Evaluation of Commercial Diagnostic Assays for the Specific Detection of Avian Influenza A (H7N9) Virus RNA Using a Quality-Control Panel and Clinical Specimens in China

A novel avian influenza A H7N9-subtype virus emerged in China in 2013 and threatened global public health. Commercial kits that specifically detect avian influenza A (H7N9) virus RNA are urgently required to prepare for the emergence and potential pandemic of this novel influenza virus. The safety and effectiveness of three commercial molecular diagnostic assays were evaluated using a quality-control panel and clinical specimens collected from over 90 patients with confirmed avian influenza A (H7N9) virus infections. The analytical performance evaluation showed that diverse influenza H7N9 viruses can be detected with high within- and between-lot reproducibility and without cross-reactivity to other influenza viruses (H1N1 pdm09, seasonal H1N1, H3N2, H5N1 and influenza B). The detection limit of all the commercial assays was 2.83 Log₁₀ copies/μl [0.7 Log₁₀TCID₅₀/mL of avian influenza A (H7N9) virus strain A/Zhejiang/DTID-ZJU01/2013], which is comparable to the method recommended by the World Health Organization (WHO). In addition, using a WHO-Chinese National Influenza Center (CNIC) method as a reference for clinical evaluation, positive agreement of more than 98% was determined for all of the commercial kits, while negative agreement of more than 99% was observed. In conclusion, our findings provide comprehensive evidence for the high performance of three commercial diagnostic assays and suggest the application of these assays as rapid and effective diagnostic tools for avian influenza A (H7N9) virus in the routine clinical practice of medical laboratories.     

Vaccination against Human Influenza A/H3N2 Virus Prevents the Induction of Heterosubtypic Immunity against Lethal Infection with Avian Influenza A/H5N1 Virus

Annual vaccination against seasonal influenza viruses is recommended for certain individuals that have a high risk for complications resulting from infection with these viruses. Recently it was recommended in a number of countries including the USA to vaccinate all healthy children between 6 and 59 months of age as well. However, vaccination of immunologically naïve subjects against seasonal influenza may prevent the induction of heterosubtypic immunity against potentially pandemic strains of an alternative subtype, otherwise induced by infection with the seasonal strains.Here we show in a mouse model that the induction of protective heterosubtypic immunity by infection with a human A/H3N2 influenza virus is prevented by effective vaccination against the A/H3N2 strain. Consequently, vaccinated mice were no longer protected against a lethal infection with an avian A/H5N1 influenza virus. As a result H3N2-vaccinated mice continued to loose body weight after A/H5N1 infection, had 100-fold higher lung virus titers on day 7 post infection and more severe histopathological changes than mice that were not protected by vaccination against A/H3N2 influenza.The lack of protection correlated with reduced virus-specific CD8+ T cell responses after A/H5N1 virus challenge infection. These findings may have implications for the general recommendation to vaccinate all healthy children against seasonal influenza in the light of the current pandemic threat caused by highly pathogenic avian A/H5N1 influenza viruses.     

In Silico Structural Homology Modelling and Docking for Assessment of Pandemic Potential of a Novel H7N9 Influenza Virus and Its Ability to Be Neutralized by Existing Anti-Hemagglutinin Antibodies

The unpredictable nature of pandemic influenza and difficulties in early prediction of pandemic potential of new isolates present a major challenge for health planners. Vaccine manufacturers, in particular, are reluctant to commit resources to development of a new vaccine until after a pandemic is declared. We hypothesized that a structural bioinformatics approach utilising homology-based molecular modelling and docking approaches would assist prediction of pandemic potential of new influenza strains alongside more traditional laboratory and sequence-based methods. The newly emerged Chinese A/Hangzhou/1/2013 (H7N9) influenza virus provided a real-life opportunity to test this hypothesis. We used sequence data and a homology-based approach to construct a 3D-structural model of H7-Hangzhou hemagglutinin (HA) protein. This model was then used to perform docking to human and avian sialic acid receptors to assess respective binding affinities. The model was also used to perform docking simulations with known neutralizing antibodies to assess their ability to neutralize the newly emerged virus. The model predicted H7N9 could bind to human sialic acid receptors thereby indicating pandemic potential. The model also confirmed that existing antibodies against the HA head region are unable to neutralise H7N9 whereas antibodies, e.g. Cr9114, targeting the HA stalk region should bind with high affinity to H7N9. This indicates that existing stalk antibodies initially raised against H5N1 or other influenza A viruses could be therapeutically beneficial in prevention and/or treatment of H7N9 infections. The subsequent publication of the H7N9 HA crystal structure confirmed the accuracy of our in-silico structural model. Antibody docking studies performed using the H7N9 HA crystal structure supported the model''s prediction that existing stalk antibodies could cross-neutralise the H7N9 virus. This study demonstrates the value of using in-silico structural modelling approaches to complement physical studies in characterization of new influenza viruses.     

A Novel Activation Mechanism of Avian Influenza Virus H9N2 by Furin

Avian influenza virus H9N2 is prevalent in waterfowl and has become endemic in poultry in Asia and the Middle East. H9N2 influenza viruses have served as a reservoir of internal genes for other avian influenza viruses that infect humans, and several cases of human infection by H9N2 influenza viruses have indicated its pandemic potential. Fortunately, an extensive surveillance program enables close monitoring of H9N2 influenza viruses worldwide and has generated a large repository of virus sequences and phylogenetic information. Despite the large quantity of sequences in different databases, very little is known about specific virus isolates and their pathogenesis. Here, we characterize a low-pathogenicity avian influenza virus, A/chicken/Israel/810/2001 (H9N2) (Israel810), which is representative of influenza virus strains that have caused severe morbidity and mortality in poultry farms. We show that under certain circumstances the Israel810 hemagglutinin (HA) can be activated by furin, a hallmark of highly pathogenic avian influenza virus. We demonstrate that Israel810 HA can be cleaved in cells with high levels of furin expression and that a mutation that eliminates a glycosylation site in HA₁ allows the Israel810 HA to gain universal cleavage in cell culture. Pseudoparticles generated from Israel810 HA, or the glycosylation mutant, transduce cells efficiently. In contrast, introduction of a polybasic cleavage site into Israel810 HA leads to pseudoviruses that are compromised for transduction. Our data indicate a mechanism for an H9N2 evolutionary pathway that may allow it to gain virulence in a distinct manner from H5 and H7 influenza viruses.     

Type 1 responses of human Vγ9Vδ2 T cells to influenza A viruses

γδ T cells are essential constituents of antimicrobial and antitumor defenses. We have recently reported that phosphoantigen isopentenyl pyrophosphate (IPP)-expanded human Vγ9Vδ2 T cells participated in anti-influenza virus immunity by efficiently killing both human and avian influenza virus-infected monocyte-derived macrophages (MDMs) in vitro. However, little is known about the noncytolytic responses and trafficking program of γδ T cells to influenza virus. In this study, we found that Vγ9Vδ2 T cells expressed both type 1 cytokines and chemokine receptors during influenza virus infection, and IPP-expanded cells had a higher capacity to produce gamma interferon (IFN-γ). Besides their potent cytolytic activity against pandemic H1N1 virus-infected cells, IPP-activated γδ T cells also had noncytolytic inhibitory effects on seasonal and pandemic H1N1 viruses via IFN-γ but had no such effects on avian H5N1 or H9N2 virus. Avian H5N1 and H9N2 viruses induced significantly higher CCL3, CCL4, and CCL5 production in Vγ9Vδ2 T cells than human seasonal H1N1 virus. CCR5 mediated the migration of Vγ9Vδ2 T cells toward influenza virus-infected cells. Our findings suggest a novel therapeutic strategy of using phosphoantigens to boost the antiviral activities of human Vγ9Vδ2 T cells against influenza virus infection.     

H7N9禽流感病毒小鼠感染动物模型的建立

目的建立H7N9禽流感病毒小鼠感染模型。方法 1×108,1×107或1×106TCID50H7N9禽流感病毒原液(A/Anhui/1/2013)滴鼻感染BALB/c小鼠。主要观测指标:临床症状、死亡率、病理变化、病毒载量和血清抗体检测。结果被感染的小鼠表现为竖毛、弓背、体重下降;病理表现为间质性肺炎,感染后第2天开始在呼吸道脱落细胞中检测到病毒;免疫组化或病毒分离方法在肺、肾、脑、肠、脾等组织检测到病毒;感染后14 d在小鼠血清中血凝抑制试验特异性抗体效价达到160;淋巴细胞减少,中性粒细胞增多。结论 H7N9感染BALB/c小鼠模型与人类禽流感感染疾病的基本特征相似,为研究该病的发病机制及药物疫苗的研发提供了工作基础。     

A human monoclonal antibody derived from a vaccinated volunteer recognizes heterosubtypically a novel epitope on the hemagglutinin globular head of H1 and H9 influenza A viruses

Most neutralizing antibodies elicited during influenza virus infection or by vaccination have a narrow spectrum because they usually target variable epitopes in the globular head region of hemagglutinin (HA). In this study, we describe a human monoclonal antibody (HuMAb), 5D7, that was prepared from the peripheral blood lymphocytes of a vaccinated volunteer using the fusion method. The HuMAb heterosubtypically neutralizes group 1 influenza A viruses, including seasonal H1N1, 2009 pandemic H1N1 (H1N1pdm) and avian H9N2, with a strong hemagglutinin inhibition activity. Selection of an escape mutant showed that the HuMAb targets a novel conformational epitope that is located in the HA head region but is distinct from the receptor binding site. Furthermore, Phe114Ile substitution in the epitope made the HA unrecognizable by the HuMAb. Amino acid residues in the predicted epitope region are also highly conserved in the HAs of H1N1 and H9N2. The HuMAb reported here may be a potential candidate for the development of therapeutic/prophylactic antibodies against H1 and H9 influenza viruses.     

Emergence of the Virulence-Associated PB2 E627K Substitution in a Fatal Human Case of Highly Pathogenic Avian Influenza Virus A(H7N7) Infection as Determined by Illumina Ultra-Deep Sequencing

Avian influenza viruses are capable of crossing the species barrier and infecting humans. Although evidence of human-to-human transmission of avian influenza viruses to date is limited, evolution of variants toward more-efficient human-to-human transmission could result in a new influenza virus pandemic. In both the avian influenza A(H5N1) and the recently emerging avian influenza A(H7N9) viruses, the polymerase basic 2 protein (PB2) E627K mutation appears to be of key importance for human adaptation. During a large influenza A(H7N7) virus outbreak in the Netherlands in 2003, the A(H7N7) virus isolated from a fatal human case contained the PB2 E627K mutation as well as a hemagglutinin (HA) K416R mutation. In this study, we aimed to investigate whether these mutations occurred in the avian or the human host by Illumina Ultra-Deep sequencing of three previously uninvestigated clinical samples obtained from the fatal case. In addition, we investigated three chicken samples, two of which were obtained from the source farm. Results showed that the PB2 E627K mutation was not present in any of the chicken samples tested. Surprisingly, the avian samples were characterized by the presence of influenza virus defective RNA segments, suggestive for the synthesis of defective interfering viruses during infection in poultry. In the human samples, the PB2 E627K mutation was identified with increasing frequency during infection. Our results strongly suggest that human adaptation marker PB2 E627K has emerged during virus infection of a single human host, emphasizing the importance of reducing human exposure to avian influenza viruses to reduce the likelihood of viral adaptation to humans.     

The viral nucleoprotein determines Mx sensitivity of influenza A viruses

Host restriction factors play a crucial role in preventing trans-species transmission of viral pathogens. In mammals, the interferon-induced Mx GTPases are powerful antiviral proteins restricting orthomyxoviruses. Hence, the human MxA GTPase may function as an efficient barrier against zoonotic introduction of influenza A viruses into the human population. Successful viruses are likely to acquire adaptive mutations allowing them to evade MxA restriction. We compared the 2009 pandemic influenza A virus [strain A/Hamburg/4/09 (pH1N1)] with a highly pathogenic avian H5N1 isolate [strain A/Thailand/1(KAN-1)/04] for their relative sensitivities to human MxA and murine Mx1. The H5N1 virus was highly sensitive to both Mx GTPases, whereas the pandemic H1N1 virus was almost insensitive. Substitutions of the viral polymerase subunits or the nucleoprotein (NP) in a polymerase reconstitution assay demonstrated that NP was the main determinant of Mx sensitivity. The NP of H5N1 conferred Mx sensitivity to the pandemic H1N1 polymerase, whereas the NP of pandemic H1N1 rendered the H5N1 polymerase insensitive. Reassortant viruses which expressed the NP of H5N1 in a pH1N1 genetic background and vice versa were generated. Congenic Mx1-positive mice survived intranasal infection with these reassortants if the challenge virus contained the avian NP. In contrast, they succumbed to infection if the NP of pH1N1 origin was present. These findings clearly indicate that the origin of NP determines Mx sensitivity and that human influenza viruses acquired adaptive mutations to evade MxA restriction. This also explains our previous observations that human and avian influenza A viruses differ in their sensitivities to Mx.     

Characterization of H9 subtype influenza viruses from the ducks of southern China: a candidate for the next influenza pandemic in humans?

A current view of the emergence of pandemic influenza viruses envisages a gene flow from the aquatic avian reservoir to humans via reassortment in pigs, the hypothetical "mixing vessel." Understanding arising from recent H5N1 influenza outbreaks in Hong Kong since 1997 and the isolation of avian H9N2 virus from humans raises alternative options for the emergence of a new pandemic virus. Here we report that H9N2 influenza viruses established in terrestrial poultry in southern China are transmitted back to domestic ducks, in which the viruses generate multiple reassortants. These novel H9N2 viruses are double or even triple reassortants that have amino acid signatures in their hemagglutinin, indicating their potential to directly infect humans. Some of them contain gene segments that are closely related to those of A/Hong Kong/156/97 (H5N1/97, H5N1) or A/Quail/Hong Kong/G1/97 (G1-like, H9N2). More importantly, some of their internal genes are closely related to those of novel H5N1 viruses isolated during the outbreak in Hong Kong in 2001. This study reveals a two-way transmission of influenza virus between terrestrial and aquatic birds that facilitates the generation of novel reassortant H9N2 influenza viruses. Such reassortants may directly or indirectly play a role in the emergence of the next pandemic virus.     

Vaccination against seasonal influenza A/H3N2 virus reduces the induction of heterosubtypic immunity against influenza A/H5N1 virus infection in ferrets

Infection with seasonal influenza viruses induces a certain extent of protective immunity against potentially pandemic viruses of novel subtypes, also known as heterosubtypic immunity. Here we demonstrate that infection with a recent influenza A/H3N2 virus strain induces robust protection in ferrets against infection with a highly pathogenic avian influenza virus of the H5N1 subtype. Prior H3N2 virus infection reduced H5N1 virus replication in the upper respiratory tract, as well as clinical signs, mortality, and histopathological changes associated with virus replication in the brain. This protective immunity correlated with the induction of T cells that cross-reacted with H5N1 viral antigen. We also demonstrated that prior vaccination against influenza A/H3N2 virus reduced the induction of heterosubtypic immunity otherwise induced by infection with the influenza A/H3N2 virus. The implications of these findings are discussed in the context of vaccination strategies and vaccine development aiming at the induction of immunity to pandemic influenza.     

The Potential of Avian H1N1 Influenza A Viruses to Replicate and Cause Disease in Mammalian Models

H1N1 viruses in which all gene segments are of avian origin are the most frequent cause of influenza pandemics in humans; therefore, we examined the disease-causing potential of 31 avian H1N1 isolates of American lineage in DBA/2J mice. Thirty of 31 isolates were very virulent, causing respiratory tract infection; 22 of 31 resulted in fecal shedding; and 10 of 31 were as pathogenic as the pandemic 2009 H1N1 viruses. Preliminary studies in BALB/cJ mice and ferrets showed that 1 of 4 isolates tested was more pathogenic than the pandemic 2009 H1N1 viruses in BALB/cJ mice, and 1 of 2 strains transmitted both by direct and respiratory-droplet contact in ferrets. Preliminary studies of other avian subtypes (H2, H3, H4, H6, H10, H12) in DBA/2J mice showed lower pathogenicity than the avian H1N1 viruses. These findings suggest that avian H1N1 influenza viruses are unique among influenza A viruses in their potential to infect mammals.     

Avian influenza H5N1: an update on molecular pathogenesis

Avian influenza A virus constitutes a large threat to human health. Recent outbreaks of highly pathogenic avian influenza H5N1 virus in poultry and in humans have raised concerns that an influenza pandemic will occur in the near future. Transmission from avian species to humans remains sporadic, but the mortality associated with human infection is very high (about 62%). To date, there are no effective therapeutic drugs or a prophylactic vaccines available, which means that there is still a long way to go before we can eradicate or cure avian influenza. This review focuses on the molecular pathogenesis of avian influenza H5N1 virus infection. An understanding of the viral pathogenesis may facilitate the development of novel treatments or effective eradication of this fatal disease.     

A Cross-Sectional Study of Avian Influenza in One District of Guangzhou, 2013

Since Feb, 2013, more than 100 human beings had been infected with novel H7N9 avian influenza virus. As of May 2013, several H7N9 viruses had been found in retail live bird markets (LBMs) in Guangdong province of southern China where several human cases were confirmed later. However, the real avian influenza virus infection status especially H7N9 in Guangzhou remains unclear. Therefore, a cross-sectional study of avian influenza in commercial poultry farms, the wholesale LBM and retail LBMs in one district of Guangzhou was conducted from October to November, 2013. A total of 1505 cloacal and environmental samples from 52 commercial poultry farms, 1 wholesale LBM and 18 retail LBMs were collected and detected using real-time RT-PCR for type A, H7, H7N9 and H9 subtype avian influenza virus, respectively. Of all the flocks randomly sampled, 6 farms, 12 vendors of the wholesale LBM and 18 retail LBMs were type A avian influenza virus positive with 0, 3 and 11 positive for H9, respectively. The pooled prevalence and individual prevalence of type A avian influenza virus were 33.9% and 7.9% which for H9 subtype was 7.6% and 1.6%, respectively. None was H7 and H7N9 subtype virus positive. Different prevalence and prevalence ratio were found in different poultry species with partridges having the highest prevalence for both type A and H9 subtype avian influenza virus. Our results suggest that LBM may have a higher risk for sustaining and transmission of avian influenza virus than commercial poultry farms. The present study also indicates that different species may play different roles in the evolution and transmission of avian influenza virus. Therefore, risk-based surveillance and management measures should be conducted in future in this area.     

Entry Properties and Entry Inhibitors of a Human H7N9 Influenza Virus

The recently identified human infections with a novel avian influenza H7N9 virus in China raise important questions regarding possible risk to humans. However, the entry properties and tropism of this H7N9 virus were poorly understood. Moreover, neuraminidase inhibitor resistant H7N9 isolates were recently observed in two patients and correlated with poor clinical outcomes. In this study, we aimed to elucidate the entry properties of H7N9 virus, design and evaluate inhibitors for H7N9 virus entry. We optimized and developed an H7N9-pseudotyped particle system (H7N9pp) that could be neutralized by anti-H7 antibodies and closely mimicked the entry process of the H7N9 virus. Avian, human and mouse-derived cultured cells showed high, moderate and low permissiveness to H7N9pp, respectively. Based on influenza virus membrane fusion mechanisms, a potent anti-H7N9 peptide (P155-185-chol) corresponding to the C-terminal ectodomain of the H7N9 hemagglutinin protein was successfully identified. P155-185-chol demonstrated H7N9pp-specific inhibition of infection with IC₅₀ of 0.19 µM. Importantly, P155-185-chol showed significant suppression of A/Anhui/1/2013 H7N9 live virus propagation in MDCK cells and additive effects with NA inhibitors Oseltamivir and Zanamivir. These findings expand our knowledge of the entry properties of the novel H7N9 viruses, and they highlight the potential for developing a new class of inhibitors targeting viral entry for use in the next pandemic.     

Homologous recombination as an evolutionary force in the avian influenza A virus

Avian influenza A viruses (AIVs), including the H5N1, H9N2,and H7N7 subtypes, have been directly transmitted to humans,raising concerns over the possibility of a new influenza pandemic.To prevent a future avian influenza pandemic, it is very importantto fully understand the molecular basis driving the change inAIV virulence and host tropism. Although virulent variants ofother viruses have been generated by homologous recombination,the occurrence of homologous recombination within AIV segmentsis controversial and far from proven. This study reports threecirculating H9N2 AIVs with similar mosaic PA genes descendedfrom H9N2 and H5N1. Additionally, many homologous recombinantsare also found deposited in GenBank. Recombination events canoccur in PB2, PB1, PA, HA, and NP segments and between lineagesof the same/different serotype. These results collectively demonstratethat intragenic recombination plays a role in driving the evolutionof AIVs, potentially resulting in effects on AIV virulence andhost tropism changes.     

Broadly Protective Adenovirus-Based Multivalent Vaccines against Highly Pathogenic Avian Influenza Viruses for Pandemic Preparedness

Recurrent outbreaks of H5, H7 and H9 avian influenza viruses in domestic poultry accompanied by their occasional transmission to humans have highlighted the public health threat posed by these viruses. Newer vaccine approaches for pandemic preparedness against these viruses are needed, given the limitations of vaccines currently approved for H5N1 viruses in terms of their production timelines and the ability to induce protective immune responses in the absence of adjuvants. In this study, we evaluated the feasibility of an adenovirus (AdV)-based multivalent vaccine approach for pandemic preparedness against H5, H7 and H9 avian influenza viruses in a mouse model. Replication-defective AdV vectors expressing hemagglutinin (HA) from different subtypes and nucleoprotein (NP) from one subtype induced high levels of humoral and cellular immune responses and conferred protection against virus replication following challenge with H5, H7 and H9 avian influenza virus subtypes. Inclusion of HA from the 2009 H1N1 pandemic virus in the vaccine formulation further broadened the vaccine coverage. Significantly high levels of HA stalk-specific antibodies were observed following immunization with the multivalent vaccine. Inclusion of NP into the multivalent HA vaccine formulation resulted in the induction of CD8 T cell responses. These results suggest that a multivalent vaccine strategy may provide reasonable protection in the event of a pandemic caused by H5, H7, or H9 avian influenza virus before a strain-matched vaccine can be produced.     

Research progress in human infection with avian influenza H7N9 virus

Since the identification of the novel reassortant avian influenza A (H7N9) virus in China in 2013, until Jun 30, 2017, the virus has caused five epidemic waves leading to a total of 1,552 human infections, with a fatality rate of about 40%. In the spring of 2017, highly pathogenic avian influenza (HPAI) H7N9 virus emerged and has caused 25 human infections. The HPAI H7N9 virus has some biological differences from the LPAI one, such as its multiple basic amino acid residues on HA leading to its independence on trypsin for replication. The pathogenicity of the HPAI H7N9 virus to experimental animals or humans is still unclear. A(H7N9) vaccine development for pandemic preparedness is ongoing, including the reassortment (H7N9/PR8) reverse genetic based vaccine, the virus like particle (VLP) vaccine, the intranasal live attenuated influenza vaccine (LAIV), the non-adjuvant Vero cell culture-derived inactivated whole-virus vaccine, the MDCK culture-derived vaccine, the H7 DNA vaccine and the recombinant replicative H7N9 virus (H7N9-53TM) vaccine. Five neuramidinase resistant sites of A(H7N9) virus isolated from patients have been reported. Some alternative drugs have been studied, such as DAS181 (Fludase), ribavirin, troglitazone and minocycline. Persistent surveillance and enhanced global control are essential to fight against human infections with A(H7N9) virus.     

Cross-recognition of avian H5N1 influenza virus by human cytotoxic T-lymphocyte populations directed to human influenza A virus

Since the number of human cases of infection with avian H5N1 influenza viruses is ever increasing, a pandemic outbreak caused by these viruses is feared. Therefore, in addition to virus-specific antibodies, there is considerable interest in immune correlates of protection against these viruses, which could be a target for the development of more universal vaccines. After infection with seasonal influenza A viruses of the H3N2 and H1N1 subtypes, individuals develop virus-specific cytotoxic T-lymphocyte responses, which are mainly directed against the relatively conserved internal proteins of the virus, like the nucleoprotein (NP). Virus-specific cytotoxic T lymphocytes (CTL) are known to contribute to protective immunity against infection, but knowledge about the extent of cross-reactivity with avian H5N1 influenza viruses is sparse. In the present study, we evaluated the cross-reactivity with H5N1 influenza viruses of polyclonal CTL obtained from a group of well-defined HLA-typed study subjects. To this end, the recognition of synthetic peptides representing H5N1 analogues of known CTL epitopes was studied. In addition, the ability of CTL specific for seasonal H3N2 influenza virus to recognize the NP of H5N1 influenza virus or H5N1 virus-infected cells was tested. It was concluded that, apart from some individual epitopes that displayed amino acid variation between H3N2 and H5N1 influenza viruses, considerable cross-reactivity exists with H5N1 viruses. This preexisting cross-reactive T-cell immunity in the human population may dampen the impact of a next pandemic.     

Genesis of pandemic influenza

During the last decade the number of reported outbreaks caused by highly pathogenic avian influenza (HPAI) in domestic poultry has drastically increased. At the same time, low pathogenic avian influenza (LPAI) strains, such as H9N2 in many parts of the Middle East and Asia and H6N2 in live bird markets in California, have become endemic. Each AI outbreak brings the concomitant possibility of poultry-to-human transmission. Indeed, human illness and death have resulted from such occasional transmissions with highly pathogenic avian H7N7 and H5N1 viruses while avian H9N2 viruses have been isolated from individuals with mild influenza. The transmission of avian influenza directly from poultry to humans has brought a sense of urgency in terms of understanding the mechanisms that lead to interspecies transmission of influenza. Domestic poultry species have been previously overlooked as potential intermediate hosts in the generation of influenza viruses with the capacity to infect humans. In this review, we will discuss molecular and epidemiological aspects that have led to the recurrent emergence of avian influenza strains with pandemic potential, with a particular emphasis on the current Asian H5N1 viruses.