Genetic and antigenic characterization of H5 and H7 avian influenza viruses isolated from migratory waterfowl in Mongolia from 2017 to 2019.

The circulation of highly pathogenic avian influenza viruses (HPAIVs) of various subtypes (e.g., H5N1, H5N6, H5N8, and H7N9) in poultry remains a global concern for animal and public health. Migratory waterfowls play important roles in the transmission of these viruses across countries. To monitor virus spread by wild birds, active surveillance for avian influenza in migratory waterfowl was conducted in Mongolia from 2015 to 2019. In total, 5000 fecal samples were collected from lakesides in central Mongolia, and 167 influenza A viruses were isolated. Two H5N3, four H7N3, and two H7N7 viruses were characterized in this study. The amino acid sequence at hemagglutinin (HA) cleavage site of those isolates suggested low pathogenicity in chickens. Phylogenetic analysis revealed that all H5 and H7 viruses were closely related to recent H5 and H7 low pathogenic avian influenza viruses (LPAIVs) isolated from wild birds in Asia and Europe. Antigenicity of H7Nx was similar to those of typical non-pathogenic avian influenza viruses (AIVs). While HPAIVs or A/Anhui/1/2013 (H7N9)-related LPAIVs were not detected in migratory waterfowl in Mongolia, sporadic introductions of AIVs including H5 and H7 viruses into Mongolia through the wild bird migration were identified. Thus, continued monitoring of H5 and H7 AIVs in both domestic and wild birds is needed for the early detection of HPAIVs spread into the country.

Pathogenicity in domestic ducks and mice of highly pathogenic H5N1 clade 2.3.2.1 influenza viruses recently circulating in Eastern Asia.

Influenza virus A (H5N1) clade 2.3.2.1 has recently caused widespread outbreaks of disease in domestic poultry and wild birds in Eastern Asia. In the current study, the antigenicity and pathogenicity of three clade 2.3.2.1 viruses (Ck/Kr/Gimje/08, Ws/Mongolia/1/09, and Ws/Mongolia/7/10) were investigated in domestic ducks and mice. The H5N1 influenza viruses in this study were antigenically similar to each other (r-values of 0.35-1.4). The three viruses replicated systemically in all tissues tested in domestic ducks, indicating high pathogenicity. However, the viruses produced different clinical signs and mortality rates: Ck/Kr/Gimje/08 and Ws/Mongolia/1/09 resulted in 100% mortality with severe neurological signs, whereas Ws/Mongolia/7/10 resulted in 50% mortality with relatively mild neurological signs. In mice, infection with Ck/Kr/Gimje/08 and Ws/Mongolia/7/10 resulted in weight loss that peaked at 4 days post-infection (22.3% and 20.8%, respectively), same MLD50 (2.2 Log10 EID50) and systemic replication. The three viruses had K deletion at the -2 position of the HA1-connecting peptide (PQRERRRK-R), which is associated with increased virulence in domestic ducks and harbored NA stalk deletion, NS1 deletion and mutation of P42S in NS1, and full length (90aa) in PB1-F2, which confer increased virulence in mice. Our study shows that clade 2.3.2.1 viruses from Korea and Mongolia are antigenically similar and highly pathogenic in both domestic ducks and mice. Moreover, we provide molecular determinants of the clade 2.3.2.1 viruses associated with the pathogenicity in domestic ducks and mice, respectively.

Highly pathogenic avian influenza virus among wild birds in Mongolia.

Mongolia combines a near absence of domestic poultry, with an abundance of migratory waterbirds, to create an ideal location to study the epidemiology of highly pathogenic avian influenza virus (HPAIV) in a purely wild bird system. Here we present the findings of active and passive surveillance for HPAIV subtype H5N1 in Mongolia from 2005-2011, together with the results of five outbreak investigations. In total eight HPAIV outbreaks were confirmed in Mongolia during this period. Of these, one was detected during active surveillance employed by this project, three by active surveillance performed by Mongolian government agencies, and four through passive surveillance. A further three outbreaks were recorded in the neighbouring Tyva Republic of Russia on a lake that bisects the international border. No HPAIV was isolated (cultured) from 7,855 environmental fecal samples (primarily from ducks), or from 2,765 live, clinically healthy birds captured during active surveillance (primarily shelducks, geese and swans), while four HPAIVs were isolated from 141 clinically ill or dead birds located through active surveillance. Two low pathogenic avian influenza viruses (LPAIV) were cultured from ill or dead birds during active surveillance, while environmental feces and live healthy birds yielded 56 and 1 LPAIV respectively. All Mongolian outbreaks occurred in 2005 and 2006 (clade 2.2), or 2009 and 2010 (clade 2.3.2.1); all years in which spring HPAIV outbreaks were reported in Tibet and/or Qinghai provinces in China. The occurrence of outbreaks in areas deficient in domestic poultry is strong evidence that wild birds can carry HPAIV over at least moderate distances. However, failure to detect further outbreaks of clade 2.2 after June 2006, and clade 2.3.2.1 after June 2010 suggests that wild birds migrating to and from Mongolia may not be competent as indefinite reservoirs of HPAIV, or that HPAIV did not reach susceptible populations during our study.

Avian influenza virus surveillance in hunter-harvested waterfowl, Texas coast, September 2009-January 2010.

Wild waterfowl are considered the natural reservoir of type A influenza viruses, and the migratory nature of many waterfowl species presents a possible vehicle for global dissemination of these infectious agents. In order to fully understand the ecology of influenza viruses, multiyear surveillance efforts are critical, particularly in understudied areas, such as waterfowl wintering areas. Herein we report results obtained during the fifth year ofa 5-yr avian influenza virus (AIV) surveillance project conducted on waterfowl wintering grounds of the Texas Coast. During year 5, the 2009-2010 hunting season (September, November-January), 655 cloacal swabs were collected from hunter-harvested waterfowl and screened for AIV by real-time RT-PCR (rRT-PCR) followed by virus isolation on all positive samples. Molecular methods were used for subtyping all AIV isolates. Sixty-five (9.5%) samples were positive for AIV by rRT-PCR, and 24 (3.7%) AIVs were isolated. Eight different hemagglutinin (H3, 4, 5, 6, 8, 9, 10, and 11) and seven different neuraminidase (N1, 2, 3, 4, 6, 8, and 9) subtypes were identified. This was the first year H8 and H9 were isolated throughout the 5-yr survey. Our results support the fact that continued multiyear surveillance of natural reservoirs, particularly in understudied areas, is needed in order to better understand the ecology of AIVs in nature.

Antigenic and genetic analysis of H3N8 influenza viruses isolated from horses in Japan and Mongolia, and imported from Canada and Belgium during 2007-2010.

A/equine/Kanazawa/1/2007 (H3N8), A/equine/Hokkaido/I828/2008 (H3N8) and A/equine/Mongolia/1/2008 (H3N8) were isolated from infected horses. A/equine/Yokohama/aq19/2009 (H3N8) and A/equine/Yokohama/aq13/2010 (H3N8) were isolated from horses imported from Canada and Belgium examined at the Animal Quarantine Service in Yokohama, Japan. In the present study, these five isolates were genetically and antigenically analyzed. Phylogenetic analysis of hemagglutinin (HA) and neuraminidase (NA) genes showed that three isolates from horses in Japan and imported from Canada belonged to the same branch, clade 1 of the Florida sublineage, while the isolates from horses in Mongolia and imported from Belgium belonged to another branch, clade 2 of the Florida sublineage. Reactivity patterns of a panel of monoclonal antibodies to the HA of A/equine/Kanazawa/1/2007 (H3N8) with the five isolates indicate that the HAs of these viruses were antigenically similar to each other and to the reference strains A/equine/La Plata/1/1993 (H3N8) and A/equine/Avesta/1/1993 (H3N8). The present findings indicate that extensive antigenic variation has not accumulated among H3N8 influenza viruses in horses.

Genetic analyses of H5N1 avian influenza virus in Mongolia, 2009 and its relationship with those of eastern Asia.

In May and August 2009, 14 highly pathogenic H5N1 isolates were obtained from migratory birds in central Mongolia. To trace the genetic lineage of the isolates, nucleotide sequences of all eight genes were determined and phylogenetically analyzed. Hemagglutinin and nucleoprotein genes were clustered in clade 2.3.2. The polymerase acidic gene was related to the isolates of South Korea and Japan obtained in 2003 and 2004 outbreaks, and a migratory duck isolate from Jiangxi, China. The neuraminidase and other internal genes were closely related to those of clade 2.3.4 viruses. The results indicate evolving genetic diversity of the hemagglutinin gene and acquisition of different polymerase acidic gene in the 2009 Mongolian isolates, likely via bird migration. Prevention of potentially wider outbreak in domestic poultry and accurate monitoring of H5N1 genetic mutation will require continuous monitoring for H5N1 in both domestic and wild birds, and will necessitate international cooperation with neighboring countries sharing migratory flyways.

Molecular epidemiology of rabies virus in Mongolia, 2005-2008.

The objective of this study was to determine the genetic diversity of rabies virus (RABV) in Mongolia based on the nucleotide sequences of viral N gene. A total of 24 rabies-positive samples from seven different domestic and wild animal species collected in western and central Mongolia between 2005 and 2008 were examined for their N gene sequences. The results showed that the endemic Mongolian RABVs could be divided into two different groups closely related to the Steppe-type and Arctic-like viruses isolated in Russia.