Characterization Of Avian Influenza Viruses Research Articles

History of Major Epidemics of Avian Influenza in Humans and Public Health Perspectives

Avian influenza (AI) has had great impacts on global health and socioeconomics in the past two decades. Among many newly emerged influenza viruses, avian influenza viruses (AIVs) have played either indirect or direct role in human infections. The continuous evolvement of AIVs through antigenic drift and genetic reassortment has led to emerging diversities of local virus strains or variants. These dynamic changes of AIVs, even low pathogenic avian influenza (LPAI) viruses, have not only affected poultry health but also resulted in severe and fatal human cases. Moreover, the persistence of these viruses at a population level may lead to outbreaks of AI occurring year after year in a vicious cycle, if they are not completely eradicated. In light of the emergence of novel H7N9 and other subtypes of AIVs in China in recent years, residents of Taiwan who live closely to this neighboring country must be well prepared in case these viruses are imported. Furthermore, the potential for interspecies transmission to humans from either of the two subtypes of avian H5N2 and H6N1 viruses increases the risk assessment of potential public health threats coming from continuous viral mutations. These two AIV subtypes are relevant in chickens and have been endemic in Taiwan for many years, and the newly emerged novel three subtypes (H5N2, H5N3, and H5N8 clade 2.3.4.4) of AIVs that have spread island-wide since January of 2015 must be expeditiously assessed for public health risks. In summary, this article discusses the history, virology, and epidemiology of avian influenza in humans. We believe that fully understanding virological and epidemiological characteristics of avian influenza viruses and the history of past pandemics or major epidemics will help strengthen the effectiveness of prevention and control measures.

Genetic characterization of a rare H12N3 avian influenza virus isolated from a green-winged teal in Japan.

This study reports on the genetic characterization of an avian influenza virus, subtype H12N3, isolated from an Eurasian green-winged teal (Anas crecca) in Japan in 2009. The entire genome sequence of the isolate was analyzed, and phylogenetic analyses were conducted to characterize the evolutionary history of the isolate. Phylogenetic analysis of the hemagglutinin and neuraminidase genes indicated that the virus belonged to the Eurasian-like avian lineage. Molecular dating indicated that this H12 virus is likely a multiple reassortant influenza A virus. This is the first reported characterization of influenza A virus subtype H12N3 isolated in Japan and these data contribute to the accumulation of knowledge on the genetic diversity and generation of novel influenza A viruses.

Molecular and epidemiological characterization of avian influenza viruses from gulls and dabbling ducks in Norway

BackgroundWild aquatic birds constitute the natural reservoir for avian influenza viruses (AIVs). Separate Eurasian and American AIV gene pools exist. Here, the prevalence and diversity of AIVs in gulls and dabbling ducks in Norway were described. The influence of host species and temporal changes on AIV prevalence was examined. Five AIVs from Norway, including three from common gull (Larus canus), were analyzed along with 10 available AIV genomes from gulls in Eurasia to search for evidence of intracontinental and intercontinental reassortment of gene segments encoding the internal viral proteins.MethodsSwabs collected from 2417 dabbling ducks and gulls in the south-west of Norway during five ordinary hunting seasons (August-December) in the period 2005–2010 were analyzed for presence of AIV. Multivariate linear regression was used to identify associations between AIV prevalence, host species and sampling time. Five AIVs from mallard (Anas platyrhynchos) (H3N8, H9N2) and common gull (H6N8, H13N2, H16N3) were full-length characterized and phylogenetically analyzed together with GenBank reference sequences.ResultsLow pathogenic AIVs were detected in 15.5% (CI: 14.1–17.0) of the samples. The overall AIV prevalence was lower in December compared to that found in August to November (p = 0.003). AIV was detected in 18.7% (CI: 16.8–20.6) of the dabbling ducks. A high AIV prevalence of 7.8% (CI; 5.9–10.0) was found in gulls. A similar temporal pattern in AIV prevalence was found in both bird groups. Thirteen hemagglutinin and eight neuraminidase subtypes were detected. No evidence of intercontinental reassortment was found. Eurasian avian (non H13 and H16) PB2 or PA genes were identified in five reference Eurasian gull (H13 and H16) AIV genomes from GenBank. The NA gene from the Norwegian H13N2 gull isolate was of Eurasian avian origin.ConclusionsThe similar temporal pattern in AIV prevalence found in dabbling ducks and gulls, the relatively high virus prevalence detected in gulls and the evidence of intracontinental reassortment in AIVs from gulls indicate that gulls that interact with dabbling ducks are likely to be mixing vessels for AIVs from waterfowl and gulls. Our results support that intercontinental reassortment is rare in AIVs from gulls in Eurasia.

Complete Genome Sequence Analysis of an H6N1 Avian Influenza Virus Isolated from Guangxi Pockmark Ducks

We report here the complete genomic sequence of a novel H6N1 avian influenza virus strain, A/Duck/Guangxi/GXd-5/2010(H6N1), isolated from pockmark ducks in Guangxi Province, Southern China. All of the 8 gene segments of A/Duck/Guangxi/GXd-5/2010(H6N1) are attributed to the Eurasian lineage; the amino acid motif of the cleavage site between HA1 and HA2 was P-Q-I-E-T-R-G. These are typical characteristics of the low-pathogenicity avian influenza virus. This study will help to understand the epidemiology and molecular characteristics of avian influenza virus in ducks.

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.

MODERN LABORATORY DIAGNOSTIC METHODS IN VETERINARY MEDICINE AND THE POSSIBILITY OF ITS APPLICATION

One of the greatest challenges of modern laboratory diagnosis is the selection of methods for fast, highly sensitive and specific detection of the infective agents. This challenge is present also in molecular diagnosing of causative agents in veterinary medicine. An example of molecular detection, the application of molecular diagnostic methods and procedures in the epizootiology of some viral infections is presented in the paper. The molecular methods play an important role in virus detection and surveillance. Out of a large number of molecular methods most frequently used are classical gel-based PCR (PCR, RT-PCR and nested PCR) and real-time PCR or RT-PCR techniques. Due to highly specificity and sensitivity these methods have been introduced as internationally recognized methods for virus detection in clinical materials. The advantage of the aforementioned molecular methods is that they are very fast and highly sensitive, and able to analyse a high number of samples. The obtained results may be used in molecular epizootiology, possibly for differentiation of filed isolates and vaccine virus strains, and for examining the samples not suitable for virus isolation. Alongside, these methods provide accurate quantification of viral particles in sample material. The detection with high sensitivity and specificity is of utmost importance in detection and typisation of the agents of highly contagious diseases and zoonoses. An example of rapid detection and sensitivity of RT-PCR and real-time RT-PCR in detection and characterization of avian influenza virus in clinical material as well as BVD virus in native bull semen is presented. Besides, molecular methods may be used for other purposes. Genome fragments amplified by PCR and RT-PCR, may be sequenced and used for classification, i.e. for virus isolate genotypisation. The results obtained in this way may be used for basic molecular research in epizootiology, that will point on the source of infections, their correlation and the prevalence of the causative agents what can help in finding the answer to the question why the diseases have occurred and what are the perspective for diseases outcome in the future. Also, these examination may help in determining pathogenicity, virulence and the spread of the pathogen agents. These information are of immeasurable importance for all the procedures in disease prevention and control. The possibilities of sequencing, molecular typisation and epizootiology of BVD virus and CSF virus isolated in Vojvodina in the last years are given as an example how this method can be used.

Molecular characterization of avian influenza virus (H7N8) isolated from poultry in Central China in the mid-1980s

The molecular and pathogenic properties of avian influenza virus (A/duck/Hubei/216/1985/H7N8) isolated from Hubei Province of China in 1985 were characterized. The hemagglutinin gene (HA) of Dk/Hb/216/85/H7N8 had the multiple amino acid sequences (-PEIPKGRG-) at the connecting peptide between HA1 and HA2, which is considered to be a distinguishing molecular characteristic of low pathogenicity. The key sites of host markers among the genes (M, NP, NS, PA and PB2) of Dk/Hb/216/85/H7N8 were similar to those of H5N1 viruses. Phylogenetic analysis showed that the genes (M, NS and PB2) of Dk/Hb/216/85/H7N8 clustered closely with those of Gs/Guangdong/1996/H5N1 (Gs/GD/96), implying that three ancient genes of Gs/GD/96-like viruses had been circulating in Central China during the 1980s. In experimental infection, Dk/Hb/216/85/H7N8 was lowly pathogenic to chickens and mice, consistent with the molecular feature of HA gene.

FAO‐OIE‐WHO Joint Technical Consultation on Avian Influenza at the Human‐Animal Interface

For the past 10 years, animal health experts and human health experts have been gaining experience in the technical aspects of avian influenza in mostly separate fora. More recently, in 2006, in a meeting of the small WHO Working Group on Influenza Research at the Human Animal Interface (Meeting report available from: http://www.who.int/csr/resources/publications/influenza/WHO_CDS_EPR_GIP_2006_3/en/index.html) in Geneva allowed influenza experts from the animal and public health sectors to discuss together the most recent avian influenza research. Ad hoc bilateral discussions on specific technical issues as well as formal meetings such as the Technical Meeting on HPAI and Human H5N1 Infection (Rome, June, 2007; information available from: http://www.fao.org/avianflu/en/conferences/june2007/index.html) have increasingly brought the sectors together and broadened the understanding of the topics of concern to each sector. The sectors have also recently come together at the broad global level, and have developed a joint strategy document for working together on zoonotic diseases (Joint strategy available from: ftp://ftp.fao.org/docrep/fao/011/ajl37e/ajl37e00.pdf). The 2008 FAO-OIE-WHO Joint Technical Consultation on Avian Influenza at the Human Animal Interface described here was the first opportunity for a large group of influenza experts from the animal and public health sectors to gather and discuss purely technical topics of joint interest that exist at the human-animal interface. During the consultation, three influenza-specific sessions aimed to (1) identify virological characteristics of avian influenza viruses (AIVs) important for zoonotic and pandemic disease, (2) evaluate the factors affecting evolution and emergence of a pandemic influenza strain and identify existing monitoring systems, and (3) identify modes of transmission and exposure sources for human zoonotic influenza infection (including discussion of specific exposure risks by affected countries). A final session was held to discuss broadening the use of tools and systems to other emerging zoonotic diseases. The meeting was structured as short technical presentations with substantial time available for facilitated discussion, to take advantage of the vast influenza knowledge and experience available from the invited expert participants. Particularly important was the identification of gaps in knowledge that have not yet been filled by either sector. Technical discussions focused on H5N1, but included other potentially zoonotic avian and animal influenza viruses whenever possible. During the consultation, the significant threat posed by subtypes other than H5N1 was continually emphasized in a variety of contexts. It was stressed that epidemiological and virological surveillance for these other viruses should be broadening and strengthened. The important role of live bird markets (LBMs) in amplifying and sustaining AIVs in some countries was also a recurring topic, and the need for better understanding of the role of LBMs in human zoonotic exposure and infection was noted. Much is understood about the contribution of various virus mutations and gene combinations to transmissibility, infectivity, and pathogenicity, although it was agreed that the specific constellation of gene types and mutations that would characterize a potentially pandemic virus remains unclear. The question of why only certain humans have become infected with H5N1 in the face of massive exposure in some communities was frequently raised during discussion of human exposure risks. It was suggested that individual-level factors may play a role. More research is needed to address this as well as questions of mode of transmission, behaviors associated with increased risk, virological and ecological aspects, and viral persistence in the environment in order to better elucidate specific human exposure risks. It became clear that great strides have been made in recent years in collaboration between the animal health and public health sectors, especially at the global level. In some countries outbreaks of H5N1 are being investigated jointly. Even greater transparency, cooperation, and information and materials exchange would allow more timely and effective responses in emergency situations, as well as in assessment and planning phases. Ensuring sustainability was also frequently emphasized, e.g. in infrastructure and capacity development and in development of tools and systems for surveillance, assessment and response. It was suggested that one way for tools and systems built or planned to address avian influenza to become more sustainable would be to make them applicable for a broader array of existing and emerging zoonotic diseases.

Genetic analysis of avian influenza virus from migratory waterfowl in Mexico

This study describes the genetic characterization of avian influenza virus from waterfowl in Mexico. Partial sequences of two H5, one H6, and one H9 hemmaglutinin (HA) genes were determined. The deduced amino acid sequences showed that they were low-pathogenic viruses (LPAI). Phylogenetic analysis of H5 and H6 HA indicates a North American lineage, closely related to contemporary Californian isolates, and H9 HA was closer to the Korean-like lineage. These results demonstrate the introduction of a diverse genetic pool of subtypes to Sonora estuaries through circulation of bird species carriers from the Pacific flyway.

Evaluation of field and laboratory protocols used to detect avian influenza viruses in wild aquatic birds

Careful selection and observance of standard field and laboratory protocols are critical for successful detection and characterization of avian influenza viruses (AIV) from wild birds. Cloacal swabs were collected from hunter-killed or nesting waterfowl and shorebirds from wildlife refuges in Alabama, Georgia, and Florida during 2006 to 2008. Swab samples were inoculated into embryonated eggs followed by hemagglutination (HA) test to determine the presence of hemagglutinating agents. Antigen capture-ELISA (AC-ELISA) and real-time reverse transcription-PCR (RRT-PCR) were used to detect AIV from both allantoic fluids (AF) and swab specimens of HA-positive samples. Hemagglutination inhibition test was used to detect Newcastle disease virus, another hemagglutinating virus common in wild birds. The HA-positive AF were sent to the National Veterinary Services Laboratory for subtyping of the isolates. Out of 825 samples tested, 19 AIV and 3 avian paramyxovirus subtypes were identified by the National Veterinary Services Laboratory. Without egg passage, AC-ELISA did not detect virus, whereas matrix gene of 13 AIV were detected using RRT-PCR. When testing was done on AF, 14 were positive for influenza A by AC-ELISA and 20 by RRT-PCR. Antigen capture-ELISA did not detect influenza A when the HA titer was lower than 125, whereas RRT-PCR detected AIV from AF with HA titer as low as 4. The highest isolation rate was from Florida, where out of 109 samples analyzed, 14 AIV were detected by RRT-PCR from AF. Real-time reverse transcription-PCR was more sensitive, specific, and cost-effective than AC-ELISA. However, to avoid false-negative results, testing should be performed on AF and not directly from cloacal swabs. Our procedures to detect AIV directly from cloacal swabs need further optimization for improved sensitivity.

Isolation and Characterization of Avian Influenza Virus Based on Macroscopic Examination of Native Chicken Embryo

The aim of this research was to isolate and characterize the virus based on macroscopic examination of native chickens embryo infected with the AI virus. This research used 10 infected native chickens from Ateuk Pahlawan and Doi villages. All chickens were examined to identify the present of AI using rapid test and in ovo virus propagation. Rapid test showed 7 chickens from Ateuk Pahlawan villageand 5 chickens from Doi village were AI positive. Propagation of the viruses in 11 days old chicken embryo in ovo showed that embryo died 5 days post inoculated whereas macroscopic examination showed dwarfed embryo, hemorrhagic, and loss of feather.

Characterization of Avian Influenza Viruses A (H5N1) from Wild Birds, Hong Kong, 2004–2008

From January 2004 through June 2008, surveillance of dead wild birds in Hong Kong, People's Republic of China, periodically detected highly pathogenic avian influenza (HPAI) viruses (H5N1) in individual birds from different species. During this period, no viruses of subtype H5N1 were detected in poultry on farms and in markets in Hong Kong despite intensive surveillance. Thus, these findings in wild birds demonstrate the potential for wild birds to disseminate HPAI viruses (H5N1) to areas otherwise free from the viruses. Genetic and antigenic characterization of 47 HPAI (H5N1) viruses isolated from dead wild birds in Hong Kong showed that these isolates belonged to 2 antigenically distinct virus groups: clades 2.3.4 and 2.3.2. Although research has shown that clade 2.3.4 viruses are established in poultry in Asia, the emergence of clade 2.3.2 viruses in nonpasserine birds from Hong Kong, Japan, and Russia raises the possibility that this virus lineage may have become established in wild birds.

Design and Validation of a Microarray for Detection, Hemagglutinin Subtyping, and Pathotyping of Avian Influenza Viruses

Continuing threats of devastating outbreaks in poultry and of human infections caused by highly pathogenic avian influenza virus (HPAIV) H5N1 emphasize the need for the further development of rapid and reliable methods of virus detection and characterization. Here we report on the design and comprehensive validation of a low-density microarray as a diagnostic tool for the detection and typing of avian influenza virus (AIV). The array consists of one probe for the conserved matrix gene and 97 probes targeting the HA(0) cleavage-site region. Following fragment amplification by a generic PCR approach, the array enables AIV detection, hemagglutinin (HA) subtyping, and pathotyping within a single assay. For validation, a panel of 92 influenza A viruses which included 43 reference strains representing all 16 HA subtypes was used. All reference strains were correctly typed with respect to their HA subtypes and pathotypes, including HPAIV H5N1/Asia, which caused outbreaks in Germany in 2006 and 2007. In addition, differentiation of strains of the Eurasian and North American lineages of the H5 and H7 subtypes was possible. The sensitivity of the microarray for the matrix gene is comparable to that of real-time reverse transcription-PCR (RT-PCR). It is, however, 10- to 100-fold lower than that of real-time RT-PCR with respect to HA subtyping and pathotyping. The specificity of the array was excellent, as no pathogens relevant for differential diagnosis yielded a positive reaction. Validation with field samples included 19 cloacal swab specimens from wild and domestic birds. Influenza A virus was verified in all samples, whereas the HA subtypes could be determined for 14 samples. The results demonstrate that the microarray assay described complements current methods and can accelerate the diagnosis and characterization of AIV.

Genetic characterization of avian influenza viruses isolated in Israel during 2000–2006

Our aim was to establish the phylogenetic and genetic relationships among avian influenza viruses (AIV) recently isolated from poultry in Israel. During this study we analyzed complete nucleotide sequences of two envelope (hemagglutinin and neuraminidase) and six internal genes (polymerase B1, polymerase B2, polymerase A, nucleoprotein, nonstructural, and matrix) of 29 selected H9N2 and six internal genes of five H5N1 viruses isolated in Israel during 2000-2006. Comparative genetic and phylogenetic analyses of these sequences revealed that the local H5N1 viruses are closely related to H5N1 viruses isolated in European, Asian, and Middle Eastern countries in 2005-2006. The H9N2 Israeli isolates, together with viruses isolated in Jordan and Saudi Arabia formed a single group. Our data support the claim that during recent years a new endemic focus of H9N2 has been formed in the Middle East. The introduction of H5N1 and co-circulation of these two subtypes of AIV in this region may augment the risk of potentially pandemic strains emergence.

Genetic characterization of avian influenza viruses isolated from waterfowl in southern part of South Korea in 2006

Aquatic birds are a reservoir of all known influenza A viruses. Avian influenza viruses have played a major role in the creation of pandemic influenza viruses in humans. In this study, we genetically characterized genes of nine isolates from waterfowl in Eulsukdo, a congregating place for migratory birds on the flyway of migration from Siberia, which is located in the southern part of South Korea. Phylogenic analysis showed that HA and NA genes of isolates belonged to Eurasian lineage, and lineage analysis showed that NS, PB1, PA, NP, and M genes of isolates clustered with Eurasian lineage, and PB2 genes of isolates belonged to North American or Eurasian lineage. Results suggest that the interregional transmission of genes of avian influenza viruses may occur in the migratory birds.

Isolation and Genetic Characterization of Avian Influenza Viruses and a Newcastle Disease Virus from Wild Birds in Barbados: 2003–2004

Zoonotic transmission of an H5N1 avian influenza A virus to humans in 2003-present has generated increased public health and scientific interest in the prevalence and variability of influenza A viruses in wild birds and their potential threat to human health. Migratory waterfowl and shorebirds are regarded as the primordial reservoir of all influenza A viral subtypes and have been repeatedly implicated in avian influenza outbreaks in domestic poultry and swine. All of the 16 hemagglutinin and nine neuraminidase influenza subtypes have been isolated from wild birds, but waterfowl of the order Anseriformes are the most commonly infected. Using 9-to-11-day-old embryonating chicken egg culture, virus isolation attempts were conducted on 168 cloacal swabs from various resident, imported, and migratory bird species in Barbados during the months of July to October of 2003 and 2004. Hemagglutination assay and reverse transcription-polymerase chain reaction were used to screen all allantoic fluids for the presence of hemagglutinating agents and influenza A virus. Hemagglutination positive-influenza negative samples were also tested for Newcastle disease virus (NDV), which is also found in waterfowl. Two influenza A viruses and one NDV were isolated from Anseriformes (40/168), with isolation rates of 5.0% (2/40) and 2.5% (1/40), respectively, for influenza A and NDV. Sequence analysis of the influenza A virus isolates showed them to be H4N3 viruses that clustered with other North American avian influenza viruses. This is the first report of the presence of influenza A virus and NDV in wild birds in the English-speaking Caribbean.

Isolation and characterization of avian influenza viruses, including highly pathogenic H5N1, from poultry in live bird markets in Hanoi, Vietnam, in 2001.

Since 1997, outbreaks of highly pathogenic (HP) H5N1 and circulation of H9N2 viruses among domestic poultry in Asia have posed a threat to public health. To better understand the extent of transmission of avian influenza viruses (AIV) to humans in Asia, we conducted a cross-sectional virologic study in live bird markets (LBM) in Hanoi, Vietnam, in October 2001. Specimens from 189 birds and 18 environmental samples were collected at 10 LBM. Four influenza A viruses of the H4N6 (n = 1), H5N2 (n = 1), and H9N3 (n = 2) subtypes were isolated from healthy ducks for an isolation frequency of over 30% from this species. Two H5N1 viruses were isolated from healthy geese. The hemagglutinin (HA) genes of these H5N1 viruses possessed multiple basic amino acid motifs at the cleavage site, were HP for experimentally infected chickens, and were thus characterized as HP AIV. These HA genes shared high amino acid identities with genes of other H5N1 viruses isolated in Asia during this period, but they were genetically distinct from those of H5N1 viruses isolated from poultry and humans in Vietnam during the early 2004 outbreaks. These viruses were not highly virulent for experimentally infected ducks, mice, or ferrets. These results establish that HP H5N1 viruses with properties similar to viruses isolated in Hong Kong and mainland China circulated in Vietnam as early as 2001, suggest a common source for H5N1 viruses circulating in these Asian countries, and provide a framework to better understand the recent widespread emergence of HP H5N1 viruses in Asia.

Characterization of avian influenza viruses isolated in Israel in 1978–1979

Influenza viruses were isolated both from migrating birds wintering in Israel (starlings and mallards) and from turkeys. Initial identification of the isolates was carried out in Israel using a complete set of monospecific antisera against all the reference hemagglutinin (HA) and neuraminidase (Nase) antigenic subtypes in HA inhibition (HI) and Nase inhibition (NI) tests. The results have shown that HA of all the isolates belong to Hav1 antigenic subtype, while the Nase of the starling isolate proved to be of Neq1 antigenic subtype and Nase of both the turkey and mallard isolates proved to be of N2 antigenic subtype. Possible epizootiological connections between the epizootic among the mallards and the influenza outbreak in the turkey farm (both occurred in close proximity and at the same period of time and in both cases antigenically identical influenza virus strains were isolated) are discussed.