Full sequence analysis of hemagglutinin and neuraminidase genes and proteins of highly pathogenic avian influenza H5N1 virus detected in Iran, 2015

Over the last two decades, the highly pathogenic avian influenza H5N1 virus has gained a lot of attention due to its zoonotic and mutative nature. Iran is among the countries significantly affected by the virus as it hosts migratory birds during seasonal migration. In this study, the molecular characterizations of hemagglutinin (HA) and neuraminidase (NA) genes and proteins of H5N1 strain A/chicken/Iran/8/2015 detected in backyard poultry, Mazandaran province, were investigated. Phylogenetic analysis classified this virus as a member of subclade 2.3.2.1c, with the cleavage site motif of “PQRERRRK-R/GLF”. HA carried a few mutations altering affinity to mammalian cells; however, the virus was categorized as avian. NA protein had the 20-amino acid deletion at aa position 49–69 similar to those isolated since 2000. Mutations of H253Y and H274Y contributing to antiviral resistance were present in NA. From this analysis, it can be concluded that the wild migratory birds flying from Western Asia to Eastern Africa are probably the main carriers of seasonal H5N1 in the country.

     

Limited susceptibility of pigeons experimentally inoculated with H5N1 highly pathogenic avian influenza viruses

An experimental infection study was performed using pigeons reared for racing or meat production in Japan and clade 2.2 and 2.3.2 isolates of H5N1 highly pathogenic avian influenza virus to evaluate the possible role of pigeons in virus transmission to poultry. In experiment 1, when 20 pigeons were intranasally inoculated with high or low viral doses, no inoculated pigeon exhibited clinical signs for 14 days. Drinking water and almost all swab samples were negative for virus isolation. Virus isolation was positive in 3 oral swab samples from 2 pigeons from day 2 through 4 postinoculation, but viral titers of positive samples were extremely low. Immunohistochemical analysis for virus detection was negative in all tissue samples. Along with seroconversion in a limited number of pigeons postinoculation, these results suggest that pigeons have limited susceptibility to the virus used for experimental infection. In experiment 2, when uninoculated chickens were housed with virus-inoculated pigeons, all pigeons and contact chickens survived for 14 days without exhibiting any clinical signs. According to serological analysis, the chickens did not exhibit seroconversion after close contact with inoculated pigeons. Our data suggest that the risk posed by pigeons with respect to the transmission of the H5N1 highly pathogenic avian influenza virus to poultry would be less than that for other susceptible avian species.     

Pathogenicity in quails and mice of H5N1 highly pathogenic avian influenza viruses isolated from ducks

In our study, the pathogenicity of H5N1 influenza A viruses circulating in waterfowls in Southern China was investigated. Three H5N1 highly pathogenic avian influenza (HPAI) viruses isolated from ducks, A/Duck/Guangdong/383/2008(DK383), A/Duck/Guangdong/378/2008(DK378) and A/Duck/Guangdong/212/2004(DK212) were inoculated at 10(6) fifty-percent egg infectious doses (EID(50)) into ducks, quails and mice and showed varying levels of pathogenicity. In ducks, the mortality rates ranged from 0 to 60% and the mean death time (MDT) was 0-6.7 days post-inoculation (DPI). While the viruses were highly pathogenic in quails, resulting in 83.3-100% mortality and the MDT of 2.3-3 DPI, they were completely lethal in mice (100% mortality). The viruses replicated in many organs of ducks and quails and were found in the brain, and kidney, lung and spleen of the mice. Phylogenetic analysis revealed that DK383 and DK378 viruses of clade 2.3.2 belonged to genotype 11, while DK212 virus of clade 9 was genotype 3. Our study illustrated H5N1 influenza viruses within Clade 2.3.2 and 9 from duck in Southern China had very highly pathogenicity to Japanese quails and BALB/c mice, but viruses within Clade 2.3.2 had more highly lethality than those of clade 9 to Muscovy ducks. Therefore, they had posed a continued challenge for disease control and public health.     

Evolution of highly pathogenic H7N3 avian influenza viruses in Mexico

Highly pathogenic H7N3 influenza A viruses have persisted in poultry in Mexico since 2012, diversifying into multiple lineages that have spread to three Mexican states, as of 2016. The H7N3 viruses segregate into three distinct clades that are geographically structured. All 2016 viruses are resistant to adamantane antiviral drugs and have an extended 24‐nucleotide insertion at the HA cleavage site that was acquired from host 28S ribosomal RNA.     

Susceptibility of laughing gulls (Larus atricilla) to H5N1 and H5N3 highly pathogenic avian influenza viruses

This investigation detailed the clinical disease, gross and histologic lesions, and distribution of viral antigen in juvenile laughing gulls (Larus atricilla) intranasally inoculated with either the A/tern/South Africa/61 (H5N3) (tern/SA) influenza virus or the A/chicken/Hong Kong/220/97 (H5N1) (chicken/HK) influenza virus, which are both highly pathogenic for chickens. Neither morbidity nor mortality was observed in gulls inoculated with either virus within the 14-day investigative period. Gross lesions resultant from infection with either virus were only mild, with the tern/SA virus causing decreased lucency of the air sacs (2/6), splenomegaly (2/6), and pancreatic mottling (1/6) and the chicken/HK virus causing only decreased lucency of the air sacs (2/8) and conjunctival edema (2/8). Histologic lesions in the tern/SA-inoculated gulls included a mild to moderate heterophilic to lymphoplasmacytic airsacculitis (6/6), mild to moderate interstitial pneumonia (3/6), and moderate necrotizing pancreatitis and hepatitis at 14 days postinoculation (DPI) (2/6). Immunohistochemical demonstration of viral antigen occurred only in association with lesions in the liver and pancreas. In contrast, viral antigen was not demonstrated in any tissues from the chicken/HK-inoculated gulls, and inflammatory lesions were confined to the air sac (3/8) and lungs (3/8). Both viruses were isolated at low titers (<10(1.68) mean embryo lethal dose) from oropharyngeal and cloacal swabs up to 7 days postinoculation (DPI), from the lung and kidney of one of two tern/SA-inoculated gulls at 14 DPI, and from the lung of one of two chicken/HK-inoculated gulls at 7 DPI. Antibodies to influenza viruses as determined with the agar gel precipitin test at 14 DPI were detected only in the two tern/SA-inoculated gulls and not in the two chicken/HK-inoculated gulls.     

Origin and evolution of highly pathogenic H5N1 avian influenza in Asia

Outbreaks of highly pathogenic avian influenza caused by H5N1 viruses were reported almost simultaneously in eight neighbouring Asian countries between December 2003 and January 2004, with a ninth reporting in August 2004, suggesting that the viruses had spread recently and rapidly. However, they had been detected widely in the region in domestic waterfowl and terrestrial poultry for several years before this, and the absence of widespread disease in the region before 2003, apart from localised outbreaks in the Hong Kong Special Autonomous Region (SAR), is perplexing. Possible explanations include limited virus excretion by domestic waterfowl infected with H5N1, the confusion of avian influenza with other serious endemic diseases, the unsanctioned use of vaccines, and the under-reporting of disease as a result of limited surveillance. There is some evidence that the excretion of the viruses by domestic ducks had increased by early 2004, and there is circumstantial evidence that they can be transmitted by wild birds. The migratory birds from which viruses have been isolated were usually sick or dead, suggesting that they would have had limited potential for carrying the viruses over long distances unless subclinical infections were prevalent. However, there is strong circumstantial evidence that wild birds can become infected from domestic poultry and potentially can exchange viruses when they share the same environment. Nevertheless, there is little reason to believe that wild birds have played a more significant role in spreading disease than trade through live bird markets and movement of domestic waterfowl. Asian H5N1 viruses were first detected in domestic geese in southern China in 1996. By 2000, their host range had extended to domestic ducks, which played a key role in the genesis of the 2003/04 outbreaks. The epidemic was not due to the introduction and spread of a single virus but was caused by multiple viruses which were genotypically linked to the Goose/GD/96 lineage via the haemagglutinin gene. The H5N1 viruses isolated from China, including the Hong Kong SAR, between 1999 and 2004 had a range of genotypes and considerable variability within genotypes. The rising incidence and widespread reporting of disease in 2003/04 can probably be attributed to the increasing spread of the viruses from existing reservoirs of infection in domestic waterfowl and live bird markets leading to greater environmental contamination. When countries in the region started to report disease in December 2003, others were alerted to the risk and disease surveillance and reporting improved. The H5N1 viruses have reportedly been eliminated from three of the nine countries that reported disease in 2003/04, but they could be extremely difficult to eradicate from the remaining countries, owing to the existence of populations and, possibly, production and marketing sectors, in which apparently normal birds harbour the viruses.     

Pathogenicity of highly pathogenic avian influenza viruses of H5N1 subtype isolated in Thailand for different poultry species

Highly pathogenic avian influenza (HPAI) viruses of the H5N1 subtype have caused several rounds of outbreaks in Thailand. In this study, we used 3 HPAI viruses isolated in Thailand in January 2004 from chicken, quail, and duck for genetic and pathogenetic studies. Sequence analysis of the entire genomes of these isolates revealed that they were genetically similar to each other. Chickens, quails, domestic ducks, and cross-bred ducks were inoculated with these isolates to evaluate their pathogenicity to different host species. A/chicken/Yamaguchi/7/04 (H5N1), an HPAI virus isolated in Japan, was also used in the chicken and quail studies for comparison. All four isolates were shown to be highly pathogenic to chickens and quails, with 100% mortality by 10(6) EID50 inoculants of the viruses. They caused sudden death in chickens and quails within 2-4 days after inoculation. The mean death times (MDT) of quails infected with the Thai isolates were shorter than those of chickens infected with the same isolates. Mortality against domestic and cross-bred ducks ranged from 50 to 75% by intranasal inoculation with the 10(6) EID50 viruses. Neurological symptoms were observed in most of the inoculated domestic ducks and appeared less severe in the cross-bred ducks. The MDTs of the ducks infected with the Thai isolates were 4.8-6 days post-inoculation. Most of the surviving ducks infected with the Thai isolates had sero-converted until 14 dpi. Our study illustrated the pathobiology of the Thai isolates against different poultry species and would provide useful information for improving control strategies against HPAI.     

Genetic variation of highly pathogenic H5N1 avian influenza viruses in Vietnam shows both species-specific and spatiotemporal associations

Domestic poultry act as a reservoir for persistent H5N1 endemicity in Vietnam, and the circulation of poultry flocks across farms and to market is thought to drive the spatial movement and evolution of avian influenza viruses. Using a dataset of complete or nearly full genomic sequences from highly pathogenic H5N1 avian influenza viruses collected in domestic poultry in Vietnam from 2003 to 2007, we explore potential differences in genetic characteristics according to species of isolation and the spatiotemporal characteristics of the viruses. Clustering algorithms and ANOVA indicate that H5N1 viruses in Vietnam show differences in the amount of genetic change that chicken viruses experience as compared to duck viruses, with duck viruses showing higher rates of molecular evolution on all eight of influenza's gene segments. There also exist distinct patterns of genetic differentiation according to the year in which they were isolated. These findings suggest that genetic evolution of avian influenza viruses is continuous through time but could also be mediated by the species in which the viruses occur, information that has implications for prevention efforts.     

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.     

The financial cost implications of the highly pathogenic notifiable avian influenza H5N1 in Nigeria

Nigeria and several other nations have recently been affected by outbreaks of the Asian H5N1 strain of highly pathogenic notifiable avian influenza (HPNAI) virus, which affects the poultry sector most heavily. This study analysed previous methods of assessing losses due to avian influenza, and used a revised economic model to calculate costs associated with the current avian influenza outbreaks. The evaluation used epidemiological data, production figures and other input parameters to determine the final costs. An infection involving 10% of the commercial bird population will cost Nigeria about $245 million and a worse scenario may lead to a loss of around $700 million. The results urge governments to invest more in measures aimed at the effective prevention of HPNAI and to consider the huge economic losses associated with the disease. Finally, an inter-disciplinary approach to managing and controlling HPNAI outbreaks is encouraged.     

The spread of highly pathogenic avian influenza (subtype H5N1) clades in Bangladesh, 2010 and 2011

Since the global spread of highly pathogenic avian influenza H5N1 during 2005–2006, control programs have been successfully implemented in most affected countries. HPAI H5N1 was first reported in Bangladesh in 2007, and since then 546 outbreaks have been reported to the OIE. The disease has apparently become endemic in Bangladesh. Spatio-temporal information on 177 outbreaks of HPAI H5N1 occurring between February 2010 and April 2011 in Bangladesh, and 37 of these outbreaks in which isolated H5N1 viruses were phylogenetically characterized to clade, were analyzed.     

Global occurrence and spread of highly pathogenic avian influenza virus of the subtype H5N1

Highly pathogenic avian influenza viruses of the subtype H5N1 (HPAIV-H5N1) have circulated continuously in Asia, Europe, and Africa since 2003. Investigations on the environmental preference and global spread processes of the virus are needed. We compiled 16 environmental variables to assess their correlation with HPAIV-H5N1 occurrences by using a niche-based model called Maxent. We found the virus had the strongest positive association with the human footprint index, as well as the presence of certain types of wetlands and mild temperature (10-30 C). Outbreaks of HPAIV-H5N1 in poultry or wild birds were also more frequent in certain major habitat types (e.g., tropical and subtropical moist broadleaf forests, temperate broadleaf and mixed forests, and flooded grasslands and savannas) and ecoregions. We conducted trend surface analysis to generate the travelling wave of the virus' global spread from 2003 to 2009, which indicated that high mountains or plateaus did not affect the spread speed and direction.     

Molecular characterization of H5N2 avian influenza viruses isolated from South African ostriches in 2006

Highly pathogenic avian influenza (HPAI) H5N2 reemerged in ostriches in South Africa during 2006, and a low-pathogenic AI H5N2 virus was also isolated. Molecular and phylogenetic characterization was performed to determine whether the outbreak strains were genetically derived from the supposedly eradicated Eastern Cape ostrich outbreak HPAI H5N2 strain of 2004. It was demonstrated that although the 2004 and 2006 South African H5N2 strains shared a common ancestor, the two outbreaks were not related. Not only were extensive reassortments with wild bird viruses involved in the evolution of the 2006 strains, but the precursor HA molecule HA0 cleavage site sequence of the 2006 HPAI H5N2 virus also contained fewer basic amino-acid insertions. Multiple transmission events occurred from wild birds to ostriches in 2006, and it appears that a reservoir of H5N2 with pathogenic potential for poultry is established in the South African wild duck population.     

Duck MDA5 functions in innate immunity against H5N1 highly pathogenic avian influenza virus infections

Melanoma differentiation-associated gene 5 (MDA5) is an important intracellular receptor that recognizes long molecules of viral double-stranded RNA in innate immunity. To understand the mechanism of duck MDA5-mediated innate immunity, we cloned the MDA5 cDNA from the Muscovy duck (Cairina moschata). Quantitative real-time PCR analysis indicates that duck MDA5 mRNA was constitutively expressed in all sampled tissues. A significant increase of MDA5 mRNA was detected in the brain, spleen and lungs of ducks after infection with an H5N1 highly pathogenic avian influenza virus (HPAIV). We investigated the role of the predicted functional domains of MDA5. The results indicate the caspase activation and recruitment domain (CARD) of duck MDA5 had a signal transmission function through IRF-7-dependent signaling pathway. Overexpression of the CARD strongly activated the chicken IFN-β promoter and upregulated the mRNA expression of antiviral molecules (such as OAS, PKR and Mx), proinflammatory cytokines (such as IL-2, IL-6, IFN-α and IFN-γ, but not IL-1β and IL-8) and retinoic acid-inducible gene I (RIG-I)-like receptors (RLR) (RIG-I and LGP2) without exogenous stimulation. We also demonstrate the NS1 of the H5N1 HPAIV inhibited the duck MDA5-mediated signaling pathway in vitro. These results suggest that duck MDA5 is an important receptor for inducing antiviral activity in the host immune response of ducks.     

Metapopulation dynamics and determinants of H5N1 highly pathogenic avian influenza outbreaks in Indonesian poultry

In 2008, the Indonesian Government implemented a revised village-level Participatory Disease Surveillance and Response (PDSR) program to gain a better understanding of both the magnitude and spatial distribution of H5N1 highly pathogenic avian influenza (HPAI) outbreaks in backyard poultry. To date, there has been considerable collection of data, but limited publically available analysis. This study utilizes data collected by the PDSR program between April 2008 and September 2010 for Java, Bali and the Lampung Province of Sumatra. The analysis employs hierarchical Bayesian occurrence models to quantify spatial and temporal dynamics in backyard HPAI infection reports at the District level in 90 day time periods, and relates the probability of HPAI occurrence to PDSR-reported village HPAI infection status and human and poultry density. The probability of infection in a District was assumed to be dependent on the status of the District in the previous 90 day time period, and described by either a colonization probability (the probability of HPAI infection in a District given there had not been infection in the previous 90 day time period) or a persistence probability (the probability of HPAI infection being maintained in the District from the previous to current 90 day period). Results suggest that the number of surveillance activities in a district had little relationship to outbreak occurrence probabilities, but human and poultry densities were found to have non-linear relationships to outbreak occurrence probabilities. We found significant spatial dependency among neighboring districts, indicating that there are latent spatial processes that are not captured by the covariates available for this study, but which nonetheless impact outbreak dynamics. The results of this work may help improve understanding of the seasonal nature of H5N1 in poultry and the potential role of poultry density in enabling endemicity to occur, as well as to assist the Government of Indonesia target scarce resources to regions and time periods when outbreaks of HPAI in poultry are most likely to occur.     

Characterization of encephalitis in wild birds naturally infected by highly pathogenic avian influenza H5N1

During the outbreak of highly pathogenic avian influenza (HPAI) H5N1 in Sweden in 2006, disease and mortality were observed in a number of wild bird species. Encephalitis was one of the most consistent and severe findings in birds submitted for postmortem examination. However, the distribution and severity of the inflammation varied among individuals. This study characterized the encephalitis and the phenotype of the cellular infiltrate in brains of 40 birds of various species naturally infected with HPAI H5N1. Brain sections stained with hematoxylin and eosin and immunostained for influenza A viral antigen were evaluated in parallel to brain sections immunostained with antibodies against T lymphocytes (CD3+), B lymphocytes (CD79a+), macrophages (Lectin RCA-1+), and astrocytes expressing glial fibrillary acidic protein. The virus showed marked neurotropism, and the neuropathology included multifocal to diffuse areas of gliosis and inflammation in the gray matter, neuronal degeneration, neuronophagia, vacuolation of the neuropil, focal necrosis, perivascular cuffing, and meningitis. Broad ranges in severity, neuroanatomical distribution, and type of cellular infiltrate were observed among the different bird species. Since neurotropism is a key feature of HPAI H5N1 infection in birds and other species and because the clinical presentation can vary, the characterization of the inflammation in the brain is important in understanding the pathogenesis of the disease and also has important diagnostic implications for sample selection.     

An outbreak of highly pathogenic avian influenza subtype H5N1 in broiler breeders, Korea

Highly pathogenic avian influenza (HPAI) was diagnosed in broiler breeders, submitted to the National Veterinary Research and Quarantine Service in South Korea. Grossly, the dead breeders had lesions consistent with HPAI, including pancreatic mottling, splenomegaly, pulmonary edema and congestion, and hemorrhages in the mucosa of the proventriculus, gizzard and small intestine, and on the serosal surface. Microscopically, there were necrotized hepatitis and pancreatitis, lymphocytic meningoencephalitis, myocarditis, and interstitial pneumonia. Influenza viral antigen was demonstrated in areas closely associated with histopathologic lesions. The AI virus was isolated from cecal tonsils, feces, trachea, and kidney of the chickens. The isolated virus was identified as the highly pathogenic H5N1, with a hemagglutinin proteolytic cleavage site deduced amino acid sequences of QREKRKKR/GLFGAGLFGAIAG. In order to determine the pathogenicity of the isolate, eight 6-week-old specific pathogen free chickens were inoculated intravenously with the virus, and all the birds died within 24 hr after inoculation. This is the first report of an outbreak of HPAI in the chickens in South Korea.     

Efficacy of vaccines in chickens against highly pathogenic Hong Kong H5N1 avian influenza.

In 1997, highly pathogenic (HP) H5N1 avian influenza virus (AIV) caused infections in poultry in Hong Kong and crossed into humans, resulting in a limited number of infections including 18 hospitalized cases and six associated deaths. The unique ability of this, AIV to infect both poultry and people raised a concern for the potential of humans to be biological as well as mechanical vectors of this AIV to poultry. The current study was undertaken to determine if existing vaccines and their technologies could be used during an outbreak to protect poultry. Commercial and experimental inactivated whole H5 AIV and baculovirus-expressed AIV H5 hemagglurinin protein vaccines provided protection from clinical signs and death in chickens after lethal challenge by human-origin HP H5N1 Hong Kong strains 156/97 and 483/97. The commercial and experimental inactivated vaccines had mean protective doses ranging from 0.25 to 0.89, which represents the milligrams of viral protein in the vaccines that provided protection from death in half of the birds. Furthermore, the vaccines reduced the ability of the challenge AIV to replicate in chickens and decreased the recovery of challenge AIV from the enteric and respiratory tracts, but the use of a vaccine will nor totally prevent AI virus replication and shedding. Existing vaccines will protect poultry from mortality and reduce virus replication from the new HP AIV strain that can infect both poultry and humans.