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.

Efficient self-assembly and protective efficacy of infectious bursal disease virus-like particles by a recombinant baculovirus co-expressing precursor polyprotein and VP4.

BACKGROUND: Virus-like particle (VLP) technology is considered one of the most promising approaches in animal vaccines, due to the intrinsic immunogenic properties as well as high safety profile of VLPs. In this study, we developed a VLP vaccine against infectious bursal disease virus (IBDV), which causes morbidity and mortality in chickens, by expressing a baculovirus in insect cells. METHODS: To improve the self-proteolytic processing of precursor polyprotein (PP), we constructed a recombinant baculovirus transfer vector that co-expresses PP and the VP4 protease gene of IBDV. RESULTS: Expression and VLP assembly of recombinant proteins and antigenicity of the VLP were examined by Western blotting, ELISA, and transmission electron microscopy. In animal experiments, vaccination with the recombinant VLP induced strong and uniform humoral immunity and provided complete protection against challenge with very virulent (vv) IBDV in SPF chickens (n = 12). As determined by the bursa of Fabricius (BF)/body weight (B/BW) ratio, the protection against post-challenge bursal atrophy was significantly higher (P < 0.001) in VLP-vaccinated birds than in non-vaccinated controls. CONCLUSIONS: Since the protective efficacy of the VLP vaccine was comparable to that of a commercially available inactivated vaccine, the recombinant VLP merits further investigation as an alternative means of protection against vvIBD.

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.

Genetic diversity of avian paramyxovirus type 4 isolates from wild ducks in Korea from 2006 to 2011.

Thirteen isolates of avian paramyxovirus type 4 (APMV-4) isolated from wild ducks in Korea from 2006 to 2011 were genetically characterized by sequence analysis of the N-terminal region of the APMV-4 fusion (F) protein gene. The results revealed that the amino acid sequence homology within Korean isolates was 97.5 % or greater. The homologies of the Korean isolates with the APMV-4/duck/HK/D3/75 and APMV-4/duck/BE/15129/07 strains were 86.9-88.0 and 95.5-96.1 %, respectively. All Korean isolates had sequence motifs of (116)DIQPR↓F(121) at the F0 cleavage site. Phylogenetic analysis based on the N-terminal region of the F protein gene of APMV-4 isolates revealed that all 2006-2011 Korean isolates formed a single genotypic cluster that was phylogenetically different from APMV-4/duck/HK/D3/75 or APMV-4/duck/BE/15129/07 strains. Korean APMV-4 isolates were more closely related to APMV-4/goose/ZA/N1468/10 (isolated in South Africa) than to the Belgium APMV-4 virus. Korean APMV-4 isolates were further divided into at least two subgroups (A and B) based on phylogenetic analysis. Subgroup A viruses were isolated throughout Korea, whereas subgroup B viruses were detected only in isolates from Cheju island in 2011, suggesting that Korean APMV-4 exhibits marked genetic diversity and differs from viruses currently circulating in Europe and other locations.

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.

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.

Geographical distribution of low pathogenic avian influenza viruses of domestic poultry in Vietnam and their genetic relevance with Asian isolates.

From the avian influenza virus (AIV) outbreaks and market surveillances in Vietnam during November 2011 and March 2012, a total of 196 AIV were isolated. Although H5N1 highly pathogenic avian influenza (HPAI) was the most prevalent subtype in Vietnam, 57 low pathogenic avian influenza (LPAI) viruses were identified from mainly domestic ducks and some chickens. Of note, various subtypes of LPAI viruses were isolated from domestic ducks in Vietnam: H3 (n = 16), H4 (n = 4), H6 (n = 24), H7 (n = 1), and H9 (n = 10). Geographically, the LPAI viruses were identified in different regions of Vietnam. Phylogenetic analysis of HA and NA genes in LPAIV in Vietnam showed that some H3 (group I) and H4 subtypes AIV clustered with the viruses of several Asian isolates from domestic poultry and wild birds. However, the H6, H9, and some H3 (group II and III) subtypes AIV were closely related to isolates from domestic poultry in Southern China. In addition, whereas the N2 and N6 subtypes AIV belonged to the Eurasian lineage, the N8 subtype AIV was classified to be both of Eurasian and American lineage. These findings revealed that the regional trade and wild birds play a key role transmission of LPAIV in domestic ducks in Vietnam. Further surveillance at the intercountry level is needed to understand the epidemiology of these viruses and to cope with emergence of novel AIV types.

Pathogenicity of H9N2 low pathogenic avian influenza viruses of different lineages isolated from live bird markets tested in three animal models: SPF chickens, Korean native chickens, and ducks.

Since its first appearance in 1996, H9N2 avian influenza virus (AIV) of the Y439 lineage persisted in Korean live bird markets (LBMs) until the last documented occurrence in 2018. However, in June 2020, the avian influenza surveillance program detected a novel H9N2 AIV belonging to the Y280 lineage, which has zoonotic potential, in a Korean native chicken (KNC) from a LBM. In this study, we infected KNCs and ducks (the 2 major species held at LBMs), as well as SPF chickens, with Y280-lineage H9N2 AIV LBM261/20 and Y439-equivalent LBM294/18 to compare pathogenicity and transmissibility. In SPF chickens, LBM261/20 replicated mostly in the respiratory tract and spread rapidly among birds. By contrast, LBM294/18 replicated preferentially in the gastrointestinal tract and transmitted more slowly than LBM261/20. LBM261/20 replicated for a longer time in KNCs than in SPF chickens, and only in the respiratory tract; by contrast, LBM294/18 was detected in the oropharynx and cloaca. Ducks did not shed either virus or seroconvert. Taken together, the data suggest that the scheme used to monitor the newly introduced H9N2 AIV of the Y280 lineage needs to be modified to place emphasis on oropharyngeal sampling. Such changes will facilitate better disease control and protect public health.

Pathogenicity of H5N8 High Pathogenicity Avian Influenza Virus in Chickens and Ducks from South Korea in 2020-2021.

During the 2020-2021 winter season, an outbreak of clade 2.3.4.4b H5N8 high pathogenicity avian influenza (HPAI) virus occurred in South Korea. Here, we evaluated the pathogenicity and transmissibility of A/mandarin duck/Korea/H242/2020 (H5N8) (H242/20(H5N8)) first isolated from this outbreak in specific pathogen-free (SPF) chickens and commercial ducks in comparison with those of A/duck/Korea/HD1/2017(H5N6) (HD1/17(H5N6)) from a previous HPAI outbreak in 2017-2018. In chickens, the 50% chicken lethal dose and mean death time of H242/20(H5N8) group were 104.5 EID50 and 4.3 days, respectively, which indicate less virulent than those of HD1/17(H5N6) (103.6 EID50 and 2.2 days). Whereas, chickens inoculated with H242/20(H5N8) survived longer and had a higher titer of viral shedding than those inoculated with HD1/17(H5N6), which may increase the risk of viral contamination on farms. All ducks infected with either HPAI virus survived without clinical symptoms. In addition, they exhibited a longer virus shedding period and a higher transmission rate, indicating that ducks may play an important role as a silent carrier of both HPAI viruses. These results suggest that the pathogenic characteristics of HPAI viruses in chickens and ducks need to be considered to effectively control HPAI outbreaks in the field.

Genetic characterization of H9N2 avian influenza virus previously unrecognized in Korea.

In this study, we describe the isolation and characterization of previously unreported Y280-lineage H9N2 viruses from two live bird markets in Korea in June 2020. Genetic analysis revealed that they were distinct from previous H9N2 viruses circulating in Korea and had highest homology to A/chicken/Shandong/1844/2019(H9N2) viruses. Their genetic constellation showed they belonged to genotype S, which is the predominant genotype in China since 2010, where genotype S viruses have infected humans and acted as internal gene donors to H5 and H7 zoonotic influenza viruses. Active surveillance and control measures need to be enhanced to protect the poultry industry and public health.

Pathogenesis and genetic characteristics of a novel reassortant low pathogenic avian influenza A(H7N6) virus isolated in Cambodia in 2019.

The first human case of zoonotic A(H7N4) avian influenza virus (AIV) infection was reported in early 2018 in China. Two months after this case, novel A(H7N4) viruses phylogenetically related to the Jiangsu isolate emerged in ducks from live bird markets in Cambodia. During active surveillance in Cambodia, a novel A(H7N6) reassortant of the zoonotic low pathogenic AIV (LPAIV) A(H7N4) was detected in domestic ducks at a slaughterhouse. Complete genome sequencing and phylogenetic analysis showed that the novel A(H7N6) AIV is a reassortant, in which four gene segments originated from Cambodia A(H7N4) viruses and four gene segments originated from LPAIVs in Eurasia. Animal infection experiments revealed that chickens transmitted the A(H7N6) virus via low-level direct contacts, but ducks did not. Although avian-origin A(H7Nx) LPAIVs do not contain the critical mammalian-adaptive substitution (E627K) in PB2, the lethality and morbidity of the A(H7N6) virus in BALB/c mice were similar to those of A(H7N9) viruses, suggesting potential for interspecies transmission. Our study reports the emergence of a new reassortant of zoonotic A(H7N4) AIVs with novel viral characteristics and emphasizes the need for ongoing surveillance of avian-origin A(H7Nx) viruses.

Multiple Reassortants of H5N8 Clade 2.3.4.4b Highly Pathogenic Avian Influenza Viruses Detected in South Korea during the Winter of 2020-2021.

During October 2020-January 2021, we isolated a total of 67 highly pathogenic avian influenza (HPAI) H5N8 viruses from wild birds and outbreaks in poultry in South Korea. We sequenced the isolates and performed phylogenetic analysis of complete genome sequences to determine the origin, evolution, and spread patterns of these viruses. Phylogenetic analysis of the hemagglutinin (HA) gene showed that all the isolates belong to H5 clade 2.3.4.4 subgroup B (2.3.4.4b) and form two distinct genetic clusters, G1 and G2. The cluster G1 was closely related to the 2.3.4.4b H5N8 HPAI viruses detected in Europe in early 2020, while the cluster G2 had a close genetic relationship with the 2.3.4.4b H5N8 viruses that circulated in Europe in late 2020. A total of seven distinct genotypes were identified, including five novel reassortants carrying internal genes of low pathogenic avian influenza viruses. Our Bayesian discrete trait phylodynamic analysis between host types suggests that the viruses initially disseminated from migratory waterfowl to domestic duck farms in South Korea. Subsequently, domestic duck farms most likely contributed to the transmission of HPAI viruses to chicken and minor poultry farms, highlighting the need for enhanced, high levels of biosecurity measures at domestic duck farms to effectively prevent the introduction and spread of HPAI.

Phylogeographic analysis of H5N1 highly pathogenic avian influenza virus isolated in Cambodia from 2018 to 2019.

Since 2004, several outbreaks of highly pathogenic avian influenza (HPAI) have been reported in Cambodia. Until 2013, all H5N1 viruses identified in Cambodia belonged to clade 1 and its subclades. H5N1 HPAI viruses belonging to clade 2.3.2.1c have been dominant since the beginning of 2014, with various genotypes (KH1-KH5) reported. Here, we isolated nine H5N1 HPAI viruses from domestic poultry farms and slaughterhouses in Cambodia during 2018-2019 and performed phylogenetic analysis of whole genome sequences. All isolates were classified as H5 clade 2.3.2.1c viruses and all harbored multi-basic amino acid sequences (PQRERRRKR/GLF) at the haemagglutinin (HA) cleavage site. Phylogenetic analysis revealed that the H5N1 isolates in this study belonged to the KH2 genotype, the dominant genotype in Cambodia in 2015. Phylogenetic analysis of the HA gene showed that the isolates were divided into two groups (A and B). The results of Bayesian discrete phylogeography analysis revealed that the viral migration pathways from Vietnam to Cambodia (Bayes factor value: 734,039.01; posterior probability: 1.00) and from Cambodia to Vietnam (Bayes factor value: 26,199.95; posterior probability: 1.00) were supported by high statistical values. These well-supported viral migrations between Vietnam and Cambodia demonstrate that viral transmission continued in both directions. Several factors may have contributed to this, including the free-grazing duck system and movement of poultry-related products. Thus, the results emphasize the need for an enhanced international surveillance program to better understand transboundary infection and evolution of H5N1 HPAI viruses, along with implementation of more stringent international trade controls on poultry and poultry products.

A novel reassortant clade 2.3.4.4 highly pathogenic avian influenza H5N6 virus identified in South Korea in 2018.

Since 2017, clade 2.3.4.4b H5N6 highly pathogenic avian influenza viruses (HPAIVs) have been detected over a broad geographic region, including Eurasia. These viruses have evolved through reassortment with Eurasian low pathogenic avian influenza viruses (LPAIVs), resulting in multiple genotypes. Here, we sequenced the full-length genome of 15 H5N6 HPAIVs collected from wild birds and poultry farms in South Korea from January to March 2018. A comparative phylogenetic analysis was then conducted. Three distinct genotypes were identified in South Korea during 2017/2018, including a novel reassortant genotype, H214. The novel reassortant H5N6 viruses isolated in this study possessed PB2, PA, and NP gene segments of Eurasian LPAIV on a genetic backbone of the H35-like genotype, which was identified in Korea and the Netherlands during 2017. Bayesian molecular clock analysis suggested that the novel reassortant viruses were generated most likely during the fall migration/wintering season of migratory waterfowl in 2017. Considering the continued emergence and spread of clade 2.3.4.4 HPAIV, enhanced surveillance of wild waterfowl is needed for early detection of HPAIV incursions.

Genetic and pathogenic characteristics of clade 2.3.2.1c H5N1 highly pathogenic avian influenza viruses isolated from poultry outbreaks in Laos during 2015-2018.

Since 2004, there have been multiple outbreaks of H5 highly pathogenic avian influenza (HPAI) viruses in Laos. Here, we isolated H5N1 HPAI viruses from poultry outbreaks in Laos during 2015-2018 and investigated their genetic characteristics and pathogenicity in chickens. Phylogenetic analysis revealed that the isolates belonged to clade 2.3.2.1c and that they differed from previous Laos viruses with respect to genetic composition. In particular, the isolates were divided into two genotypes, each of which had a different NS segments. The results of possible migration analysis suggested a high likelihood that the Laos isolates were introduced from neighbouring countries, particularly Vietnam. The recent Laos isolate, A/Duck/Laos/NL-1504599/2018, had an intravenous pathogenicity index score of 3.0 and showed a 50% chicken lethal dose of 102.5 EID50 /0.1 ml, indicating high pathogenicity. The isolated viruses exhibited no critical substitution in the markers associated with mammalian adaptation, but possess markers related to neuraminidase inhibitor resistance. These results emphasize the need for ongoing surveillance of circulating influenza virus in South-East Asia, including Laos, to better prepare for and mitigate global spread of H5 HPAI.

Genetic characteristics and pathogenesis of H5 low pathogenic avian influenza viruses from wild birds and domestic ducks in South Korea.

H5 and H7 subtypes of low pathogenic avian influenza viruses (LPAIVs) can mutate to highly pathogenic forms and are therefore subject to stringent controls. We characterized H5 LPAIVs isolated from wild-bird habitats and duck farms in South Korea from 2010 to 2017. Through nationwide active surveillance for AIVs, 59 H5 LPAIVs were isolated from wild-bird habitats (a mean annual rate of 5.3% of AIV isolations). In 2015, one LPAI H5N3 strain was isolated on a duck farm. Phylogenetic analysis revealed that the hemagglutinin (HA) gene of H5 isolates belonged to the Eurasian lineage, classified into three subgroups (HA-II, HA-III, and HA-IV). The H5 LPAIVs of the HA-III and HA-IV subgroups appeared in 2015 and 2017 in unusually high proportions (13.1% and 14.4%, respectively). In gene-constellation analysis, H5 LPAIVs isolated from 2015 to 2017 constituted ≥ 35 distinct genotypes, representing high levels of genetic diversity. Representative strains of three HA subgroups replicated restrictively in specific-pathogen-free chickens. Among the 11 isolates that were tested, 10 infected and replicated in mice without prior adaptation. The frequency of recent H5 LPAIV isolates with high genetic diversity indicates the importance of continued surveillance in both wild birds and poultry to monitor genetic and pathobiological changes.

Pathogenicity of clade 2.3.4.4 H5N6 highly pathogenic avian influenza virus in three chicken breeds from South Korea in 2016/2017.

In 2016, novel H5N6 highly pathogenic avian influenza virus emerged in Korea. During the outbreak, the virus caused the largest culling, especially in brown chicken lines. We determined the pathogenicity and transmissibility of the virus in 2 white chicken lines of the specific pathogen-free chickens, broilers and brown chicken line of Korean native chicken (KNC). A KNC had a longer virus shedding period and longer mean death time than others. Our study showed that this characteristic in the KNC might have contributed to a farm-to-farm transmission of the brown chicken farms.

Genetic evidence for the intercontinental movement of avian influenza viruses possessing North American-origin nonstructural gene allele B into South Korea.

Avian influenza viruses (AIVs) are genetically separated by geographical barriers, resulting in the independent evolution of North American and Eurasian lineages. In the present study, to determine whether AIVs possessing the North American-origin nonstructural (NS) gene were previously introduced into South Korea, we performed a genetic analysis of AIVs isolated from fecal samples of migratory birds. We detected seven viruses possessing the North American-origin NS allele B among 413 AIV-positive samples obtained during AI surveillance between 2012 and 2017. We found evidence for the intercontinental transmission of at least three genetically distinct clusters of the B allele of the North American-origin NS gene into Eurasia at a low frequency. The host species of three viruses were identified as the greater white-fronted goose (Anser albifrons) using a DNA barcoding technique. Moreover, we used GPS-CDMA-based telemetry to determine the migration route of the greater white-fronted goose between the Far East of Russia and South Korea and found that this species may play an important role as an intermediate vector in the intercontinental transmission of AIVs. To improve our understanding of the role of wild birds in the ecology of AIVs, advanced AIV surveillance is required in the Far East of Russia as well as in Alaska region of Beringia accompanied by host identification and wild bird tracking.

Novel reassortants of clade 2.3.4.4 H5N6 highly pathogenic avian influenza viruses possessing genetic heterogeneity in South Korea in late 2017.

Novel H5N6 highly pathogenic avian influenza viruses (HPAIVs) were isolated from duck farms and migratory bird habitats in South Korea in November to December 2017. Genetic analysis demonstrated that at least two genotypes of H5N6 were generated through reassortment between clade 2.3.4.4 H5N8 HPAIVs and Eurasian low pathogenic avian influenza virus in migratory birds in late 2017, suggesting frequent reassortment of clade 2.3.4.4 H5 HPAIVs and highlighting the need for systematic surveillance in Eurasian breeding grounds.