Molecular and phylogenetic analysis of the H5N1 avian influenza virus caused the first highly pathogenic avian influenza outbreak in poultry in the Czech Republic in 2007

On 19th July 2007 re-occurrence of the H5N1 highly pathogenic avian influenza (HPAI) virus was noticed in Europe. The index strain of this novel H5N1 lineage was identified in the Czech Republic where it caused historically the first HPAI outbreak in commercial poultry. In the present study we performed molecular and phylogenetic analysis of the index strain of the re-emerging H5N1 virus lineage along with the Czech and the Slovak H5N1 strains collected in 2006 and established the evolutionary relationships to additional viruses circulated in Europe in 2005-2006. Our analysis revealed that the Czech and the Slovak H5N1 viruses collected during 2006 were separated into two sub-clades 2.2.1 and 2.2.2, which predominated in Europe during 2005-2006. On the contrary the newly emerged H5N1 viruses belonged to a clearly distinguishable sub-clade 2.2.3. Within the sub-clade 2.2.3 the Czech H5N1 strains showed the closest relationships to the simultaneously circulated viruses from Germany, Romania and Russia (Krasnodar) in 2007 and were further clustered with the viruses from Afghanistan and Mongolia circulated in 2006. The origin of the Czech 2007 H5N1 HPAI strains was also discussed.     

Surveillance and identification of influenza A viruses in wild aquatic birds in the Crimea, Ukraine (2006-2008)

The ecology of avian influenza (AI) viruses in wild aquatic birds of Asia is poorly understood, especially for the H5N1 high pathogenicity AI (HPAI) viruses. From March 2006 through November 2008, 20 AI viruses were isolated in the Crimea region of Ukraine with an overall frequency of virus recovery of 3.3%. All the viruses were isolated from three species of dabbling ducks: mallard (Anas platyrhynchos), wigeon (Anas penelope), and garganey (Anas querquedula), making the frequency of virus recovery for dabbling ducks 6.3%. The viruses were predominantly isolated during the fall sampling period. All viruses were genetically and antigenically characterized. No H5N1 HPAI viruses were isolated, but other HA and NA subtypes were identified including H3N1 (2), H3N6 (3), H3N8 (4), H4N6 (6), H5N2 (3), H7N8 (1), and H10N6 (1) subtypes. All isolates were of low pathogenicity, as determined by the intravenous pathogenicity index of 0.00. For H5N2 and H7N8 isolates, the HA gene was sequenced and the phylogenetic analysis revealed possible ecologic connections of the Crimea region with AI viruses from Siberia and Europe. No influenza A isolates were recovered from other Anseriformes (diving ducks [two species of pochards] and graylag geese), Columbiformes (collared doves), Gruiformes (coot), and Galliformes (gray partridges).     

Susceptibility to and transmission of H5N1 and H7N1 highly pathogenic avian influenza viruses in bank voles (Myodes glareolus)

The study of influenza type A (IA) infections in wild mammals populations is a critical gap in our knowledge of how IA viruses evolve in novel hosts that could be in close contact with avian reservoir species and other wild animals. The aim of this study was to evaluate the susceptibility to infection, the nasal shedding and the transmissibility of the H7N1 and H5N1 highly pathogenic avian influenza (HPAI) viruses in the bank vole (Myodes glareolus), a wild rodent common throughout Europe and Asia. Two out of 24 H5N1-infected voles displayed evident respiratory distress, while H7N1-infected voles remained asymptomatic. Viable virus was isolated from nasal washes collected from animals infected with both HPAI viruses, and extra-pulmonary infection was confirmed in both experimental groups. Histopathological lesions were evident in the respiratory tract of infected animals, although immunohistochemistry positivity was only detected in lungs and trachea of two H7N1-infected voles. Both HPAI viruses were transmitted by direct contact, and seroconversion was confirmed in 50% and 12.5% of the asymptomatic sentinels in the H7N1 and H5N1 groups, respectively. Interestingly, viable virus was isolated from lungs and nasal washes collected from contact sentinels of both groups. The present study demonstrated that two non-rodent adapted HPAI viruses caused asymptomatic infection in bank voles, which shed high amounts of the viruses and were able to infect contact voles. Further investigations are needed to determine whether bank voles could be involved as silent hosts in the transmission of HPAI viruses to other mammals and domestic poultry.     

Public health risk from avian influenza viruses

Since 1997, avian influenza (AI) virus infections in poultry have taken on new significance, with increasing numbers of cases involving bird-to-human transmission and the resulting production of clinically severe and fatal human infections. Such human infections have been sporadic and are caused by H7N7 and H5N1 high-pathogenicity (HP) and H9N2 low-pathogenicity (LP) AI viruses in Europe and Asia. These infections have raised the level of concern by human health agencies for the potential reassortment of influenza virus genes and generation of the next human pandemic influenza A virus. The presence of endemic infections by H5N1 HPAI viruses in poultry in several Asian countries indicates that these viruses will continue to contaminate the environment and be an exposure risk with human transmission and infection. Furthermore, the reports of mammalian infections with H5N1 AI viruses and, in particular, mammal-to-mammal transmission in humans and tigers are unprecedented. However, the subsequent risk for generating a pandemic human strain is unknown. More international funding from both human and animal health agencies for diagnosis or detection and control of AI in Asia is needed. Additional funding for research is needed to understand why and how these AI viruses infect humans and what pandemic risks they pose.     

Development of vaccine strains of H5 and H7 influenza viruses

To establish vaccine strains of H5 and H7 influenza viruses, A/duck/Hokkaido/Vac-1/04 (H5N1) [Vac-1/04 (H5N1)], A/duck/Hokkaido/Vac-3/07 (H5N1) [Vac-3/07 (H5N1)], and A/duck/Hokkaido/ Vac-2/04 (H7N7) [Vac-2/04 (H7N7)] were generated from non-pathogenic avian influenza viruses isolated from migratory ducks. Vac-1/04 (H5N1) and Vac-3/07 (H5N1) were generated by genetic reassortment between H5N2 or H5N3 virus as an HA gene provider and H7N1 or H6N1 viruses as an NA gene provider. Vac-2/04 (H7N7) was a genetic reassortant obtained using H7N7 and H9 N2 viruses to give high growth character of the H9N2 virus in chicken embryonated eggs. The results of sequence analyses and experimental infections revealed that these H5N1 and H7N7 reassortant viruses were non-pathogenic in chickens and embryos, and had good growth potential in embryonated eggs. These viruses should be useful to develop vaccines against H5 and H7 highly pathogenic avian influenza viruses.     

Lessons from emergence of A/goose/Guangdong/1996-like H5N1 highly pathogenic avian influenza viruses and recent influenza surveillance efforts in southern China

Southern China is proposed as an influenza epicentre. At least two of the three pandemics in the last century, including 1957 and 1968 influenza pandemics, originated from this area. In 1996, A/goose/Guangdong/1/1996 (H5N1), the precursor of currently circulating highly pathogenic H5N1 avian influenza viruses (HPAIVs) was identified in farmed geese in southern China. These H5N1 HPAIVs have been spread across Asia, Europe and Africa and poses a continuous threat to both animal and human health. However, how and where this H5N1 HPAIV emerged are not fully understood. In the past decade, many influenza surveillance efforts have been carried out in southern China, and our understanding of the genetic diversity of non-human influenza A viruses in this area has been much better than ever. Here, the historical and first-hand experimental data on A/goose/Guangdong/1/1996(H5N1)-like HPAIVs are reviewed within the context of the findings from recent surveillance efforts on H5N1 HPAIVs and other non-human influenza A viruses. Such a retrospective recapitulation suggests that long-term and systematic surveillance programmes should continue to be implemented in southern China that the wet markets on the animal-human interface shall be the priority area and that the surveillance on the animal species bridging the interface between wildlife and domestic animal populations and the interface between the aquatics and territories shall be the strengthened.     

Molecular characterization of the hemagglutinin and neuraminidase genes of H5N1 influenza A viruses isolated from poultry in Vietnam from 2004 to 2005

Highly pathogenic H5N1 avian influenza A viruses have been spreading among domestic poultry, wild aquatic birds, and humans in many Asian countries since 2003. The largest number of patients, to date, infected with the H5N1 viruses are in Vietnam, where these viruses continue to cause outbreaks in domestic poultry. Here, we molecularly characterized the hemagglutinin and neuraminidase genes of nine H5N1 viruses isolated between January 2004 and August 2005 from domestic poultry in Vietnam. We found that several groups of highly pathogenic H5N1 avian influenza viruses are circulating among these birds, which suggests that H5N1 viruses of different lineages have been introduced into Vietnam multiple times.     

Pathogenicity of H5N1 influenza A viruses isolated in Vietnam between late 2003 and 2005

Since late 2003, highly pathogenic H5N1 influenza A viruses have spread among poultry and wild aquatic birds in Asian countries. Transmission of these viruses to humans can be lethal. Most human cases of infection with H5N1 viruses have occurred in Vietnam. Therefore, to understand the pathogenicity in mammals of these H5N1 viruses, we took viruses isolated from poultry (5 strains) and humans (2 strains) in Vietnam and tested their virulence in mice. The results showed that the H5N1 viruses from humans were pathogenic in mice and that one avian isolate was also pathogenic. These findings suggested that the H5N1 viruses circulating in poultry adapted during replication in humans or that strains pathogenic in mice were transmitted directly to humans.     

Pathogenicity of H5N8 virus in chickens from Korea in 2014

In 2014, two genetically distinct H5N8 highly pathogenic avian influenza (HPAI) viruses were isolated from poultry and wild birds in Korea. The intravenous pathogenicity indices for the two representative viruses were both 3.0. Mortality of chickens intranasally inoculated with the two H5N8 viruses was 100% with a mean death times of 2.5 and 4.5 days. Mortality rates of the contact groups for the two H5N8 viruses were 33.3% and 66.6%. Our study showed that transmissibility of the novel H5N8 viruses was different from that of previously identified H5N1 HPAI viruses, possibly due to genetic changes.     

H5N1亚型禽流感病毒对鸭的致病性研究

为评估H5N1亚型禽流感病毒(AIV)在实验室环境下对鸭的致病力,本研究以无特殊病原(SPF)鸭为模型,对我国近年分离的7株病毒进行了致病力分析。结果发现其中4株病毒对鸭致死率为100%,2株病毒对鸭的致死率分别为60%和80%,另外1株病毒,A/goose/Hubei/51/05(GS/HB/51/05),对鸭无致病力。本研究还发现,与高致病力毒株一样,GS/HB/51/05也可在鸭体内呈全身性复制,并且可通过喉头和泻殖腔向外排泄。我们推测GS/HB/51/05可能是中国南方出现的其他对鸭呈高致病力的H5N1病毒的祖先,对这些病毒的系统研究,可揭示H5N1亚型AIV对鸭的致病力遗传机制。     

Genetic diversity of H9N2 influenza viruses from pigs in China: a potential threat to human health?

Pandemic strains of influenza A virus might arise by genetic reassortment between viruses from different hosts. Pigs are susceptible to both human and avian influenza viruses and have been proposed to be intermediate hosts or mixing vessels, for the generation of pandemic influenza viruses through reassortment or adaptation to the mammalian host. In this study, we summarize and report for the first time the coexistence of 10 (A-J) genotypes in pigs in China by analyzing the eight genes of 28 swine H9N2 viruses isolated in China from 1998 to 2007. Swine H9N2 viruses in genotype A and B were completely derived from Y280-like and Shanghai/F/98-like viruses, respectively, which indicated avian-to-pig interspecies transmission of H9N2 viruses did exist in China. The other eight genotype (C-J) viruses might be double-reassortant viruses, in which six genotype (E-J) viruses possessed 1-4 H5-like gene segments indicating they were reassortants of H9 and H5 viruses. In conclusion, genetic diversity of H9N2 influenza viruses from pigs in China provides further evidence that avian to pig interspecies transmission of H9N2 viruses did occur and might result in the generation of new reassortant viruses by genetic reassortment with swine H1N1, H1N2 and H3N2 influenza viruses, therefore, these swine H9N2 influenza viruses might be a potential threat to human health and continuing to carry out swine influenza virus surveillance in China is of great significance.     

Genomic characterization of H1N2 swine influenza viruses in Italy

Three subtypes (H1N1, H1N2, and H3N2) are currently diffused worldwide in pigs. The H1N2 subtype was detected for the first time in Italian pigs in 1998. To investigate the genetic characteristics and the molecular evolution of this subtype in Italy, we conducted a phylogenetic analysis of whole genome sequences of 26 strains isolated from 1998 to 2010. Phylogenetic analysis of HA and NA genes showed differences between the older (1998-2003) and the more recent strains (2003-2010). The older isolates were closely related to the established European H1N2 lineage, whereas the more recent isolates possessed a different NA deriving from recent human H3N2 viruses. Two other reassortant H1N2 strains have been detected: A/sw/It/22530/02 has the HA gene that is closely related to H1N1 viruses; A/sw/It/58769/10 is an uncommon strain with an HA that is closely related to H1N1 and an NA similar to H3N2 SIVs. Amino acid analysis revealed interesting features: a deletion of two amino acids (146-147) in the HA gene of the recent isolates and two strains isolated in 1998; the presence of the uncommon aa change (N66S), in the PB1-F2 protein in strains isolated from 2009 to 2010, which is said to have contributed to the increased virulence. These results demonstrate the importance of pigs as mixing vessels for animal and human influenza and show the presence and establishment of reassortant strains involving human viruses in pigs in Italy. These findings also highlighted different genomic characteristics of the NA gene the recent Italian strains compared to circulating European viruses.     

A serological survey of canine H3N2, pandemic H1N1/09 and human seasonal H3N2 influenza viruses in dogs in China

Influenza viruses have been isolated from dogs in China; however, the extent of influenza infection among dogs is not yet clear. Here, we examined the seroprevalence of avian-origin canine H3N2, pandemic H1N1/09 and human seasonal H3N2 influenza viruses in pet dogs in China during January 2012 to June 2013. The seropositivity rate of canine H3N2, H1N1/09 and human H3N2 were 3.5%, 1.5%, and 1.2%, respectively. Dogs aged 2–5 years were most commonly seropositive to canine H3N2 virus. It is worth noting that two serum samples were positive against both canine H3N2 and H1N1/09 viruses, suggesting the possibility of coinfection with both viruses. Our findings emphasize the necessity for continued surveillance of influenza viruses in dogs in China.     

Serological Evidence of Pandemic H1N1 Influenza Virus Infections in Greek Swine

The introduction of the 2009 pandemic H1N1 (pH1N1) influenza virus in pigs changed the epidemiology of influenza A viruses (IAVs) in swine in Europe and the rest of the world. Previously, three IAV subtypes were found in the European pig population: an avian‐like H1N1 and two reassortant H1N2 and H3N2 viruses with human‐origin haemagglutinin (HA) and neuraminidase proteins and internal genes of avian decent. These viruses pose antigenically distinct HAs, which allow the retrospective diagnosis of infection in serological investigations. However, cross‐reactions between the HA of pH1N1 and the HAs of the other circulating H1 IAVs complicate serological diagnosis. The prevalence of IAVs in Greek swine has been poorly investigated. In this study, we examined and compared haemagglutination inhibition (HI) antibody titres against previously established IAVs and pH1N1 in 908 swine sera from 88 herds, collected before and after the 2009 pandemic. While we confirmed the historic presence of the three IAVs established in European swine, we also found that 4% of the pig sera examined after 2009 had HI antibodies only against the pH1N1 virus. Our results indicate that pH1N1 is circulating in Greek pigs and stress out the importance of a vigorous virological surveillance programme.     

Molecular epidemiology of avian influenza viruses circulating among healthy poultry flocks in farms in northern Vietnam

Repeated epizootics of highly pathogenic avian influenza (HPAI) virus subtype H5N1 were reported from 2003 to 2005 among poultry in Vietnam. More than 200 million birds were killed to control the spread of the disease. Human cases of H5N1 infection have been sporadically reported in an area where repeated H5N1 outbreaks among birds had occurred. Subtype H5N1 strains are established as endemic among poultry in Vietnam, however, insights into how avian influenza viruses including the H5N1 subtype are maintained in endemic areas is not clear. In order to determine the prevalence of different avian influenza viruses (AIVs), including H5N1 circulating among poultry in northern Vietnam, surveillance was conducted during the years 2006-2009. A subtype H5N1 strain was isolated from an apparently healthy duck reared on a farm in northern Vietnam in 2008 and was identified as an HPAI. Although only one H5N1 virus was isolated, it supports the view that healthy domestic ducks play a pivotal role in maintaining and transmitting H5N1 viruses which cause disease outbreaks in northern Vietnam. In addition, a total of 26 AIVs with low pathogenicity were isolated from poultry and phylogenetic analysis of all the eight gene segments revealed their diverse genetical backgrounds, implying that reassortments have occurred frequently among strains in northern Vietnam. It is, therefore, important to monitor the prevalence of influenza viruses among healthy poultry between epidemics in an area where AIVs are endemic.     

Effect of species,breed and route of virus inoculation on the pathogenicity of H5N1 highly pathogenic influenza (HPAI) viruses in domestic ducks

H5N1 highly pathogenic avian influenza (HPAI) viruses continue to be a threat to poultry in many regions of the world. Domestic ducks have been recognized as one of the primary factors in the spread of H5N1 HPAI. In this study we examined the pathogenicity of H5N1 HPAI viruses in different species and breeds of domestic ducks and the effect of route of virus inoculation on the outcome of infection. We determined that the pathogenicity of H5N1 HPAI viruses varies between the two common farmed duck species, with Muscovy ducks (Cairina moschata) presenting more severe disease than various breeds of Anas platyrhynchos var. domestica ducks including Pekin, Mallard-type, Black Runners, Rouen, and Khaki Campbell ducks. We also found that Pekin and Muscovy ducks inoculated with two H5N1 HPAI viruses of different virulence, given by any one of three routes (intranasal, intracloacal, or intraocular), became infected with the viruses. Regardless of the route of inoculation, the outcome of infection was similar for each species but depended on the virulence of the virus used. Muscovy ducks showed more severe clinical signs and higher mortality than the Pekin ducks. In conclusion, domestic ducks are susceptible to H5N1 HPAI virus infection by different routes of exposure, but the presentation of the disease varied by virus strain and duck species. This information helps support the planning and implementation of H5N1 HPAI surveillance and control measures in countries with large domestic duck populations.     

Induction of a cross-reactive antibody response to influenza virus M2 antigen in pigs by using a Sendai virus vector

Protecting pigs from simultaneous infection with avian, swine, and human influenza viruses would be an effective strategy to prevent the emergence of reassortants with pandemic potential. M2 protein is a candidate antigen for so-called 'universal vaccines,' which confer cross-protection to different influenza viruses in a strain- and subtype-independent manner. We tested whether a recombinant F gene-deleted Sendai virus vector that contained an M2 gene derived from an H5N1 avian influenza virus (SeV/ΔF/H5N1M2) could induce a cross-reactive antibody response to the extracellular domain of M2 protein (M2e) in pigs. SeV/ΔF/H5N1M2 induced an antibody response to M2e when the vector was inoculated intramuscularly. The antibodies induced by SeV/ΔF/H5N1M2 cross-reacted with M2e derived from different avian, swine, and human influenza viruses. In mice, however, SeV/ΔF/H5N1M2 did not confer cross-protection to challenge with a heterologous H3N2 influenza virus. Our results confirm those of other groups indicating that antibodies to M2e do not mediate protection to influenza viruses in pigs.     

Hemagglutinin glycosylation modulates the pathogenicity and antigenicity of the H5N1 avian influenza virus

The location and number of glycosylation in HA proteins exhibit large variations among H5 subtype avian influenza viruses (AIVs). To investigate the effect of glycosylation in the globular head of HA on the pathogenicity and antigenicity of H5N1 AIVs, seven rescued AIVs differing in their glycosylation patterns (144N, 158N and 169N) within the HA globular head of A/Mallard/Huadong/S/2005 were generated using site directed mutagenesis. Results showed that loss of glycosylation 158N was the prerequisite for H5 AIV binding to the α2,6-linked receptor. Only in conjunction with the removal of the 158N glycosylation, the H5 AIVs harboring both 144N and 169N glycosylations obtained an optimal binding preference to the α2,6-linked receptor. Compared with the wild-type virus, growth of viruses lacking glycosylation at either 158N or 169N was significantly reduced both in MDCK and A549 cells, while replication of viruses with additional glycosylation 144N was significantly promoted. Mutant viruses with loss of 158N or 169N glycosylation sites showed increased pathogenicity, systemic spread and pulmonary inflammation in mice compared to the wild-type H5N1 virus. In addition, chicken studies demonstrated that inactivated de-glycosylation 169N mutant induced cross-reaction HI and neutralization antibody against various clades of H5N1 AIVs. Moreover, this type of glycan pattern vaccine virus provided better cross-protection in chickens compared to wild-type vaccine virus. Thus, the glycosylation alteration of HA should be considered in the global surveillance and vaccine design of H5 subtype AIVs.     

The characterization of low pathogenic avian influenza viruses isolated from wild birds in northern Vietnam from 2006 to 2009

Due to concerns that wild birds could possibly spread H5N1 viruses, surveillance was conducted to monitor the types of avian influenza viruses circulating among the wild birds migrating to or inhabiting in northern Vietnam from 2006 to 2009. An H5N2 virus isolated from a Eurasian woodcock had a close phylogenetic relationship to H5 viruses recently isolated in South Korea and Japan, suggesting that H5N2 has been shared between Vietnam, South Korea, and Japan. An H9N2 virus isolated from a Chinese Hwamei was closely related to two H9N2 viruses that were isolated from humans in Hong Kong in 2009, suggesting that an H9N2 strain relevant to the human isolates had been transmitted to and maintained among the wild bird population in Vietnam and South China. The results support the idea that wild bird species play a significant role in the spread and maintenance of avian influenza and that this also occurs in Vietnam.     

Characterization of H5N1 influenza viruses isolated from migratory birds in Qinghai province of China in 2006

Avian influenza H5N1 viruses pose a significant threat to human health because of their ability to infect humans directly. In the paper, three highly pathogenic H5N1 influenza viruses were isolated from three species of migratory birds in Qinghai Province of China in 2006. The analysis of the genome sequences indicated that the three isolates shared high homology with each other (94% to 99%). Three isolates shared a common ancestor and were closest to strains isolated from Qinghai and Siberia in 2005, but distinct from poultry viruses found in Southeast Asia. In experimental infection, all three viruses were highly pathogenic to chickens and mice. The results suggest that highly pathogenic avian influenza H5N1 viruses still exist in the migratory birds and could spread to other regions with wild bird migration.