Newcastle disease virus (NDV) causes significant economic losses to the poultry industry in Southeast Asia. In the present study, 12 field isolates of NDV were recovered from dead village chickens in Vietnam between 2007 and 2012, and were characterized. All the field isolates were classified as velogenic. Based on the sequence analysis of the F variable region, two distinct genetic groups (Vietnam genetic groups G1 and G2) were recognized. Phylogenetic analysis revealed that all the 12 field isolates fell into the class II genotype VII cluster. Ten of the field isolates, classified as Vietnam genetic group G1, were closely related to VIIh viruses that had been isolated from Indonesia, Malaysia, and Cambodia since the mid-2000s, while the other two field isolates, of Vietnam genetic group G2, clustered with VIId viruses, which were predominantly circulating in China and Far East Asia. Our results indicate that genotype VII viruses, especially VIIh viruses, are predominantly responsible for the recent epizootic of the disease in Vietnam.

Application of real-time reverse transcription polymerase chain reaction to the detection the matrix, H5 and H7 genes of avian influenza viruses in field samples from South Korea.

BACKGROUND: The rapid and accurate identification of the H5 and H7 subtypes of avian influenza (AI) virus is an important step for the control and eradication of highly pathogenic AI outbreaks and for the surveillance of AI viruses that have the potential to undergo changes in pathogenicity in poultry and wild birds. Currently, real-time reverse transcription polymerase chain reaction (RRT-PCR) is routinely used for the rapid detection of the H5 and H7 genes, but misidentification is frequent for emergent isolates and viruses isolated from diverse regions due to the high sequence variation among AI viruses. FINDINGS: In this study, an RRT-PCR method was tested for the detection of matrix, H5 and H7 genes from diverse subtypes of AI viruses and from field samples obtained through AI surveillance in South Korea over the last four years. Both RRT-PCR and conventional experiment (virus isolation using egg inoculation followed by reverse transcription polymerase chain reaction) agreed on the virus-positive samples. And the comparison of the results with 174 clinical samples showed a high level of agreement without decreasing the specificity and sensitivity. CONCLUSIONS: This assay could be useful tool for the rapid detection of AI using the field samples from domestic poultry and wild birds in South Korea, and continuous regional updates is needed to validate primer sets as the AI virus evolves.

Molecular epidemiological investigation of velogenic Newcastle disease viruses from village chickens in Cambodia.

Three isolates of Newcastle disease virus (NDV) were isolated from tracheal samples of dead village chickens in two provinces (Phnom Penh and Kampong Cham) in Cambodia during 2011-2012. All of these Cambodian NDV isolates were categorized as velogenic pathotype, based on in vivo pathogenicity tests and F cleavage site motif sequence ((112)RRRKRF(117)). The phylogenetic analysis and the evolutionary distances based on the sequences of the F gene revealed that all the three field isolates of NDV from Cambodia form a distinct cluster (VIIh) together with three Indonesian strains and were assigned to the genotype VII within the class II. Further phylogenetic analysis based on the hyper-variable region of the F gene revealed that some of NDV strains from Malaysia since the mid-2000s were also classified into the VIIh virus. This indicates that the VIIh NDVs are spreading through Southeast Asia. The present investigation, therefore, emphasizes the importance of further surveillance of NDV in neighboring countries as well as throughout Southeast Asia to contain further spreading of these VIIh viruses.

Genetic and antigenic variation of shedding viruses from vaccinated chickens after challenge with virulent Newcastle disease virus.

Newcastle disease virus (NDV) isolation was attempted from La Sota-vaccinated or unvaccinated chickens exposed to the virulent NDV variant E347Kmt. Shedding viruses were purified by plaque assay and then were sequenced for HN and F genes. The amino acid sequences of the F gene of all shedding viruses were identical to the sequence of the challenge virus. However, amino acid substitution occurred at four positions (70, 347, 466, and 517) in the HN protein among shedding viruses from vaccinated and challenged chickens but not from unvaccinated and challenged chickens. Amino acid substitution occurred more frequently at position 347 (K to G or V) in the HN protein compared with the other positions. There was minor antigenic variation between some of mutant viruses shed and challenge virus. However, none of mutant viruses had a significantly lower antigenic R value with La Sota virus compared with challenge virus E347Kmt. Our findings indicate that vaccinal immunity might facilitate an evolutional event through antigenic selection, genetic mutation among virulent virus populations shed from vaccinated flocks, or both.

Transmission and persistence of methicillin-resistant Staphylococcus aureus in milk, environment, and workers in dairy cattle farms.

The aim of this study was to determine the presence and persistence of methicillin-resistant Staphylococcus aureus (MRSA) in milk, farm environment, and farmers on 22 dairy cattle farms in Korea during 2008-2009. Genetic relatedness among the MRSA isolates was also investigated. Of 1146 samples examined, 35 of 559 (6.3%) quarter milk samples from 371 cows, four of 86 (4.7%) hand and nose samples from 43 farmers, and 6 of 501 (1.2%) farm environment samples were MRSA positive. Except for three isolates, all MRSA were classified into ST72-spa t324-SCCmec IV with PVL negative, the most predominant clonal type among community-associated MRSA in South Korea. All 35 MRSA-positive milk samples from 19 cows were obtained from a single farm (Farm G) out of 22 (4.5%) farms tested. The farm G was revisited 1 year later and milk samples were collected for examination of MRSA again. Two of six previous MRSA-positive cattle that had been kept on the farm still harbored MRSA genetically identical to MRSA strains, which were isolated from the same farm a year ago. The results of this study provide the evidence of transmission of MRSA among cattle, farm environment, and farmers and also long-term persistence of MRSA in animals.

Restriction fragment length polymorphism analysis of multiple genome regions of Korean isolates of infectious laryngotracheitis virus collected from chickens.

This study was conducted to characterize infectious laryngotracheitis (ILT) viruses isolated from poultry in South Korea using RFLP analysis of PCR products. Seven wild-type Korean isolates from commercial chicken farms collected between 1986 and 2012 were compared with 3 imported commercial vaccine strains [LT Blen (Hudson strain, United States), Laryngo Vac (Cover strain, United States), and Nobilis ILT (Serva strain, France)] and a Korean chicken embryo origin (CEO) vaccine strain [ILT-VAC (Gyeonggi97 strain, Korea)]. Six of the field isolates were highly virulent viruses, and the Kr12AD37 isolate was considered an attenuated type according to Han's RFLP method. These virulent Korean ILT viruses were divided into 3 classes (class I, II, and III). The Kr12AD37 isolate was found to have the same RFLP pattern as the Korean CEO vaccine strain, and both of these strains were different from the 3 foreign vaccine strains. The results suggest that the Korean CEO vaccine strain has been responsible for recent outbreaks, and the characterization of ILT viruses by RFLP was useful for diagnosis by providing epidemiological information.

Phylogenetic analysis and genetic characterization of chicken anemia virus isolates from Cambodia.

Three chicken anemia viruses (CAV) were detected by PCR during screening of field samples from village chickens collected in Cambodia in 2011/2012. Nearly full-length VP1 viral structural protein genes (nt 1-1,293) from the 3 CAV were sequenced and characterized. Phylogenetic analysis revealed that all 3 of the Cambodian CAV were clustered with CAV strains belonging to genotype II and were most closely related to CAV strains from Guangdong province, China. On the amino acid level, major substitutions were observed at 12 residues in the VP1 protein (positions 22, 75, 97, 125, 139, 144, 254, 287, 290, 370, 376, and 413) when compared with published reference CAV strains. In motifs associated with virulence, all Cambodian CAV had virulence-associated motifs composed of 75I, 89T, 125I, 139Q, 141Q, 144Q, and 394Q, which are commonly found in highly virulent genotype II viruses and some genotype III viruses. This is the first report of CAV isolated from village chickens in Southeast Asia as well as Cambodia.