Isolation and characterization of H6N1 and H9N2 avian influenza viruses from Ducks in Hanoi, Vietnam

We report the genetic characterization of low pathogenic avian influenza (LPAI) viruses isolated from domestic ducks in northern Vietnam in 2009. In total, 22 influenza A viruses consisting of 21 H6N1 subtypes and one H9N2 subtype were isolated from 1488 ducks collected in February, March, and April 2009, accounting the overall virus isolation rate for 1.5%. No H5N1 strain was isolated in this study. Phylogenetic analysis indicated that all the eight genes of the H6N1 and H9N2 subtypes analyzed in this study were similar to those isolated in Korea, southeast China and northern Japan, and wild birds which migrate along the coastal East Asian Flyway are estimated to transmit these viruses. There was no evidence that the H6N1 and H9N2 subtypes share the gene segments with H5N1 subtypes. However, it is important to monitor the prevalence and genetical backgrounds of LPAI viruses among poultry in an area where several different influenza A subtypes are in circulation.     

Genetic analysis of H9N2 avian influenza viruses isolated from India

H9N2 avian influenza viruses are endemic in domestic poultry in Asia and are grouped into three major sublineages represented by their prototype strains A/Duck/Hong Kong/Y280/97 (Y280-like), A/Quail/Hong Kong/G1/97 (G1-like) and A/Chicken/Korea/38349-p96323/96 (Korean-like). To understand the genetic relationship of Indian viruses, we determined the partial nucleotide sequence of five H9N2 avian influenza viruses isolated from chicken in India during 2003-2004 and compared them with H9N2 sequences available in GenBank. Deduced amino acid sequence analysis revealed that four isolates shared an R-S-S-R/G motif at the cleavage site of HA, representing low pathogenicity in chickens, while one virus harbors an R-S-N-R/G motif at the same position. All the viruses maintained the human-like motif 226Lysine (H3 numbering) at the HA receptor binding site. Phylogenetic analysis showed that 50% of the genes (HA, NA, NP and M) were similar to G1-like viruses, whereas the remaining genes of the Indian isolates formed a separate, not yet defined, sublineage in the Eurasian lineage. Our finding provides evidence of a novel reassortant H9N2 genotype of G1-like viruses circulating in India.     

Characterization of H5N1 influenza A viruses isolated from domestic green-winged teal

Two avian influenza virus strains, A/domestic green-winged teal/Hunan/67/2005 (H5N1) (D-GWT/67) and A/domestic green-winged teal/Hunan/79/2005 (H5N1) (D-GWT/79), were isolated from healthy domestic green-winged teals (Anas crecca) in Hunan Province, South China. Genomic analysis showed that both isolates were reassortants. The hemagglutinin (HA) genes of the two isolates were closely related to that of an H5N1 strain isolated from tree sparrow (A/tree sparrow/Henan/1/04). The neuraminidase (NA) genes and the internal protein genes of both isolates were closely related to those from A/chicken/Shantou/4231/2003-like (H5N1) viruses, with exception of the matrix (M) gene of D-GWT/79, which was closely related to that of the H7N3 strain A/mallard/Netherlands/12/2000 isolated from wild mallard duck. The virulence of the two isolates was examined in chickens, ducks, and mice. Both strains were found to be highly pathogenic in chickens and ducks, but showed low pathogenicity in mice. These findings contribute to the realization that domestic green-winged teals carrying the H5N1 virus may play an important role in transmitting the virus among birds.     

Genetic characterization of highly pathogenic H5N1 avian influenza viruses isolated from poultry farms in Egypt

Twenty-four avian influenza viruses were collected from poultry farms in three different governorates in Egypt during the years 2006–2009 and genetically characterized. All the isolates were confirmed to be type A and subtype H5 influenza virus by chromatographic strip test and hemagglutination inhibition assay. The sequence and phylogenetic data revealed that all Egyptian isolates cluster together and belong to subclade 2.2.1 of the H5N1 virus of Eurasian origin. Within the clade, Egyptian isolates were classified into three major groups (A, B, and C) based on genetic similarity and chronology of the isolation. The majority of the recent isolates belonged to subgroup A. Interestingly, four strains, which were isolated from the same farm with two of the samples collected on the same day, were located in separate subgroups. In addition, the difference in topology between HA and NS phylogenetic trees, which indicates possible difference in dynamics of genetic evolution in two genes, was observed. Genetic characterization data of H5N1 isolates obtained from farms with different vaccination histories indicate the vaccines currently being used in Egypt do not provide adequate level of protection. Our study provides additional evidence for the need for updated vaccine and warrants continuous monitoring of H5N1 influenza virus in Egypt.     

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

Virus Genes - 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...     

Genetic characterization of H1 avian influenza viruses isolated from migratory birds and domestic ducks in Korea

H1 avian influenza viruses (AIVs) isolated from migratory birds and domestic ducks from 2003 to 2007 were analyzed to determine their genetic relationship. Phylogenic analysis with nucleotide sequences of all eight gene segments showed that 13 H1 AIVs from migratory birds and domestic ducks belonged to Eurasian avian lineages and were closely related to each other. Compared with H1 influenza viruses of swine or human origin in Korea, there was no evidence of reassortment among the human, swine, and avian hosts. Our results show that H1 AIVs isolated in Korea from 2003 to 2007 were genetically stable. However, continued surveillance is needed considering the role of migratory birds and domestic duck as a source of AIVs.     

Genetic characterization of H5N1 influenza A viruses isolated from zoo tigers in Thailand

The H5N1 avian influenza virus outbreak among zoo tigers in mid-October 2004, with 45 animals dead, indicated that the avian influenza virus could cause lethal infection in a large mammalian species apart from humans. In this outbreak investigation, six H5N1 isolates were identified and two isolates (A/Tiger/Thailand/CU-T3/04 and A/Tiger/Thailand/CU-T7/04) were selected for whole genome analysis. Phylogenetic analysis of the 8 gene segments showed that the viruses clustered within the lineage of H5N1 avian isolates from Thailand and Vietnam. The hemagglutinin (HA) gene of the viruses displayed polybasic amino acids at the cleavage site, identical to those of the 2004 H5N1 isolates, which by definition are highly pathogenic avian influenza (HPAI). In addition, sequence analyses revealed that the viruses isolated from tigers harbored few genetic changes compared with the viruses having infected chicken, humans, tigers and a leopard isolated from the early 2004 H5N1 outbreaks. Sequence analyses also showed that the tiger H5N1 isolated in October 2004 was more closely related to the chicken H5N1 isolated in July than that from January. Interestingly, all the 6 tiger H5N1 isolates contained a lysine substitution at position 627 of the PB2 protein similar to the human, but distinct from the original avian isolates.     

Molecular aspects of avian influenza (H5N1) viruses isolated from humans

In 1997, 18 human infections with H5N1 influenza type A were identified in Hong Kong and six of the patients died. There were concomitant outbreaks of H5N1 infections in poultry. The gene segments of the human H5N1 viruses were derived from avian influenza A viruses and not from circulating human influenza A viruses. In 1999 two cases of human infections caused by avian H9N2 virus were also identified in Hong Kong. These events established that avian influenza viruses can infect humans without passage through an intermediate host and without acquiring gene segments from human influenza viruses. The likely origin of the H5N1 viruses has been deduced from molecular analysis of these and other viruses isolated from the region. The gene sequences of the H5N1 viruses were analysed in order to identify the molecular basis for the ability of these avian viruses to infect humans.     

Characterization of highly pathogenic avian influenza H5N8 virus from Egyptian domestic waterfowl in 2017

In 2016, the highly pathogenic avian influenza (HPAI) H5N8 virus was detected in wild birds for the first time in Egypt. In the present study, we identified the HPAI virus H5N8 of clade 2.3.4.4 from domestic waterfowl in Egypt, suggesting its transmission to the domestic poultry from the migratory birds. Based on partial haemagglutinin gene sequence, this virus has a close genetic relationship with subtype H5N8 viruses circulating in Asia and Europe. Pathologically, H5N8 virus in hybrid duck induced nervous signs accompanied by encephalomalacia, haemorrhages, nonsuppurative encephalitis and nonsuppurative vasculitis. The granular layer of cerebellum showed multifocal areas of hydropic degeneration and the Purkinje cell neurons were necrotized or lost. Additionally, the lung, kidney and spleen were congested, and necrotizing pancreatitis was also observed. The co-circulation of both HPAI H5N1 and H5N8 subtypes with the low pathogenic avian influenza H9N2 subtype complicate the control of avian influenza in Egypt with the possibility of emergence of new reassortant viruses. Therefore, continuous monitoring with implementation of strict control measures is required.

Research highlights

• HPAI H5N8 virus clade 2.3.4.4 was detected in domestic ducks and geese in Egypt in 2017.

• Phylogenetically, the virus was closely related to HPAI H5N8 viruses identified in Asia and Europe

• Nonsuppurative encephalitis was widely observed in HPAI H5N8 virus-infected ducks.

• Degeneration of the cerebellar granular layer was found in most of the brain tissues examined.

     

Differential susceptibility of different cell lines to swine-origin influenza A H1N1, seasonal human influenza A H1N1, and avian influenza A H5N1 viruses

BackgroundThe novel swine-origin influenza A H1N1 virus (S-OIV) causes the current pandemic. Its tissue tropism and replication in different cell lines are not well understood.ObjectiveCompare the growth characteristics of cell lines infected by S-OIV, seasonal influenza A H1N1 (sH1N1) and avian influenza A H5N1 (H5N1) viruses and the effect of temperature on viral replication.Study designCytopathic effect (CPE), antigen expression by immunofluorescence (IF) and viral load profile by quantitative RT-PCR in 17 cell lines infected by S-OIV, sH1N1 and H5N1 were examined. Comparison of their replication efficiency in chick embryo was performed. The effect of temperature on viral replication in Madin–Darby canine kidney (MDCK) cells was determined by TCID₅₀ at 33 °C, 37 °C and 39 °C for 5 consecutive days.ResultsS-OIV replicated in cell lines derived from different tissues or organs and host species with comparable viral load to sH1N1. Among 13 human cell lines tested, Caco-2 has the highest viral load for S-OIV. S-OIV showed a low viral load with no CPE or antigen expression in pig kidney cell PK-15, H5N1 demonstrated the most diverse cell tropism by CPE and antigen expression, and the highest viral replication efficiency in both cell lines and allantoic fluid. All three viruses demonstrated best growth at 37 °C in MDCK cells.ConclusionCell line growth characteristics of S-OIV, sH1N1 and H5N1 appear to correlate clinically and pathologically with involved anatomical sites and severity. Low replication of S-OIV in PK-15 suggests that this virus is more adapted to human than swine.     

Phylogenetic characterization of H5N1 avian influenza viruses isolated in Indonesia from 2003-2007

The wide distribution of H5N1 highly pathogenic avian influenza viruses is a global threat to human health. Indonesia has had the largest number of human infections and fatalities caused by these viruses. To understand the enzootic conditions of the viruses in Indonesia, twenty-four H5N1 viruses isolated from poultry from 2003 to 2007 were phylogenetically characterized. Although previous studies exclusively classified the Indonesian viruses into clades 2.1.1-2.1.3, our phylogenetic analyses showed a new sublineage that did not belong to any of the present clades. In addition, novel reassortant viruses were identified that emerged between this new sublineage and other clades in 2005-2006 on Java Island. H5N1 viruses were introduced from Java Island to Sulawesi, Kalimantan, and Sumatra Island on multiple occasions from 2003-2007, causing the geographical expansion of these viruses in Indonesia. These findings identify Java Island as the epicenter of the Indonesian H5N1 virus expansion.     

Genetic characterization of H5N1 influenza viruses isolated from chickens in Indonesia in 2010

Since 2003, highly pathogenic H5N1 avian influenza viruses have caused outbreaks among poultry in Indonesia every year, producing the highest number of human victims worldwide. However, little is known about the H5N1 influenza viruses that have been circulating there in recent years. We therefore conducted surveillance studies and isolated eight H5N1 viruses from chickens. Phylogenic analysis of their hemagglutinin and neuraminidase genes revealed that all eight viruses belonged to clade 2.1.3. However, on the basis of nucleotide differences, these viruses could be divided into two groups. Other viruses genetically closely related to these two groups of viruses were all Indonesian isolates, suggesting that these new isolates have been evolving within Indonesia. Among these viruses, two distinct viruses circulated in the Kalimantan islands during the same season in 2010. Our data reveal the continued evolution of H5N1 viruses in Indonesia.     

Pathogenicity of two Egyptian H5N1 highly pathogenic avian influenza viruses in domestic ducks

Domestic ducks have been implicated in the dissemination and evolution of H5N1 highly pathogenic avian influenza (HPAI) viruses. In this study, two H5N1 HPAI viruses belonging to clade 2.2.1 isolated in Egypt in 2007 and 2008 were analyzed for their pathogenicity in domestic Pekin ducks. Both viruses produced clinical signs and mortality, but the 2008 virus was more virulent, inducing early onset of neurological signs and killing all ducks with a mean death time (MDT) of 4.1 days. The 2007 virus killed 3/8 ducks with a MDT of 7 days. Full-genome sequencing and phylogenetic analysis were used to examine differences in the virus genes that might explain the differences observed in pathogenicity. The genomes differed in 49 amino acids, with most of the differences found in the hemagglutinin protein. This increase in pathogenicity in ducks observed with certain H5N1 HPAI viruses has implications for the control of the disease, since vaccinated ducks infected with highly virulent strains shed viruses for longer periods of time, perpetuating the virus in the environment and increasing the possibility of transmission to susceptible birds.     

Characterization of avian H3N3 and H1N1 influenza A viruses isolated from pigs in Canada

H3N3 and H1N1 influenza A viruses were isolated from Canadian pigs in 2001 and 2002. These viruses are phylogenetically related to waterfowl viruses and antigenically distinct from reference swine influenza viruses. The isolation of these viruses reemphasizes the potential for interspecies transmission of influenza viruses from waterfowl to pigs in North America.     

Characterization of avian influenza viruses isolated from domestic ducks in Vietnam in 2009 and 2010

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.     

Genetic characterization of subtype H1 avian influenza viruses isolated from live poultry markets in Zhejiang Province, China, in 2011

Nine avian influenza A viruses (AIVs), H1N2 (n = 2) and H1N3 (n = 7), were isolated from domestic ducks in live poultry markets in Zhejiang Province, Eastern China, in 2011. All viruses were characterized by whole genome sequencing with subsequent phylogenetic analysis and genetic comparison. Phylogenetic analysis of all eight viral genes showed that the viruses clustered in the Eurasian lineage of influenza A viruses. The hemagglutinin cleavage site of all viruses displayed features of a monobasic cleavage site. Although there was no evidence of re-assortment in subtype H1 AIVs among the avian species and mammalian hosts in this study, continued surveillance is needed considering the important role of the domestic duck in the dissemination and re-assortment of AIVs.     

Genetic and biological characterization of avian influenza H5N1 viruses isolated from wild birds and poultry in Western Siberia

Three viruses included in the study were isolated from dead birds (A/duck/Omsk/1822/2006, A/chicken/Reshoty/02/2006, and A/duck/Tuva/01/2006), whereas the virus A/common gull/Chany/P/2006 was isolated from an apparently healthy gull during outbreaks of highly pathogenic avian influenza in Russia in 2006. The intravenous pathogenicity index (IVPI) of viruses A/duck/Omsk/1822/2006, A/chicken/Reshoty/02/2006, and A/duck/Tuva/01/2006 ranged from 2.7 to 3.0, while the virus A/common gull/Chany/P/2006 had a markedly lower IVPI of 1.7. The virus A/common gull/Chany/P/2006 had a unique pattern of six amino acid substitutions in the regions of viral proteins crucial for virulence of H5N1 viruses. We hypothesize that these substitutions may affect the pathogenicity of A/common gull/Chany/P/2006.     

Real-time RT-PCR assay to differentiate clades of H5N1 avian influenza viruses circulating in Vietnam

Continued circulation and geographical expansion of highly pathogenic avian influenza H5N1 virus have led to the emergence of numerous clades in Vietnam. Although viral RNA sequencing and phylogenetic analysis are the gold standard for H5N1 HA clade designation, limited sequencing capacity in many laboratories precludes rapid H5N1 clade identification and detection of novel viruses. Therefore, a Taqman real-time RT-PCR assay for rapid differentiation of the four major H5N1 clades detected in Vietnam was developed. Using HA sequence alignments of clades 1.1, 2.3.2.1, 2.3.4, and 7 viruses, primers and FAM-labeled probes were designed to target conserved regions characteristic of each clade. The assay was optimized and evaluated using circulating clades of H5N1 collected in Vietnam from 2007 to 2012 and shown to be both sensitive and specific for the differentiation of the four H5N1 clades. The assay provides a useful tool for screening of large specimen collections for HA gene sequencing and phylogenetic analysis and for the rapid identification of molecular clade signatures to support outbreak investigations and surveillance activities. Finally, this assay may be useful to monitor for the emergence of novel or variant clades of H5N1 in Vietnam in the future or in other countries where these particular clades may circulate.