Influenza Virus Of Subtype H5N1 Research Articles

Use of “ARRIAH-AviFluVac” vaccine in turkeys, geese and ducks

“ARRIAH-AviFluVac” vaccine against H5 avian influenza was demonstrated to be effective for ducks, geese and turkeys both in the laboratory and production environment. When administered to ducks at 0.5; 1.0 and 1.5 cm3, the vaccine provided 100%-effective protection of birds against the disease and death after challenge with the relevant high pathogenicity avian influenza virus of subtype H5N1, clade 2.3.4.4b. Singular 0.5–1.5 cm3 inoculation induced formation of antibodies, which were detected in the hemagglutination inhibition test at the titres that ranged from 4.3 to 6.1 log2. The vaccine facilitated 9–26-fold decrease in the virulent virus shedding by the ducks. Protection of turkeys vaccinated at the dose of 1.0 cm3 was maintained at the level of 87.5% after challenge with high pathogenicity avian influenza virus of subtype H5N1, clade 2.3.4.4b. The vaccine induced formation of antibodies at the titres of 4.9 and 5.5 log2 in turkeys after singular and double vaccination at the dose of 1.0 cm3, respectively. It was demonstrated, that after double administration of 1.0 cm3 of “ARRIAH-AviFluVac” vaccine, the post-vaccinal avian influenza antibody level exceeded 5.0 log2 in 75.9–90.0% of the geese population. The most appropriate way of the vaccine use in turkeys, ducks and geese involves at least its double administration at the double commercial dose. Higher species resistance of ducks to the challenge with avian influenza virus of subtype H5, clade 2.3.4.4bas compared to turkeys was also demonstrated.

Molecular analysis of hemagglutinin-1 fragment of avian influenza H5N1 viruses isolated from chicken farms in Indonesia from 2008 to 2010

Highly pathogenic avian influenza virus of subtype H5N1 (AIV-H5N1) has been circulating in Indonesia since 2003. To understand the genetic diversity of these viruses, and to predict vaccine efficacy, the hemaglutinin-1 (HA-1) fragment of viruses isolated from chicken farms in Indonesia from 2008 to 2010 was sequenced and analyzed. The effects of these molecular changes were investigated in challenge experiments and HI assays of homologous and heterologous strains. Molecular analysis showed that these AIV-H5N1 isolates had evolved into three distinct sub-lineages from an ancestor circulating since 2003. Although no significant positive selection of residues was detected, 12 negatively selected sites were identified (p<0.05). Moreover, four sites showed evidence of significant episodic diversifying selection. The findings indicated complete protectivity and high HI titers with homologous strains, compared with protectivity ranging from 40 to 100% and lower HI titers with heterologous strains resulting from polymorphisms at antigenic sites. Our findings provide valuable insight into the molecular evolution of AIV and have important implications for vaccine efficacy and future vaccination strategies.

Molecular dynamics simulation of the effects of single (S221P) and double (S221P and K216E) mutations in the hemagglutinin protein of influenza A H5N1 virus: a study on host receptor specificity

Avian influenza viruses of subtype H5N1 circulating in animals continue to pose threats to human health. The binding preference of the viral surface protein hemagglutinin (HA) to sialosaccharides of receptors is an important area for understanding mutations in the receptor binding site that could be the cause for avian-to-human transmission. In the present work, we studied the effect of two receptor binding site mutations, S221P singly and in combination with another mutation K216E in the HA protein of influenza A H5N1 viruses. Docking of sialic acid ligands corresponding to both avian and human receptors and molecular dynamics simulations of the complexes for wild and mutant strains of H5N1 viruses were carried out. The H5N1 strain possessing the S221P mutation indicated decreased binding to α2,3-linked sialic acids (avian receptor, SAα2,3Gal) when compared to the binding of the wild-type strain that did not possess the HA-221 mutation. The binding to α2,6-linked sialic acids (human receptor, SAα2,6Gal) was found to be comparable, indicating that the mutant strain shows limited dual receptor specificity. On the other hand, the S221P mutation in synergism with the K216E mutation in the binding site, resulted in increased binding affinity for SAα2,6Gal when compared to SAα2,3Gal, indicative of enhanced binding to human receptors. The in-depth study of the molecular interactions in the docked complexes could explain how co-occurring mutations in the HA viral protein can aid in providing fitness advantage to the virus, in the context of host receptor specificity in emerging variants of H5N1 influenza viruses.

A model for early onset of protection against lethal challenge with highly pathogenic H5N1 influenza virus

Highly pathogenic avian influenza viruses of subtype H5N1 sporadically cause severe disease in humans and involve the risk of inducing a pandemic by gaining the ability for human-to-human transmission. In naïve poultry, primarily gallinaceous birds, the virus induces fatal disease and the used inactivated vaccines occasionally are unable to provide efficient and early onset of protection. Therefore, optimized vaccines must be developed and evaluated in model systems. In our study, we tested a novel H5 neuraminidase-deleted influenza A virus variant to analyze the induction of a very early onset of immunity. Ferrets, mice and chickens were each immunized with a single vaccine dose seven, three and one day before lethal challenge infection, respectively. Sound protection was conferred in 100% of animals immunized seven days prior to challenge infection. In these animals, no clinical signs were observed, and no challenge virus RNA was detected by real-time RT-PCR analyses of swabs, nasal washings, and organ samples. Moreover, the attenuated modified-live virus variant protected all chickens, mice, and ferrets as early as three days after vaccination against severe clinical signs. Chickens and ferrets developed hemagglutinin-specific antibodies after seven days, but no neuraminidase-specific antibodies, making this kind of neuraminidase-negative strain suitable for the DIVA (“differentiating vaccinated from infected animals”) strategy.

ClassyFlu: classification of influenza A viruses with Discriminatively trained profile-HMMs.

Accurate and rapid characterization of influenza A virus (IAV) hemagglutinin (HA) and neuraminidase (NA) sequences with respect to subtype and clade is at the basis of extended diagnostic services and implicit to molecular epidemiologic studies. ClassyFlu is a new tool and web service for the classification of IAV sequences of the HA and NA gene into subtypes and phylogenetic clades using discriminatively trained profile hidden Markov models (HMMs), one for each subtype or clade. ClassyFlu merely requires as input unaligned, full-length or partial HA or NA DNA sequences. It enables rapid and highly accurate assignment of HA sequences to subtypes H1–H17 but particularly focusses on the finer grained assignment of sequences of highly pathogenic avian influenza viruses of subtype H5N1 according to the cladistics proposed by the H5N1 Evolution Working Group. NA sequences are classified into subtypes N1–N10. ClassyFlu was compared to semiautomatic classification approaches using BLAST and phylogenetics and additionally for H5 sequences to the new “Highly Pathogenic H5N1 Clade Classification Tool” (IRD-CT) proposed by the Influenza Research Database. Our results show that both web tools (ClassyFlu and IRD-CT), although based on different methods, are nearly equivalent in performance and both are more accurate and faster than semiautomatic classification. A retraining of ClassyFlu to altered cladistics as well as an extension of ClassyFlu to other IAV genome segments or fragments thereof is undemanding. This is exemplified by unambiguous assignment to a distinct cluster within subtype H7 of sequences of H7N9 viruses which emerged in China early in 2013 and caused more than 130 human infections. http://bioinf.uni-greifswald.de/ClassyFlu is a free web service. For local execution, the ClassyFlu source code in PERL is freely available.

Host Cytokine Responses of Pigeons Infected with Highly Pathogenic Thai Avian Influenza Viruses of Subtype H5N1 Isolated from Wild Birds

Highly pathogenic avian influenza virus (HPAIV) of the H5N1 subtype has been reported to infect pigeons asymptomatically or induce mild symptoms. However, host immune responses of pigeons inoculated with HPAIVs have not been well documented. To assess host responses of pigeons against HPAIV infection, we compared lethality, viral distribution and mRNA expression of immune related genes of pigeons infected with two HPAIVs (A/Pigeon/Thailand/VSMU-7-NPT/2004; Pigeon04 and A/Tree sparrow/Ratchaburi/VSMU-16-RBR/2005; T.sparrow05) isolated from wild birds in Thailand. The survival experiment showed that 25% of pigeons died within 2 weeks after the inoculation of two HPAIVs or medium only, suggesting that these viruses did not cause lethal infection in pigeons. Pigeon04 replicated in the lungs more efficiently than T.sparrow05 and spread to multiple extrapulmonary organs such as the brain, spleen, liver, kidney and rectum on days 2, 5 and 9 post infection. No severe lesion was observed in the lungs infected with Pigeon04 as well as T.sparrow05 throughout the collection periods. Encephalitis was occasionally observed in Pigeon04- or T.sparrow05-infected brain, the severity, however was mostly mild. To analyze the expression of immune-related genes in the infected pigeons, we established a quantitative real-time PCR analysis for 14 genes of pigeons. On day 2 post infection, Pigeon04 induced mRNA expression of Mx1, PKR and OAS to a greater extent than T.sparrow05 in the lungs, however their expressions were not up-regulated concomitantly on day 5 post infection when the peak viral replication was observed. Expressions of TLR3, IFNα, IL6, IL8 and CCL5 in the lungs following infection with the two HPAIVs were low. In sum, Pigeon04 exhibited efficient replication in the lungs compared to T.sparrow05, but did not induce excessive host cytokine expressions. Our study has provided the first insight into host immune responses of pigeons against HPAIV infection.

Differential host gene responses in mice infected with two highly pathogenic avian influenza viruses of subtype H5N1 isolated from wild birds in Thailand

In Thailand, highly pathogenic avian influenza (HPAI) viruses of subtype H5N1 had been isolated from various wild birds during the HPAI outbreak in poultries. In this study, we examined the pathogenicity of two wild bird isolates (A/Pigeon/Thailand/VSMU-7-NPT/2004; Pigeon04 and A/Tree sparrow/Ratchaburi/VSMU-16-RBR/2005; T.sparrow05) in mice. They showed similar replication in several organs and lethal outcome. However, on day 3 post-infection, Pigeon04 induced mRNA expression of proinflammatory cytokines (IL6 and TNFα) and MIP-2, neutrophil chemoattractant, in the lungs, resulting in severe pneumonia that was accompanied by neutrophil infiltration. In contrast, on day 7 post-infection, T.sparrow05 induced the expression of several cytokines to a greater extent than Pigeon04; it also potently induced mRNA expression of several cytokines in brains of the infected mice that triggered frequent inflammatory events. In sum, our study demonstrated that two HPAI viruses induced different host responses, despite having similar replications, resulting in lethal outcome in mice.

Genetic Characterization of Variant Strains of Highly Pathogenic Avian Influenza H5N1 That Escaped Detection by Real-Time Reverse Transcriptase–PCR Diagnostic Tests

The highly pathogenic avian influenza virus of subtype H5N1 that caused serious outbreaks in Egypt in 2006 was efficiently detected using a commercially available real-time reverse transcriptase-PCR (RRT-PCR) for the type A specific matrix (M) gene in field samples of cloacal and tracheal swabs. RRT-PCR was also used for subtyping and confirmation of H5 subtype. During late 2007 the National Laboratory for Veterinary Quality Control on Poultry Production detected five field cases that were positive for avian influenza virus (AIV) based on the M gene RRT-PCR. Three different commercial H5 RRT-PCRs were used for identification of the H5 subtype, as well as a published World Organization for Animal Health (OIE) H5 RRT-PCR that had been previously carefully validated. The five cases had positive results for the H5 gene using the published OIE H5 RRT-PCR, but the three commercial H5 RRT-PCRs tests only returned two to four positive results out of the five positive cases. The hemagglutinin gene (HA) sequencing analysis of these five isolates showed multiple nucleotide substitution mutations, suggesting genetic variation that could affect the H5 primer and/or probe binding sequences. These data highlight the importance of continued monitoring of RRT-PCR primers and probes to ensure that sensitivity and specificity are maintained. The use of conventional methods in national and reference AIV laboratories, including virus isolation, serologic subtyping, and alternative RRT-PCR primers, is necessary to detect the newly emerging variant H5N1 strains that affect diagnostic performance.

Avian influenza viruses in mammals

Highly pathogenic avian influenza viruses of subtype H5N1 are remarkable because of their expanding non-avian host range and wide tissue tropism. They have caused severe or fatal respiratory and extra-respiratory disease in seven naturally infected species of carnivore. However, they are not unique in their ability to cross the species barrier, to cause clinical disease and mortality, or to replicate in extra-respiratory organs. Low pathogenic avian influenza viruses have crossed from birds to swine, horses, harbour seals, whales and mink; have resulted in severe respiratory disease and mortality; and may have spread beyond the respiratory tract in some of these species. They are also transmitted from mammal to mammal in most species, and have become endemic in swine and horse populations, demonstrating their ability to adapt to and become sustained in mammals. Until now, highly pathogenic avian influenza viruses H5N1 have not acquired this ability, but there are concerns that they may adapt to mammalian species and, thus, could spark an influenza pandemic in humans.

Epidemiological and Ornithological Aspects of Outbreaks of Highly Pathogenic Avian Influenza Virus H5N1 of Asian Lineage in Wild Birds in Germany, 2006 and 2007

In Germany, two distinct episodes of outbreaks of highly pathogenic avian influenza virus of subtype H5N1 (HPAIV H5N1) in wild birds occurred at the beginning of 2006, and in summer 2007. High local densities of wild bird populations apparently sparked clinically detectable outbreaks. However, these remained restricted in (i) number of birds, (ii) species found to be affected, (iii) time, and (iv) location despite the presence of several hundred thousands of susceptible wild birds and further stressors (food shortage, harsh weather conditions and moulting). Northern and southern subpopulations of several migratory anseriform species can be distinguished with respect to their preference for wintering grounds in Germany. This corroborates viral genetic data by Starick et al. (2008) demonstrating the introduction of two geographically restricted virus subpopulations of Qinghai-like lineage (cluster 2.2.A and 2.2.B) into northern and southern Germany, respectively, in 2006. The incursion of virus emerging in 2007, found to be distinct from the clusters detected in 2006 (Starick et al., 2008), may have been associated with moulting movements. Intensive past-outbreak investigations with negative results of live and dead wild birds and of terrestrial scavengers excluded continued circulation of virus on a larger scale. However, persistence of virus in small pockets of local wild bird populations could not be ruled out resiliently. 1.5% of investigated sera originating from cats sampled at the epicentres of the Ruegen 2006-outbreak contained H5-antibodies. Passive monitoring was found to be highly superior to live bird surveillance when aiming at the detection of HPAIV H5N1 in wild birds (P < 0.0001).

Human and Avian Influenza Viruses Target Different Cells in the Lower Respiratory Tract of Humans and Other Mammals

Viral attachment to the host cell is critical for tissue and species specificity of virus infections. Recently, pattern of viral attachment (PVA) in human respiratory tract was determined for highly pathogenic avian influenza virus of subtype H5N1. However, PVA of human influenza viruses and other avian influenza viruses in either humans or experimental animals is unknown. Therefore, we compared PVA of two human influenza viruses (H1N1 and H3N2) and two low pathogenic avian influenza viruses (H5N9 and H6N1) with that of H5N1 virus in respiratory tract tissues of humans, mice, ferrets, cynomolgus macaques, cats, and pigs by virus histochemistry. We found that human influenza viruses attached more strongly to human trachea and bronchi than H5N1 virus and attached to different cell types than H5N1 virus. These differences correspond to primary diagnoses of tracheobronchitis for human influenza viruses and diffuse alveolar damage for H5N1 virus. The PVA of low pathogenic avian influenza viruses in human respiratory tract resembled that of H5N1 virus, demonstrating that other properties determine its pathogenicity for humans. The PVA in human respiratory tract most closely mirrored that in ferrets and pigs for human influenza viruses and that in ferrets, pigs, and cats for avian influenza viruses.

Viral Tropism and the Pathogenesis of Influenza in the Mammalian Host

Influenza viruses, single-stranded negative-sense RNA viruses within the family Orthomyxoviridae, remain an important cause of human morbidity and mortality worldwide. Despite effective vaccines, more than 200,000 individuals in the United States are hospitalized each year for influenza, and approximately 35,000 succumb to disease.1 Seasonal epidemics arise through antigenic variation and recombination events from existing and currently circulating human strains, which initially target the upper respiratory tract with extension to a bronchointerstitial pneumonia in a subset of affected individuals. Disease is often more severe in older and debilitated patients, producing a characteristic distribution in age-related mortality within the affected population.2 In contrast to seasonal epidemics, pandemics arise when antigenically novel viruses emerge and are readily transmissible within the naive human population. Such events are rare and occur after zoonotic transmission of viruses through recombination events between established human and avian strains or possibly through direct adaptation of avian strains for efficient human to human transmission.3 Concerns over an avian influenza pandemic have risen since 1997 with the recognition of H5N1 as a cause of fulminant disease in humans.4,5 Pathologically fatal H5N1 causes diffuse alveolar damage and progression to multiple organ dysfunction with a case fatality rate estimated at more than 50%. Although H5N1 disease is severe, transmission rates have been low, and viral adaptation to efficient human to human transmission has not occurred. Understanding viral and host barriers that prevent transmission may be critical in establishing rational control measures as well as predicting and stratifying risk for individual strains of influenza. In this issue of The American Journal of Pathology, van Riel et al6 examine the viral binding distribution in pulmonary tissue of a number of avian and human influenza strains to elucidate the role of tropism in transmission and pathogenesis.

Ecologic immunology of avian influenza (H5N1) in migratory birds.

The claim that migratory birds are responsible for the long-distance spread of highly pathogenic avian influenza viruses of subtype H5N1 rests on the assumption that infected wild birds can remain asymptomatic and migrate long distances unhampered. We critically assess this claim from the perspective of ecologic immunology, a research field that analyzes immune function in an ecologic, physiologic, and evolutionary context. Long-distance migration is one of the most demanding activities in the animal world. We show that several studies demonstrate that such prolonged, intense exercise leads to immunosuppression and that migratory performance is negatively affected by infections. These findings make it unlikely that wild birds can spread the virus along established long-distance migration pathways. However, infected, symptomatic wild birds may act as vectors over shorter distances, as appears to have occurred in Europe in early 2006.

Molecular analysis of highly pathogenic avian influenza virus of subtype H5N1 isolated from wild birds and mammals in northern Germany

Analysis of the full-length sequences of all eight segments of the German wild-bird H5N1 highly pathogenic avian influenza virus index isolate, A/Cygnus cygnus/Germany/R65/2006, and an H5N1 isolate from a cat (A/cat/Germany/R606/2006) obtained during an outbreak in February 2006 revealed a very high similarity between these two sequences. One amino acid substitution in the PA gene, encoding a protein involved in virus RNA replication, and one amino acid substitution in the haemagglutinin (HA) protein were observed. Phylogenetic analyses of the HA and neuraminidase nucleotide sequences showed that avian influenza H5N1 isolates from the Astrakhan region located in southern Russia were the closest relatives. Reassortment events could be excluded in comparison with other 'Qinghai-like' H5N1 viruses. In addition, an H5N1 isolate originating from a single outbreak in poultry in Germany was found to be related closely to the H5N1 viruses circulating at that time in the wild-bird population.