Surveillance and Genetic Analysis of Low-Pathogenicity Avian Influenza Viruses Isolated from Feces of Wild Birds in Mongolia, 2021 to 2023.

The introduction of novel highly pathogenic (HPAI) viruses into Korea has been attributed to recombination events occurring at breeding sites in the Northern Hemisphere. This has increased interest in monitoring and genetically analyzing avian influenza viruses (AIVs) in northern regions, such as Mongolia, which share migratory bird flyways with Korea. AIVs in Mongolia were monitored by analyzing 10,149 fecal samples freshly collected from wild birds from April to October in 2021 to 2023. The prevalence of AIVs in wild birds was 1.01%, with a total of 77 AIVs isolated during these 3 years. These 77 AIVs included hemagglutinin (HA) subtypes H1, H2, H3, H4, H6, H10 and H13 and neuraminidase (NA) subtypes N1, N2, N3, N6, N7 and N8. The most frequently detected subtype combinations were H3N8 (39.0%) and H4N6 (19.5%), although HPAI viruses were not detected. Genetic analysis indicated that theses AIVs isolated from Mongolian samples were closely related to AIVs in wild birds in Korea, including those of Eurasian lineage. These findings indicate the necessity of continuous AIV surveillance and monitoring, as HPAI viruses introduced into Korea may derive from strains in Mongolia.

Comparison of the Whole-Genome Sequence of the African Swine Fever Virus from a Mongolian Wild Boar with Genotype II Viruses from Asia and Europe.

African swine fever (ASF) is a highly contagious and severe viral hemorrhagic disease in domestic and wild pigs. ASF seriously affects the global swine industry as the mortality rate can reach 100% with highly virulent strains. In 2007, ASF was introduced into the Caucasus and spread to Russia and later into other European and Asian countries. This study reported the first whole-genome sequence (WGS) of the ASF virus (ASFV) that was detected in a Mongolian wild boar. This sequence was then compared to other WGS samples from Asia and Europe. Results show that the ASFV Genotype II from Mongolia is similar to the Asian Genotype II WGS. However, there were three nucleotide differences found between the Asian and European genome sequences, two of which were non-synonymous. It was also observed that the European Genotype II ASFV WGS was more diverse than that of the Asian counterparts. The study demonstrates that the ASFV Genotype II variants found in wild boars and domestic pigs are highly similar, suggesting these animals might have had direct or indirect contact, potentially through outdoor animal breeding. In conclusion, this study provides a WGS and mutation spectrum of the ASFV Genotype II WGS in Asia and Europe and thus provides important insights into the origin and spread of ASFV in Mongolia.

Whole genome sequencing and phylogenetic analysis of African swine fever virus detected in a backyard pig in Mongolia, 2019.

African swine fever (ASF) is a highly contagious and fatal disease affecting domestic and wild pigs caused by the African swine fever virus (ASFV). Since the first outbreak in China in August 2018, ASF has spread rapidly in Asia. and the first case in Mongolia was confirmed in January 2019. In this study, we report the first whole genome sequence of an ASFV (ASFV SS-3/Mongolia/2019) detected from a backyard pig in Mongolia in February 2019 using whole genome sequencing. We analyzed their phylogenetic relationship with other genotype II ASFVs from Eurasia. The ASFV SS-3/Mongolia/2019 belonged to genotype II (p72 and p54), serogroup 8 (CD2v), Tet-10a variant (pB602L), and IGRIII variant (intergenic region between the I73R/I329L genes). A total of five amino acid substitutions were observed in MGF 360-10L, MGF 505-4R, MGF 505-9R, NP419L, and I267L genes compared to the ASFV Georgia 2007/1 virus. ML phylogenetic analysis of the whole genome sequence showed that the virus shares a high nucleotide sequence identity with ASFVs recently identified in Eastern Europe and Asia and clustered with the ASFV/Zabaykali/WB5314/2020|Russia|2020 virus which was identified at the border between the Russian Federation and Mongolia in 2020. Our results suggest that trans boundary spread of ASF occurred through close geographic proximity.

Intense Innate Immune Responses and Severe Metabolic Disorders in Chicken Embryonic Visceral Tissues Caused by Infection with Highly Virulent Newcastle Disease Virus Compared to the Avirulent Virus: A Bioinformatics Analysis.

The highly virulent Newcastle disease virus (NDV) isolates typically result in severe systemic pathological changes and high mortality in Newcastle disease (ND) illness, whereas avirulent or low-virulence NDV strains can cause subclinical disease with no morbidity and even asymptomatic infections in birds. However, understanding the host's innate immune responses to infection with either a highly virulent strain or an avirulent strain, and how this response may contribute to severe pathological damages and even mortality upon infection with the highly virulent strain, remain limited. Therefore, the differences in epigenetic and pathogenesis mechanisms between the highly virulent and avirulent strains were explored, by transcriptional profiling of chicken embryonic visceral tissues (CEVT), infected with either the highly virulent NA-1 strain or the avirulent vaccine LaSota strain using RNA-seq. In our current paper, severe systemic pathological changes and high mortality were only observed in chicken embryos infected with the highly virulent NA-1 strains, although the propagation of viruses exhibited no differences between NA-1 and LaSota. Furthermore, virulent NA-1 infection caused intense innate immune responses and severe metabolic disorders in chicken EVT at 36 h post-infection (hpi), instead of 24 hpi, based on the bioinformatics analysis results for the differentially expressed genes (DEGs) between NA-1 and LaSota groups. Notably, an acute hyperinflammatory response, characterized by upregulated inflammatory cytokines, an uncontrolled host immune defense with dysregulated innate immune response-related signaling pathways, as well as severe metabolic disorders with the reorganization of host-cell metabolism were involved in the host defense response to the CEVT infected with the highly virulent NA-1 strain compared to the avirulent vaccine LaSota strain. Taken together, these results indicate that not only the host's uncontrolled immune response itself, but also the metabolic disorders with viruses hijacking host cell metabolism, may contribute to the pathogenesis of the highly virulent strain in ovo.

Highly Pathogenic Avian Influenza Clade 2.3.4.4 Subtype H5N6 Viruses Isolated from Wild Whooper Swans, Mongolia, 2020.

We identified clade 2.3.4.4 highly pathogenic avian influenza A(H5N6) viruses from whooper swans (Cygnus cygnus) found dead in Mongolia. The identification of these infections in wild birds in this area is of concern because of the potential for virus dissemination during fall migration.

Identification and a full genome analysis of novel camel hepatitis E virus strains obtained from Bactrian camels in Mongolia.

Hepatitis E virus (HEV) infects humans and a wide variety of other mammalian hosts. Recently, HEV strains belonging to genotype 8 (G8) within the Orthohepevirus A species of the Hepeviridae family, were identified in Bactrian camels (Camelus bactrianus) in China. The Bactrian camel (also known as the Mongolian camel) is native to the steppes of Central Asia. However, the HEV strains of Mongolian camels have not been examined. Among 200 serum samples from domestic Bactrian camels raised on 6 farms, in 6 soums in 3 provinces; 71 (35.5 %) were positive for anti-HEV IgG, with prevalence differing by farm (soum) (4.2-75.0 %); and 2 camels (1.0 %) that had been raised in Bogd, Bayankhongor Province, which had the highest seroprevalence among the six studied areas, were positive for HEV RNA. The two HEV strains (BcHEV-MNG140 and BcHEV-MNG146) obtained from the viremic camels in the present study shared 97.7 % nucleotide identity. They were closest to the reported G8 Chinese camel HEV strains but differed from them by 13.9-14.3 % over the entire genome, with a nucleotide difference of 24.0-26.5 % from the reported G1-G7 HEV strains. A phylogenetic tree indicated that the BcHEV-MNG140 and BcHEV-MNG146 strains were located upstream of a clade consisting of the Chinese camel HEV strains and formed a cluster with them, with a bootstrap value of 100 %, suggesting that they may represent a novel subtype within G8. These results indicate a high prevalence of HEV infection in Mongolian camels and suggest that the variability of camel HEV genomes is markedly high.

Putative Novel Serotypes '33' and '35' in Clinically Healthy Small Ruminants in Mongolia Expand the Group of Atypical BTV.

Between 2015 and 2018, we identified the presence of three so-far-unknown Bluetongue virus (BTV) strains (BTV-MNG1/2018, BTV-MNG2/2016, and BTV-MNG3/2016) circulating in clinical healthy sheep and goats in Mongolia. Virus isolation from EDTA blood samples of BTV-MNG1/2018 and BTV-MNG3/2016 was successful on the mammalian cell line BSR using blood collected from surveillance. After experimental inoculation of goats with BTV-MNG2/2016 positive blood as inoculum, we observed viraemia in one goat and with the EDTA blood of the experimental inoculation, the propagation of BTV-MNG2/2016 in cell culture was successful on mammalian cell line BSR as well. However, virus isolation experiments for BTV-MNG2/2016 on KC cells were unsuccessful. Furthermore, we generated the complete coding sequence of all three novel Mongolian strains. For atypical BTV, serotyping via the traditional serum neutralization assay is not trivial. We therefore sorted the 'putative novel atypical serotypes' according to their segment-2 sequence identities and their time point of sampling. Hence, the BTV-MNG1/2018 isolate forms the 'putative novel atypical serotype' 33, the BTV-MNG3/2016 the 'putative novel atypical serotype' 35, whereas the BTV-MNG2/2016 strain belongs to the same putative novel atypical serotype '30' as BTV-XJ1407 from China.

Introduction of Avian Influenza A(H6N5) Virus into Asia from North America by Wild Birds.

An avian influenza A(H6N5) virus with all 8 segments of North American origin was isolated from wild bird feces in South Korea. Phylogenetic analysis suggests that this virus may have been introduced into Asia by wild birds, highlighting the role of wild birds in the dispersal of these viruses.

The first isolation and identification of canine parvovirus (CPV) type 2c variants during 2016-2018 genetic surveillance of dogs in Mongolia.

Canine parvovirus type 2 (CPV-2) causes a highly contagious and fatal disease, developing into acute hemorrhagic enteritis and myocarditis, in dogs. CPV-2 has evolved, generating antigenic variants CPV-2a/2b/2c that are globally distributed. However, investigating molecular characterization of CPV-2 among dog populations in Mongolia has been limited. Herein, 42 stool samples were collected from dogs with clinical signs of infection, and conventional PCR assays were employed to detect CPV-2 in 23. Our results indicated that during 2016-2018, the new CPV-2a and 2c subtypes were detected in 34.7% of the samples, and the new CPV-2b subtype was detected in 30.4% of samples. VP2 protein sequence analysis and next-generation sequencing of the complete viral genome confirmed these antigenic types. However, sequence analysis indicated new and unreported mutations, Pro580Thr, and Tyr584His in the CPV-2c subtype. From a PCR-positive sample, CPV-2c was successfully isolated, and we performed an immunofluorescence assay for antigen detection. Additionally, we performed genetic characterization and phylogenetic analysis to investigate genetic diversity among isolates from the region, resulting in high CPV-2 genetic diversity in the Mongolian dog population. Striking similarities were also observed between sequences of the strains isolated from Mongolia and China over a similar time span.

Molecular characterization and genetic diversity of avian paramyxovirus type 4 isolated in South Korea from 2013 to 2017.

In recent years, avian paramyxovirus type 4 (APMV-4) frequently isolated from wild and domestic bird populations particularly waterfowls worldwide. However, molecular characteristics and genetic diversity of APMV-4 are uncertain, owing to the limited availability of sequence information. A total of 11 APMV-4 strains from 9850 fecal, swab, and environmental samples were isolated during the surveillance program in wintering seasons of 2013-2017 in South Korea. We performed genetic characterization and phylogenetic analysis to investigate the genetic diversity and relatedness between isolates from the region. We report high APMV-4 genetic diversity (multiple genotypes and sub-genotypes) among wild bird and poultry populations in Korea and that the potential virus exchange occurs between neighboring countries via wild bird migration. Furthermore, our study results suggest the possibility of transcontinental transmission of APMV-4 between Asia and Europe.

Molecular Characterization of Avian Paramyxovirus Types 4 and 8 Isolated from Wild Migratory Waterfowl in Mongolia.

Avian paramyxoviruses (APMVs) constitute some of the most globally prevalent avian viruses and are frequently isolated from wild migratory bird species. Using 1,907 fresh fecal samples collected during the 2012 avian influenza surveillance program, we identified two serotypes of APMV: APMV-4 ( n=10) and APMV-8 ( n=1). Sequences for these isolates phylogenetically clustered with Asian APMV-4 and APMV-8 recently isolated from wild birds in Korea, Japan, China, and Kazakhstan. Analysis by DNA barcoding indicated that the Mongolian APMV-4 and APMV-8 strains were isolated from Anseriformes species including Mallards ( Anas platyrhynchos) and Whooper Swans ( Cygnus cygnus). The close genetic relatedness to Asian isolates, and to similar host species, suggested that wild bird species in the Anatidae family might play an important role as a natural reservoir in the spread of APMV-4 and APMV-8. However, we did not find conclusive evidence to support this hypothesis owing to the limited number of strains that could be isolated. Enhanced surveillance of poultry and wild bird populations in Asia is therefore crucial for the understanding of global AMPV transmission, ecology, evolution, and epidemiology.

Complete Genome Sequence of an Avian Paramyxovirus Type 4 Strain Isolated from Domestic Duck at a Live Bird Market in South Korea.

We report here the first full-genome sequence of an avian paramyxovirus type 4 (APMV-4) strain isolated from a domestic mallard duck at a live bird market in South Korea. Phylogenetic analyses provide genetic information on a new genetic clade, APMV-4, isolated from a domestic duck and evidence of APMV-4 exchange between poultry and wild birds.

Experimental infection of dogs with highly pathogenic avian influenza virus (H5N8).

During the highly pathogenic avian influenza (HPAI) H5N8 virus outbreak in Korea, a dog in layer farm contaminated by H5N8 was reported seropositive for HPAI H5N8. To investigate the possibility of adaptation and transmission of HPAI H5N8 to dogs, we experimentally inoculated dogs with H5N8. Viral genes were weakly detected in nasal swabs and seroconversions in inoculated and contact dogs. Although the H5N8 virus did not induced severe clinical signs to dogs, the results suggest that surveillance of farm dogs should continue as a species in which the avian influenza virus may acquire infectivity to mammals through frequent contact with the virus.

Experimental Infection of Chickens with Intercontinental Reassortant H9N2 Influenza Viruses from Wild Birds.

The H9N2 subtype of low pathogenic avian influenza (LPAI) virus is the most prevalent LPAI in domestic poultry. We previously reported the natural reassortant H9N2 viruses between North American and Eurasian lineages isolated from wild birds in Korea. These viruses were identified in China and Alaska, providing evidence of intercontinental dispersal. In this study, we evaluated the infectivity, transmissibility, and pathogenic potential of these H9N2 viruses and Eurasian H9N2 virus identified from wild birds using specific-pathogen-free chickens. Three-week-old chickens were infected intranasally. All of these reassortant H9N2 viruses could not be replicated and transmitted in chickens. On the other hand, three out of eight chickens inoculated with the Eurasian H9N2 virus shed detectable levels of virus and showed seroconversion but did not show contact transmission of the virus. Although all reassortant H9N2 viruses could not be replicated and transmitted in chickens, and although there are no reports on reassortant H9N2 virus infection in poultry farms until now, monitoring of reassortant H9N2 viruses should be continued to prepare for the advent and evolution of these viruses.

Pre-immune state induced by chicken interferon gamma inhibits the replication of H1N1 human and H9N2 avian influenza viruses in chicken embryo fibroblasts.

BACKGROUND: Interferon gamma (IFN-γ), an immunoregulatory cytokine, is known to control many microbial infections. In a previous study, chicken interferon gamma (chIFN-γ) was found to be up-regulated following avian influenza virus (AIV) infection in specific pathogen-free chickens. We aimed to investigate whether the pre-immune state induced by chIFN-γ could generate an antiviral response against influenza virus. METHODS: We generated a chIFN-γ-expressing plasmid and transfected it into chicken embryo fibroblasts (CEFs) and then infected the cells with human origin H1N1 or avian origin H9N2 influenza viruses. Viral titers of culture medium were evaluated in MDCK cell and the viral RNA and IFN-stimulated genes (ISGs) were then quantified by real-time reverse transcriptase polymerase. To further evaluate the role of the antiviral effect of chIFN-γ by using a backward approach, synthetic small interfering RNAs (siRNA) targeting chIFN-γ were used to suppress chIFN-γ. RESULTS: The chIFN-γ-stimulated CEFs inhibited the replication of viral RNA (vRNA) and showed a mild decrease in the infectious virus load released in the culture medium. Compared to the mock-transfected control, the messenger RNA (mRNA) levels of type I IFNs and IFN-stimulated genes were up-regulated in the cells expressing chIFN-γ. After treatment with the siRNA, we detected a higher expression of viral genes than that observed in the mock-transfected control. CONCLUSIONS: Our results suggest that apart from the important role played by chIFN-γ in the antiviral state generated against influenza virus infection, the pre-immune state induced by chIFN-γ can be helpful in mitigating the propagation of influenza virus.

Application of Diagnostic Microarray Technique in Subtyping and Pathotyping of Avian Influenza Viruses Isolated in Mongolia

Asian-lineage H5 highly pathogenic avian influenza (HPAI) viruses have caused continuous outbreaks in poultry and wild birds. Development of rapid and accurate diagnostic methods is needed for preventing further spread of the virus and reducing the time required for eradication of the virus. We developed a low-density microarray for the rapid detection and identification of avian influenza virus subtypes H5, H7, and H9 and their pathotypes in a previous study. In the present study, we evaluated previously developed diagnostic microarray using avian influenza viruses isolated in Mongolia, including H5 HPAI viruses. All H5 HPAI viruses isolated in Mongolia were shown as H5-specific and highly pathogenic pattern in the microarray. H2, H3 and H12 viruses isolated in Mongolia used in this study did not show any H5, H7 and H9 patterns. These results indicated that this diagnostic microarray has enormous potential for the rapid subtyping and pathotyping of influenza viruses, including viruses isolated in Mongolia.

Viscerotropic velogenic Newcastle disease virus replication in feathers of infected chickens

Newcastle disease viruses (NDVs) cause systemic diseases in chickens with high mortality. However, little is known about persistence of NDVs in contaminated tissues from infected birds. In this study, we examined viral replication in the feather pulp of chickens inoculated with viscerotropic velogenic NDV (vvNDV) genotype VII. Reverse transcription real-time PCR and immunohistochemistry were used to investigate viral persistence in the samples. vvNDV was detected in the oropharynx and cloaca and viral antigens were detected in the feathers, suggesting that feathers act as sources of viral transmission.

Virus-like particle vaccine confers protection against a lethal newcastle disease virus challenge in chickens and allows a strategy of differentiating infected from vaccinated animals.

In this study, we developed Newcastle disease virus (NDV) virus-like particles (VLPs) expressing NDV fusion (F) protein along with influenza virus matrix 1 (M1) protein using the insect cell expression system. Specific-pathogen-free chickens were immunized with oil emulsion NDV VLP vaccines containing increasing dosages of VLPs (0.4, 2, 10, or 50 µg of VLPs/0.5-ml dose). Three weeks after immunization, the immunogenicity of the NDV VLP vaccines was determined using a commercial enzyme-linked immunosorbent assay (ELISA) kit, and a lethal challenge using a highly virulent NDV strain was performed to evaluate the protective efficacy of the NDV VLP vaccines. NDV VLP vaccines elicited anti-NDV antibodies and provided protection against a lethal challenge in a dose-dependent manner. Although the VLP vaccines containing 0.4 and 2 µg of VLPs failed to achieve high levels of protection, a single immunization with NDV VLP vaccine containing 10 or 50 µg could fully protect chickens from a lethal challenge and greatly reduced challenge virus shedding. Furthermore, we could easily differentiate infected from vaccinated animals (DIVA) using the hemagglutination inhibition (HI) test. These results strongly suggest that utilization of NDV VLP vaccine in poultry species may be a promising strategy for the better control of NDV.