key: cord-1037986-7m3dh8hu authors: Nomura, Naoki; Sakoda, Yoshihiro; Endo, Mayumi; Yoshida, Hiromi; Yamamoto, Naoki; Okamatsu, Masatoshi; Sakurai, Kenji; Hoang, Nam Van; Nguyen, Long Van; Chu, Huy Duc; Tien, Tien Ngoc; Kida, Hiroshi title: Characterization of avian influenza viruses isolated from domestic ducks in Vietnam in 2009 and 2010 date: 2011-11-09 journal: Arch Virol DOI: 10.1007/s00705-011-1152-3 sha: 436dc38d42e3cab8af470f486936cb1f077247b8 doc_id: 1037986 cord_uid: 7m3dh8hu In the surveillance of avian influenza in Vietnam, 26 H9N2, 1 H3N2, 1 H3N8, 7 H4N6, 3 H11N3, and 1 H11N9 viruses were isolated from tracheal and cloacal swab samples of 300 domestic ducks in April 2009, and 1 H9N6 virus from 300 bird samples in March 2010. Out of the 27 H9 virus isolates, the hemagglutinins of 18 strains were genetically classified as belonging to the sublineage G1, and the other nine belonged to the Korean sublineage. Phylogenetic analysis revealed that one of the 27 H9 viruses was a reassortant in which the PB2 gene belonged to the Korean sublineage and the other seven genes belonged to the G1 sublineage. Three representative H9N2 viruses were intranasally inoculated into ducks, chickens, pigs, and mice. On the basis of experimental infection studies, it was found that each of the three viruses readily infected pigs and replicated in their upper respiratory tracts, and they infected chickens with slight replication. Viruses were recovered from the lungs of mice inoculated with two of the three isolates. The present results reveal that H9 avian influenza viruses are prevailing and genetic reassortment occurs among domestic ducks in Vietnam. It is recommended that careful surveillance of swine influenza with H9 viruses should be performed to prepare for pandemic influenza. Avian influenza viruses of various subtypes are circulating in poultry in Asian countries [1, 15, 20, 30, 40] . In particular, H9N2 influenza virus is present in poultry in Eurasian countries [9] [10] [11] 25] . Since H9N2 viruses were isolated from quails in Hong Kong in 1988, they have become prevalent in live-bird markets and poultry farms in Asia [8, 34] . H9N2 virus infections have greatly affected not only the poultry industry but also public health [8, 40] . The hemagglutinin (HA) genes of Eurasian H9N2 viruses have been phylogenetically divided into G1, Y280, and Korean sublineages [10] . H9N2 viruses do not usually cause severe disease in poultry, but co-infection of H9N2 viruses with bacteria such as Staphylococcus aureus, Haemophilus paragallinarum, or attenuated coronavirus vaccine exacerbates the disease [12, 21] . H9N2 viruses were also isolated from domestic pigs in China [39] and Korea, and from humans with febrile respiratory illness in Hong Kong in 1998 Kong in , 1999 Kong in , 2003 Kong in , 2008 Kong in , and 2009 . Thus, it is postulated that H9N2 virus may cause pandemic influenza in humans. In our laboratory, avian influenza has been surveyed in Japan, Alaska, Siberia, Mongolia, and Australia since 1977 [14, 22, 27, 31, 36, 41] . The isolates were antigenically and phylogenetically analyzed and assessed for pathogenicity in birds and mammals by experimental infection [22, 27, 36, 41] . In the present study, a surveillance of avian influenza was carried out in Vietnam in domestic ducks and wild birds in 2009 and 2010, and the isolates were antigenically and phylogenetically analyzed and their pathogenicity in birds and mammals was assessed. Viruses A/duck/Hong Kong/Y280/1997 (H9N2), A/chicken/Hong Kong/ G9/1997 (H9N2) , and A/duck/Hong Kong/W213/ 1998 (H9N2) of the Y280 sublineage and A/quail/Hong Kong/G1/1997 (H9N2) of the G1 sublineage were provided by Dr. K. F. Shortridge, the University of Hong Kong, China. A/turkey/Wisconsin/1/1966 (H9N2) of the North American lineage was provided by Dr. R. G. Webster, St. Jude Children's Research Hospital, United States of America. A/duck/Hokkaido/49/1998 (H9N2) and A/duck/ Hokkaido /13/2000 (H9N2) of Korean sublineage were isolated from ducks under surveillance in our laboratory [27, 31] . Viruses isolated from domestic ducks in Vietnam in 2009 and 2010 were grown in 10-day-old embryonated chicken eggs, and infectious allantoic fluids were stored at -80°C until use. One hundred tracheal and cloacal swab samples that were viral gene positive from 600 domestic ducks and 207 wild birds (night heron, Nycticorax nycticorax; grey heron, Ardea cinerea; purple heron, Ardea purpurea; chinese pond heron, Ardeola bacchus; chinese egret, Egretta eulophotes; little egret, Egretta garzetta; intermediate egret, Egretta intermedia; cormorant, Phalacrocorax carbo; little cormorant, Microcarbo niger; Japanese bush warbler, Cettia diphone; black-browed reed warbler, Acrocephalus bistrigiceps; olive bulbul, Iole virescens; black capped kingfisher, Halcyon pileata; collared kingfisher, Halcyon chloris; racket tailed treepie, Crypsirina temia; oriental magpie robin, Copsychus saularis; tiger shrike, Lanius tigrinus; yellow bittern, Ixobrychus sinensis; indian cuckoo, Cuculus micropterus; common koel, Eudynamys scolopacea; and black collared starling, Sturnus nigricollis) in April 2009 and March 2010 in southern Vietnam were inoculated into the allantoic cavities of 10-day-old embryonated chicken eggs. Viral RNA was detected by the reverse transcription loop-mediated isothermal amplification (RT-LAMP) method described previously [42] as a screening test for virus isolation. Viral RNAs were extracted from the allantoic fluids of chicken embryos infected with viruses by TRIzol LS Reagent (Invitrogen, CA, USA) and reverse-transcribed using the Uni12 primer [13] and M-MLV reverse transcriptase (Invitrogen). Polymerase chain reaction for amplification of the viral genes was performed using a PTC-200 thermal cycler (Bio-Rad, CA, USA). Direct sequencing of the viral genes was performed using an autosequencer CEQ 2000XL (Beckman Coulter, CA, USA). For phylogenetic analysis, sequence data for these genes together with those from public database were analyzed by the neighbor-joining method [35] using MEGA 5.0 software (http://www.megasoftware.net/). Accession numbers of the gene sequences of the isolates in the present study are as follows: AB545593, AB545594, AB639351-AB639356 (OIE-2313), AB621343, AB639024-AB639030 (OIE-2326), AB545591, AB545592, AB571519-AB571524 (OIE-2327), AB571525-AB571532 (OIE-2328), AB638754-AB638761 (OIE-2390), AB638722-AB638729 (OIE-2576), AB638746-AB638753 (OIE-2581), AB638730-AB638737 (OIE-2582), AB571533-AB571539, AB572587 (OIE-2583), AB638738-AB638745 (OIE-2584), AB638603-AB638610 (OIE-2587), AB638320-AB638327 (OIE-2592), AB638312-AB638319 (OIE-2593), and AB636530-AB636537 (OIE-2595). Subtypes of influenza virus isolates were identified by hemagglutination-inhibition (HI) and neuraminidase-inhibition tests using chicken antisera to the reference strains of influenza viruses [17] . Four-week-old Chelly Valley ducks were purchased from Takikawa Shinseien (Hokkaido, Japan). Four-week-old Boris brown chickens were purchased from Hokuren Co. (Hokkaido, Japan). Three-week-old crossbred (Landrace 9 Duroc 9 Yorkshire) specific-pathogen-free pigs were purchased from Yamanaka Chikusan (Hokkaido, Japan). Four-week-old female BALB/c mice were purchased from Japan SLC, Inc. (Shizuoka, Japan). All procedures were performed according to the animal experiment guidelines of Graduate school of Veterinary Medicine, Hokkaido University. (Dk/VN/OIE-2583 ) were inoculated intranasally into three ducks (100 ll/duck), six chickens (100 ll/chicken), two pigs (1 ml/pig), and ten mice (30 ll/mouse) at a 50% egg infectious dose (EID 50 ) of 10 5.8 EID 50 , 10 5.8 EID 50 , 10 6.8 EID 50 , and 10 5.0 EID 50 , respectively. After the inoculation of each influenza virus into three ducks, laryngopharyngeal and cloacal swabs were collected in minimal essential medium (MEM; Nissui, Tokyo, Japan) with antibiotics (penicillin G potassium, streptomycin sulfate, gentamicin sulfate, and nystatin) daily from 1 to 7 days post-infection (d.p.i.). All ducks were clinically observed for 14 days after inoculation with influenza viruses. After the inoculation of each influenza virus into six chickens, three chickens were sacrificed at 3 d.p.i., and the brain, trachea, lung, and colon were collected and homogenized to make 10% (w/v) suspensions in MEM. The remaining three chickens were clinically observed for 14 days after inoculation. After the inoculation of each influenza virus into two pigs, nasal swabs from these pigs were collected in MEM from 1 to 7 d.p.i. daily, and two pigs were clinically observed for 14 days after inoculation. After the inoculation of each influenza virus into ten mice, five mice were sacrificed at 3 d.p.i., and the lungs were collected and homogenized to make 10% (w/v) suspensions in MEM. The other five mice were clinically observed and their body weight was monitored for 14 days after inoculation. Virus titers in the supernatants of the swabs and the tissue homogenates were determined in 10-day-old embryonated chicken eggs and expressed as the EID 50 /ml and g of tissue, respectively. Antibody responses to the inoculated viruses in ducks, chickens, and pigs at 14 d.p.i. were examined by HI test or enzyme-linked immunosorbent assay (ELISA) [18] . In the present study, surveillance of avian influenza was carried out in Vinh Loi district, Bac Lieu town, and Hoa Binh district in Vietnam in April 2009 and March 2010. Twelve strains (1 H3N2, 1 H3N8, 6 H4N6, 2 H9N2, 1 H11N3, and 1 H11N9) were isolated from 34 RT-LAMPpositive tracheal and cloacal swab samples from 240 domestic ducks in Vinh Loi district. Nine strains (7 H9N2 and 2 H11N3) were isolated from 38 RT-LAMP-positive swab samples from 160 domestic ducks in Bac Lieu town. Nineteen strains (1 H4N6, 17 H9N2, and 1 H9N6) were isolated from 28 RT-LAMP-positive swab samples of 200 domestic ducks in Hoa Binh district ( Table 1 ). All of the viruses were isolated from domestic ducks in households, live-bird markets, and slaughterhouses in Vinh Loi district, Bac Lieu town, and Hoa Binh district in Vietnam ( The sequence data of the HA genes of 27 H9 isolates, including reference strains of three different sublineages, were phylogenetically analyzed by the neighbor-joining method (Fig. 2) . All of the H9 HA genes were classified as belonging to the Eurasian lineage, and the HA genes of 19 and 8 isolates were grouped into the G1 and Korean sublineage, respectively. The partial nucleotide sequence of each gene segment of the isolates was analyzed phylogenetically (Fig. 2) . Gene constellations of the H9N2 virus isolates were divided into three patterns. H9N2 viruses belonging to the G1 sublineage were isolated from domestic ducks in households A and E. On the other hand, H9N2 viruses belonging to the Korean sublineage were isolated in live-bird market G. Furthermore, one of these H9N2 viruses of the G1 sublineage of the HA gene was isolated in live-bird market G, and this virus also possessed a PB2 gene of the Antigenic analysis of the HAs of H9 influenza viruses H9 influenza viruses isolated from domestic ducks in Vietnam were analyzed by the HI test ( Table 2 ). All of the H9 isolates tested reacted with antisera against the H9 viruses of the Korean and G1 sublineages. However, the isolates of the Korean sublineage showed low cross-reactivity to antisera against the H9 viruses of the G1 sublineage, and all isolates in this study showed moderate and low cross-reactivity to antisera against the H9 viruses of the Y280 sublineage and North American lineage, respectively. This suggests that the antigenicity of the H9 isolates of the Korean sublineage is different from that of viruses of the G1 sublineage. It was also found that reactivity patterns of H9 isolates belonging to the G1 and Korean sublineage in the present study were the same as those of the reference strains. Clinical signs were not observed during the experiment in any of the pigs. Viruses were recovered from the nasal swabs of pigs inoculated with each of the three H9N2 isolates, and anti-H9 HA antibodies were detected in the sera of pigs at 14 d.p.i (Table 4 ). Antibodies were not detected in the sera of pig #1 inoculated with Dk/VN/OIE-2327/2009, indicating that the pig was not infected with influenza viruses. Body weight fell in mice inoculated with Dk/VN/OIE-2583/2009, with 15-20% loss from 4 to 8 d.p.i (Fig. 3) . Viruses were recovered from the lungs of mice inoculated with Dk/VN/OIE-2328/2009 and Dk/VN/OIE-2583/2009 at 3 d.p.i. (Table 5) , and anti-H9 HA antibodies were detected in the sera of all mice at 14 d.p.i (Table 6 ). Recently, H9N2 viruses of the G1, Y280, and Korean sublineages have been isolated from wild birds and poultry worldwide [2, 3, 8, 29, 40] . H9N2 viruses have been isolated from pigs and humans in China [4, 39] and Korea, suggesting that the H9N2 virus is a candidate to cause pandemic influenza in humans. Live-bird markets provide an ideal environment for genetic reassortment and interspecies transmission of influenza viruses [24, 26, 28, 38] . In Asia, H9N2 influenza viruses had been isolated only from feral ducks until 1988 [37] , but since then, H9N2 viruses have been isolated from domestic ducks and chickens [5] . H9N3 viruses belonging to the Korean sublineage have been isolated from domestic ducks in Vietnam [30] . In the present study, it was found that H9 viruses belonging to the Korean and G1 sublineages are circulating in domestic ducks in Vietnam, and one of these H9N2 viruses, belonging to the G1 sublineage but possessing the PB2 gene of the Korean sublineage, was isolated from domestic ducks. Thus, genetic reassortment has occurred between viruses of the G1 and Korean sublineages in the poultry population in Vietnam. In this study, H9N2 viruses did not replicate well in chickens and ducks. It has been reported that H9N2 viruses isolated from ducks replicate slightly in chickens [34] , suggesting that the similar results in this study were due to the low susceptibility of chickens to H9N2 viruses. It has also been reported that H9N2 viruses isolated from ducks replicate in only some of the organs in ducks, and viruses of low titer are recovered from tracheal and cloacal swabs [11, 32] . The present results were similar to those of the previous reports. In this animal experiment, we collected These factors might affect the titer of recovered virus. In mice, H9N2 viruses replicate in the lungs, and body weight losses are observed [16, 28] . In this experiment, viruses replicated efficiently in the lungs of mice The genetic analysis suggested that the PB2 genes may be responsible for the higher replication rate in mice, since the sublineages of the PB2 gene are different in these two viruses. Further study is needed to clarify the pathogenicity of H9N2 viruses in mice. Experimental infection studies revealed that pigs are highly susceptible to infection with avian influenza viruses of each of the known HA subtypes, and genetic reassortment can take place in pigs [19] . Thus, pigs have been suggested to serve as intermediate hosts to generate genetic reassortants [19] . Three H9N2 viruses were recovered from swabs from pigs in this experiment, and the results were similar to those of previous reports [6, 19] . Especially, viruses were recovered efficiently from nasal swabs from pigs inoculated with Dk/VN/OIE-2328/2009 and Dk/VN/OIE-2583/2009, which belong to the G1 sublineage and replicate efficiently in mice. In addition, H9N2 viruses isolated from humans in Hong Kong were genetically classified as belonging to the G1 sublineage [4, 7, 23, 33, 43] , suggesting that H9N2 viruses belonging to the G1 sublineage have the potential to replicate efficiently in mammals. The findings indicate that H9N2 virus is one of the candidates for pandemic influenza in humans. Surveillance of influenza in wild birds, domestic birds, and pigs is important in order to prepare for pandemic influenza in humans. 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This work was supported by J-GRID; the Japan Initiative for Global Research Network on Infectious Diseases of Ministry of Education, Culture, Sports, Science and Technology of Japan. This work was also supported by Japan Science and Technology Agency Basic Research Programs. We are grateful for the support of the Global Center of Excellence (GCOE) Program of Hokkaido University.