Pathogenicity of the Korean H5N8 highly pathogenic avian influenza virus in commercial domestic poultry species

In 2014, the highly pathogenic avian influenza (HPAI) virus H5N8 triggered outbreaks in wild birds and poultry farms in South Korea. In the present study, we investigated the pathogenicity of the H5N8 HPAI virus, belonging to the clade 2.3.4.4, in different species of poultry. For this, we examined clinical signs and viral shedding levels following intranasal inoculation of the virus in 3-week-old commercial layer chickens and quails, 10-week-old Korean native chickens, and 8-week-old Muscovy ducks. Intranasal inoculation with 10^6.0 viruses at 50% egg-infective dose resulted in 100% mortality in the layer chickens (8/8) and quails (4/4), but 60% and 0% deaths in the Korean native chickens (3/5) and Muscovy ducks (0/4), respectively. In addition, transmission of the inoculated virus to contact-exposed birds was evident in all the species used in this study. Based on our results, we conclude that the H5N8 HPAI virus has lower pathogenicity and transmissibility in poultry species compared with previously reported H5N1 HPAI viruses.     

High prevalence of amantadine-resistance influenza a (H3N2) in six prefectures, Japan, in the 2005-2006 season

Substantial increase in amantadine-resistant influenza A (H3N2) was reported in Asia and North America in 2005. In this study the frequency and genetic characteristics of amantadine-resistant influenza A, circulated in Japan in 2005-2006 season, were investigated. Isolates were tested by amantadine susceptibility test (TCID(50)/0.2 ml method), and sequencing of the M2 gene to identify mutations that confer resistance. Additionally, the hemagglutinin (HA) and neuraminidase (NA) genes of the viruses were examined. In total, 415 influenza A isolates from six prefectures were screened, and 231 (65.3%) of 354 influenza A (H3N2) were amantadine-resistant, with a serine to asparagine (S31N) change in the M2 gene. However, none of 61 A (H1N1) isolates were resistant. In addition, genetic analyses of the HA gene showed all amantadine-resistant viruses clustered in one (named clade N), possessing specific double mutations at 193, serine to phenylalanine (S193F), and at 225, asparatic acid to asparagine (D225N), and sensitive viruses belonged to another group (clade S). The clinical presentations at the clinical visit did not differ between patients shedding clade N virus and those shedding clade S virus. None of the patients had received previous treatment with amantadine. The results indicate an unusually high prevalence and wide circulation of the amantadine-resistance influenza A (H3N2) in Japan in the 2005-2006 season. These strains had the characteristic double mutations in the HA, in addition to the M2 mutation responsive for resistance. Antiviral resistance monitoring should be intensified and maintained for rapid feedback into treatment strategies, and selection of alternative therapeutic agents.     

Protection of Chinese painted quails (Coturnix chinensis) against a highly pathogenic H5N1 avian influenza virus strain after vaccination

Chinese painted quails immunized with a single dose (6 μg HA) of inactivated H5N1 (clade 1) influenza vaccine NIBRG-14 and challenged with 100 LD₅₀ of the heterologous A/Swan/Nagybaracska/01/06(H5N1) (clade 2.2) strain were protected, whereas unvaccinated quails died after challenge. No viral antigens or RNA were detected in cloacal swabs from immunized animals. Sera obtained post-immunization gave low titres in serological assays against the vaccine and the challenge viruses. Our results demonstrate the protective efficacy of the NIBRG-14 strain against the challenge virus and the usefulness of these small birds in protection studies of influenza vaccines.     

Intracontinental and intercontinental dissemination of Asian H5 highly pathogenic avian influenza virus (clade 2.3.4.4) in the winter of 2014–2015

Asian H5 highly pathogenic avian influenza viruses (HPAIVs) that possess the clade 2.3.4.4 HA gene have been identified in wild birds and poultry since late 2014 in both Europe and North America (N. America). Clade 2.3.4.4 H5 HPAIVs of the H5N8 subtype have been isolated in both regions, whereas reassortment viruses with NA N1 and N2 subtypes of the North American (N. American). avian lineage have only been identified in N. America. The HA genes of those isolates were closely related to genes of the HPAIVs that have caused massive outbreaks in poultry in Korea since January 2014. The outbreaks caused by those viruses and the genetic relatedness of their HA and NA genes are reviewed in this study. Although the illegal movement of poultry and poultry products cannot be ruled out as a cause of intercontinental and intracontinental dissemination of clade 2.3.4.4 H5 HPAIVs during the winter of 2014–2015, transmission of the viruses by infected migratory birds appears to be a more plausible mechanism for their dissemination. In particular, the involvement of migratory birds in HPAIV transmission between Asia and N. America is highly likely because of the reassortments between H5N8 HPAIV and the N. American lineage avian influenza viruses. Copyright © 2015 John Wiley & Sons, Ltd.     

Genetic characterization and pathogenicity assessment of highly pathogenic H5N1 avian influenza viruses isolated from migratory wild birds in 2011, South Korea

The continued spread of a highly pathogenic avian influenza (HPAI) H5N1 virus among wild birds and poultry has posed a potential threat to human public health. In the present study, we report the isolation of HPAI H5N1 viruses (A/Md/Korea/W401/11 and A/Md/Korea/W404/11) from fecal samples of migratory birds. Genetic and phlyogenetic analyses demonstrated that these viruses are genetically identical possessing gene segments from avian virus origin and showing highest sequence similarities (as high as 99.8%) to A/Ws/Hokkaido/4/11 and 2009-2010 Mongolian-like clade 2.3.2 isolates rather than previous Korean H5N1 viruses. Both viruses possess the polybasic motif (QRERRRK/R) in HA but other genes did not bear additional virulence markers. Pathogenicity of A/Md/Korea/W401/11 was assessed and compared with a 2006 clade 2.2 HPAI H5N1 migratory bird isolate (A/EM/Korea/W149/06) in chickens, ducks, mice and ferrets. Experimental infection in these hosts showed that both viruses have high pathogenic potential in chickens (2.3-3.0 LD(50)s) and mice (3.3-3.9 LD(50)s), but A/Md/Korea/W401/11 was less pathogenic in duck and ferret models. Despite recovery of both infection viruses in the upper respiratory tract, efficient ferret-to-ferret transmission was not observed. These data suggest that the 2011 Korean HPAI wild bird H5N1 virus could replicate in mammalian hosts without pre-adaptation but could not sustain subsequent infection. This study highlights the role of migratory birds in the perpetuation and spread of HPAI H5N1 viruses in Far-East Asia. With the changing pathobiology caused by H5N1 viruses among wild and poultry birds, continued surveillance of influenza viruses among migratory bird species remains crucial for effective monitoring of high-pathogenicity or pandemic influenza viruses.     

The genetic and antigenic diversity of avian influenza viruses isolated from domestic ducks, muscovy ducks, and chickens in northern and southern Vietnam, 2010–2012

To estimate the prevalence of avian influenza virus infection in Vietnam, surveillance was conducted in domestic and wild birds from households, live-bird markets, slaughtering sites, and bird sanctuaries in Vietnam between October 2010 and October 2012. Of the 4,550 samples collected, 226 influenza A virus isolates were obtained from domestic ducks, muscovy ducks, and chickens. Of these, 25 and 22 H5N1 highly pathogenic avian influenza viruses (HPAIVs) were isolated from apparently healthy domestic ducks in live-bird markets and slaughtering sites in northern and southern Vietnam, respectively. The HA genes of H5 viruses isolated from birds in northern Vietnam phylogenetically belonged to the genetic clade 2.3.2.1 and those in southern Vietnam belonged to the genetic clade 1.1. In addition, 39 H3, 12 H4, 1 H5, 93 H6, 2 H7, 18 H9, 3 H10, and 11 H11 viruses were isolated. Phylogenetic and antigenic analyses of the H6 and H9 viruses revealed that they were closely related to the isolates obtained from domestic poultry in China. Phylogenetic analyses of internal gene segments of these isolates revealed that these viruses were circulating in both domestic and wild birds in Asia and reassortment events had occurred frequently. Therefore, it will be important to continue the surveillance and strict controls over the movement and trade of poultry and poultry products in order to eradicate H5N1 HPAIV from Asia.     

Genetic characterization of highly pathogenic H5N1 avian influenza virus from live migratory birds in Bangladesh

Since the first outbreak of highly pathogenic avian influenza virus (HPAIV) subtype H5N1 in Bangladesh in 2007, the virus has been circulating among domestic poultry causing severe economic losses. To investigate the presence of HPAIV H5N1 in migratory birds and their potential role in virus spread, 205 pools of fecal samples from live migratory birds were analyzed. Here, the first virus isolation and genome characterization of two HPAIV H5N1 isolates from migratory birds (A/migratory bird/Bangladesh/P18/2010 and A/migratory bird/Bangladesh/P29/2010)are described. Full-length amplification, sequencing, and a comprehensive phylogenetic analysis were performed for HA, NA, M, NS, NP, PA, PB1, and PB2 gene segments. The selected migratory bird isolates belong to clade 2.3.2.1 along with recent Bangladeshi isolates from chickens, ducks, and crows which grouped in the same cluster with contemporary South and South-East Asian isolates. The studied isolates were genetically similar to other H5N1 isolates from different species within the respective clade although some unique amino acid substitutions were observed among them. Migratory birds remain a real threat for spreading pathogenic avian influenza viruses across the continent and introduction of new strains into Bangladesh.     

Characterization of a highly pathogenic H5N1 avian influenza virus isolated from ducks in Eastern China in 2011

This study describes the characterization of seven H5N1 avian influenza viruses from domestic ducks in Eastern China in 2011. Phylogenetic analysis showed these viruses were closely related to an H5N1 virus circulating in wild birds in Hong Kong. Some characteristics of these viruses were similar to those of an H5N1 strain that circulated in China and Vietnam (2003-2004). The virulence of three isolates was examined in chickens and mice, and they were found to be highly pathogenic in chickens but showed low pathogenicity in mice. These results suggest that continued H5N1 surveillance in poultry should be used as an early warning system for avian influenza outbreaks.     

High prevalence of amantadine-resistant influenza A virus isolated in Gyeonggi Province, South Korea, during 2005–2010

Amantadine resistance among influenza A viruses was investigated in South Korea in 2005–2010. Of 308 influenza A viruses examined, 229 had the S31N substitution in the M2 protein. The frequency of amantadine resistance was 30 %, 100 %, and 76 % in influenza A/H1N1, pandemic A/H1N1 2009(A/H1N1pdm), and A/H3N2 subtypes, respectively. The amantadine-resistant influenza A/H1N1pdm and A/H3N2 viruses were circulating continuously from 2008 to 2009 and from 2005 to 2006, respectively. Amantadine resistance among influenza A viruses increased dramatically during the 5-year study period, and this has diminished the usefulness of this class of drugs.     

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 a highly pathogenic avian influenza H5N1 virus sublineage in poultry seized at ports of entry into Vietnam

Highly pathogenic avian influenza H5N1 virus was detected in poultry seized at two ports of entry located in Lang Son Province, Vietnam. Sequence analysis of the hemagglutinin (HA) genes from five H5N1 virus isolates and ten PCR amplicons from chicken cloacal samples revealed their close phylogenetic relationship to clade 7 H5N1 HA genes. However, these HA genes exhibited extensive genetic divergence at both the nucleotide and amino acid levels in comparison to previously described clade 7 viruses; e.g., A/chicken/Shanxi/2/2006. In addition, hemagglutination inhibition tests revealed antigenic differences between these and previously isolated H5N1 viruses from Vietnam. These results indicate that viruses with clade 7 HA are evolving rapidly in poultry in Southeast Asia.     

Pathogenicity of a highly pathogenic avian influenza virus, A/chicken/Yamaguchi/7/04 (H5N1) in different species of birds and mammals

Summary. Outbreaks of highly pathogenic avian influenza (HPAI) have been occurring in domestic poultry in Asia since 1996. In the beginning of 2004, HPAI outbreaks were caused by H5N1 virus in two farms and a group of pet chickens in different areas of Japan. In the present study, the pathogenicity of A/chicken/Yamaguchi/7/04 (H5N1), which had been isolated from a dead chicken during the first outbreak in Japan, was assessed in chickens, quails, budgerigars, ducklings, mice, and miniature pigs by experimental infection. The virus was highly pathogenic to all the birds tested. Mice were susceptible to infection with a low mortality rate and miniature pigs were resistant to infection with the virus.     

Investigating a crow die-off in January–February 2011 during the introduction of a new clade of highly pathogenic avian influenza virus H5N1 into Bangladesh

We investigated unusual crow mortality in Bangladesh during January-February 2011 at two sites. Crows of two species, Corvus splendens and C. macrorhynchos, were found sick and dead during the outbreaks. In selected crow roosts, morbidity was ~1 % and mortality was ~4 % during the investigation. Highly pathogenic avian influenza virus H5N1 clade 2.3.2.1 was isolated from dead crows. All isolates were closely related to A/duck/India/02CA10/2011 (H5N1) with 99.8 % and A/crow/Bangladesh/11rs1984-15/2011 (H5N1) virus with 99 % nucleotide sequence identity in their HA genes. The phylogenetic cluster of Bangladesh viruses suggested a common ancestor with viruses found in poultry from India, Myanmar and Nepal. Histopathological changes and immunohistochemistry staining in brain, pancreas, liver, heart, kidney, bursa of Fabricius, rectum, and cloaca were consistent with influenza virus infection. Through our limited investigation in domesticated birds near the crow roosts, we did not identify any samples that tested positive for influenza virus A/H5N1. However, environmental samples collected from live-bird markets near an outbreak site during the month of the outbreaks tested very weakly positive for influenza virus A/H5N1 in clade 2.3.2.1-specific rRT-PCR. Continuation of surveillance in wild and domestic birds may identify evolution of new avian influenza virus and associated public-health risks.     

Antigenic, genetic, and pathogenic characterization of H5N1 highly pathogenic avian influenza viruses isolated from dead whooper swans (Cygnus cygnus) found in northern Japan in 2008

In April and May 2008, whooper swans (Cygnus cygnus) were found dead in Hokkaido in Japan. In this study, an adult whooper swan found dead beside Lake Saroma was pathologically examined and the identified H5N1 influenza virus isolates were genetically and antigenically analyzed. Pathological findings indicate that the swan died of severe congestive edema in the lungs. Phylogenetic analysis of the HA genes of the isolates revealed that they are the progeny viruses of isolates from poultry and wild birds in China, Russia, Korea, and Hong Kong. Antigenic analyses indicated that the viruses are distinguished from the H5N1 viruses isolated from wild birds and poultry before 2007. The chickens vaccinated with A/duck/Hokkaido/Vac-1/2004 (H5N1) survived for 14 days after challenge with A/whooper swan/Hokkaido/1/2008 (H5N1), although a small amount of the challenge virus was recovered from the tissues of the birds. These findings indicate that H5N1 highly pathogenic avian influenza viruses are circulating in wild birds in addition to domestic poultry in Asia and exhibit antigenic variation that may be due to vaccination.     

Active reassortment of H9 influenza viruses between wild birds and live-poultry markets in Korea

Surveillance of H9 avian influenza viruses in Korean live-poultry markets from September 2004 through October 2007 was carried out to investigate active reassortment between wild migratory birds and domestic poultry in Korea. Antigenic and phylogenetic analyses showed that most of the isolates belong to the previous Korean H9N2-like lineage and differ from the southeastern Chinese strains. Interestingly, the Ck/Korea/LPM77/06 group (genotype B) and Dk/Korea/LPM248/07 group (genotype C) showed unique properties distinct from those of other Korean H9N2 strains. Although the HA genes of these two groups belong to Korean H9N2-like lineage, the PA genes closely resemble those of the Chinese Y280-like lineage. In addition, the PB2 genes of the Dk/Korea/LPM248/07 group were closely related to those isolated from migratory birds. Several other isolates also clustered within the H9N2 B genotype, an indication that there are at least two predominant H9N2 influenza genotypes in Korea. Another isolate, Dk/Korea/LPM71/06, was identified as an H9N1 subtype, the first ever discovered in Korean live-poultry markets. These findings reveal that reassortment of Korean H9 influenza viruses has occurred frequently in live-poultry markets and may have been mediated by introduction of genetic material from viruses circulating among migratory wild birds to domestic birds. Consequently, the new dominant H9N2 genotypes have become established in Korean live-poultry markets through continued reassortment.     

Genetic analysis of nonstructural genes (NS1 and NS2) of H9N2 and H5N1 viruses recently isolated in Israel

The avian influenza virus subtype H9N2 affects wild birds, domestic poultry, swine, and humans; it has circulated amongst domestic poultry in Israel during the last 6 years. The H5N1 virus was recorded in Israel for the first time in March 2006. Nonstructural (NS) genes and NS proteins are important in the life cycle of the avian influenza viruses. In the present study, NS genes of 21 examples of H9N2 and of two examples of H5N1 avian influenza viruses, isolated in Israel during 2000–2006, were completely sequenced and phylogenetically analyzed. All the H9N2 isolates fell into a single group that, in turn, was subdivided into three subgroups in accordance with the time of isolation; their NS1 and NS2 proteins possessed 230 and 121 amino acids, respectively. The NS1 protein of the H5N1 isolates had five amino acid deletions, which was typical of highly pathogenic H5N1 viruses isolated in various countries during 2005–2006. Comparative analysis showed that the NS proteins of the H9N2 Israeli isolates contained few amino acid sequences associated with high pathogenicity or human host specificity.     

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.     

Emergence of amantadine-resistant H3N2 avian influenza A virus in South Korea

We found a relatively high frequency of unique amantadine-resistant H3N2 and H9N2 avian influenza viruses (Val27Ile on M2 protein) isolated from live poultry markets in South Korea and confirmed that a Val27Ile single substitution in the M2 protein is enough to acquire the amantadine resistance phenotype by using reverse-genetically created human-avian reassortant viruses.     

The pathogenesis of influenza in humans

The rapid evolution of influenza A and B viruses contributes to annual influenza epidemics in humans. In addition, pandemics of influenza are also caused by influenza A viruses, whereas influenza B does not have the potential to cause pandemics because there is no animal reservoir of the virus. Study of the genetic differences between influenza A and influenza B viruses, which are restricted to humans, may be informative in understanding the factors that govern mammalian adaptation of influenza A viruses. Aquatic birds provide the natural reservoir for influenza A viruses, but in general, avian influenza is asymptomatic in feral birds. Occasionally, however, highly pathogenic strains of influenza cause serious systemic infections in domestic poultry. The pathogenicity of these strains is related to the presence of a polybasic cleavage sequence in the precursor of the surface glycoprotein haemagglutinin, which makes the glycoprotein susceptible to activation by ubiquitous proteases such as furin and PC6. However, the mechanism of pathogenicity may differ in highly pathogenic strains of human influenza, such as the H1N1 pandemic strain of 1918 and the H5N1 strain involved in the outbreak in Hong Kong in 1997. Binding of host proteases by the viral neuraminidase to assist activation of the haemagglutinin, shortening of the neuraminidase and substitutions in the polymerase gene, PB2, have all been suggested as alternative molecular correlates of pathogenicity of human influenza viruses. Additionally, systemic spread in humans of pathogenic subtypes has not been demonstrated and host factors such as interferons may be crucial in preventing the spread of the virus outside the respiratory tract.