Polymerase trapping as the mechanism of H5 highly pathogenic avian influenza virus genesis.

Introduction of novel inactivated oil-emulsion vaccines against different strains of prevailing and emerging low pathogenic avian influenza (LPAI) viruses is not an economically viable option for poultry. Engineering attenuated Salmonella Gallinarum (S. Gallinarum) vaccine delivering H5 LPAI antigens can be employed as a bivalent vaccine against fowl typhoid and LPAI viruses, while still offering economic viability and sero-surveillance capacity. In this study, we developed a JOL1814 bivalent vaccine candidate against LPAI virus infection and fowl typhoid by engineering the attenuated S. Gallinarum to deliver the globular head (HA1) domain of hemagglutinin protein from H5 LPAI virus through pMMP65 constitutive expression plasmid. The important feature of the developed JOL1814 was the delivery of the HA1 antigen to cytosol of peritoneal macrophages. Immunization of chickens with JOL1814 produced significant level of humoral, mucosal, cellular and IL-2, IL-4, IL-17 and IFN-γ cytokine immune response against H5 HA1 and S. Gallinarum antigens in the immunized chickens. Post-challenge, only the JOL1814 immunized chicken showed significantly faster clearance of H5N3 virus in oropharyngeal and cloacal swabs, and 90% survival rate against lethal challenge with a wild type S. Gallinarum. Furthermore, the JOL1814 immunized were differentiated from the H5N3 LPAI virus infected chickens by matrix (M2) gene-specific real-time PCR. In conclusion, the data from the present showed that the JOL1814 can be an effective bivalent vaccine candidate against H5N3 LPAI and fowl typhoid infection in poultry while still offering sero-surveillance property against H5 avian influenza virus.

To the Editor: Wild migratory birds are reservoirs for low pathogenic avian influenza (LPAI) viruses (1), but their role in transmitting highly pathogenic avian influenza (HPAI) viruses is hotly debated and unclear (2–4). Beginning in July 2005, a clade of HPAI (H5N1) viruses rapidly expanded from an apparent focus in western People’s Republic of China and spread to the Middle East, Africa, and Europe (5). Genetic analysis of HPAI virus isolates from dead wild birds along major flyways indicated that the strains were closely related to the Qinghai H5N1 A/bar-headed goose/Qinghai/65/2005 virus (clade II) (GenBank accession no. {type:entrez-nucleotide,attrs:{text:DQ095622,term_id:70955434,term_text:DQ095622}}DQ095622). In addition to transmission to domestic poultry, HPAI (H5N1)–infected mute swans have been implicated in direct transmission to humans in Azerbaijan (6). The US Naval Medical Research Unit No. 3 and the Ministry of Environment of Egypt have collaborated since 2003 in obtaining samples from migratory birds to detect circulating influenza viruses. During the 2005–06 migratory birds season, 1,304 migratory birds were sampled from either live bird markets or cage birds trapped by fishermen in Port Said, Damietta, Fayoum, Arish, and Sharm el Sheikh (Appendix Figure, panel A. A total of 203 cloacal swab samples were positive for influenza A virus matrix gene when tested by real-time PCR, and 2 were also positive for the hemagglutinin 5 (H5) gene by using specific primers (7). Of the 2 migratory birds positive for the H5 gene, the first was a common teal (Anas crecca) captured in the Nile Delta region of Damietta in October 2005 (Appendix Figure, panel A). Sequencing of the H5 gene showed that this virus was an LPAI most closely related to strain A/mallard/Bavaria/1/2005(H5N2) (GenBank accession no. {type:entrez-nucleotide,attrs:{text:DQ387854,term_id:88656455,term_text:DQ387854}}DQ387854 (2). In January 2006, an influenza A H5 virus (weak positive result) was detected in another common teal (trapped in a cage by a fisherman) sampled from the Damietta region in December 2005 (Appendix Figure, panel A). The low viral load, coupled with the failure to isolate the virus, precluded the laboratory from conducting sequence analysis at the time on the basis of insufficient template material. After the outbreak of influenza A (H5N1) in poultry and humans in Egypt in February 2006, additional retrospective attempts to concentrate RNA were used to assess potential introduction scenarios. After multiple RNA extractions were conducted and the RNA was concentrated, this specimen was found to be positive for the neuraminidase 1 (N1) gene by real-time PCR. The hemagglutinin gene from both teal strains was sequenced (≈1,596 bp). Sequences were aligned with other influenza A (H5N1) strains from Egypt (9 from humans, 5 from chickens). Twenty other strains with high similarity and from different locations were selected by using a GenBank search algorithm and included in the alignment. A phylogenetic analysis was conducted by using the Kimura 2-parameter model. The LPAI H5 virus strain was used as an outgroup in a neighbor-joining phylogenetic tree. Bootstrap analysis with 500 replicates of sequence data was also conducted by using MEGA 3.1 software (8). Phylogenetic analysis showed clustering of the HPAI (H5N1) strains collected from 1 geographic region (country) (Appendix Figure, panel B). All HPAI (H5N1) strains from Egypt from humans or chickens analyzed clustered with a bootstrap support value of 98%. Furthermore, the A/Teal/Egypt/14051-NAMRU3/2006 (H5N1) strain (collected in December 2005; Appendix Figure, panel A) is an HPAI and is closely related to the parent of the group of viruses isolated in the early 2006 Egypt outbreak, with an average identity of 99.4% with all other strains from Egypt and a bootstrap support value of 96% (Appendix Figure, panel B). Despite the rapid spread of this clade (Qinghai-like strain) to many countries, since late 2005, strains analyzed in this study showed low-level genetic variation (<2%). Brown et al. reported that species can vary greatly in their response to HPAI (9). At least in ducks, it appears that viral shedding is highest in birds with clinical signs of infection, and lowest, as seen in the common teal infected with the HPAI strain in this study, in birds with subclinical infections. These subclinical infections may be due to flock immunity from previous exposure to LPAI H5 virus or genetic factors. This suggestion is conceivable in light of the LPAI H5 virus detected in the other teal a few months earlier. Such naturally resistant wild birds might serve as vectors for introduction of HPAI viruses into new locations. Data presented herein suggest that an HPAI virus may have been introduced into Egypt through a migratory bird. Whether poultry were infected before mid-February or the teal was infected with influenza A (H5N1) virus by a domesticated species is not unknown. The low degree of viral shedding indicates that detection of any influenza A (H5N1) virus in wild birds in a new region should be immediately followed up with efforts to characterize the virus to control the spread of new subtypes/strains of HPAI into new locations.

The emergence and evolution of avian influenza A viruses (AIVs) pose significant challenges to both public health and animal husbandry worldwide. Here, we characterized a novel reassortant highly pathogenic avian influenza virus (HPAIV), clade 2.3.4.4b H5N6, that was isolated from a mandarin duck in South Korea in December 2023. Phylogenetic and molecular analyses show that the hemagglutinin (HA) gene of the 23‐JBN‐F12‐36/H5N6 virus clustered with HPAIV clade 2.3.4.4b H5N1 viruses, which were circulating in South Korea and Japan in 2022–2023. The M and polymerase acidic (PA) genes also revealed a close association with the HPAIV clade 2.3.4.4b H5N1 AIV that was identified previously in South Korea during November 2022. Notably, the neuraminidase (NA) gene of the 23‐JBN‐F12‐36/H5N6 virus was estimated to have its origins in the HPAIV clade 2.3.4.4h H5N6 prevalent in poultry in China, and it is clustered with the AIVs that are associated with human infection cases. Taken together, these results show that the virus has been produced by reassortment with H5N1 HPAIV, which is prevalent in wild birds; H5N6 HPAIV, which is circulated in poultry in China; and the internal genes of low pathogenic avian influenza viruses (LPAIVs). In light of the reassortment of HPAIVs circulating in existing wild birds and HPAIVs circulating in poultry in China within the 2.3.4.4b H5Nx clade, it is imperative to strengthen active surveillance across wild bird populations, poultry farms, and live poultry markets, and to inform for the effective design of improved prevention and control strategies.

Low pathogenicity avian influenza (LPAI) viruses of the H7 subtype generally cause mild disease in poultry. However the evolution of a LPAI virus into highly pathogenic avian influenza (HPAI) virus results in the generation of a virus that can cause severe disease and death. The classification of these two pathotypes is based, in part, on disease signs and death in chickens, as assessed in an intravenous pathogenicity test, but the effect of LPAI viruses in turkeys is less well understood. During an investigation of LPAI virus infection of turkeys, groups of three-week-old birds inoculated with A/chicken/Italy/1279/99 (H7N1) showed severe disease signs and died or were euthanised within seven days of infection. Virus was detected in many internal tissues and organs from culled birds. To examine the possible evolution of the infecting virus to a highly pathogenic form in these turkeys, sequence analysis of the haemagglutinin (HA) gene cleavage site was carried out by analysing multiple cDNA amplicons made from swabs and tissue sample extracts employing Sanger and Next Generation Sequencing. In addition, a RT-PCR assay to detect HPAI virus was developed. There was no evidence of the presence of HPAI virus in either the virus used as inoculum or from swabs taken from infected birds. However, a small proportion (<0.5%) of virus carried in individual tracheal or liver samples did contain a molecular signature typical of a HPAI virus at the HA cleavage site. All the signature sequences were identical and were similar to HPAI viruses collected during the Italian epizootic in 1999/2000. We assume that the detection of HPAI virus in tissue samples following infection with A/chicken/Italy/1279/99 reflected amplification of a virus present at very low levels within the mixed inoculum but, strikingly, we observed no new HPAI virus signatures in the amplified DNA analysed by deep-sequencing.

A differentiating infected from vaccinated animals (DIVA) vaccine is an ideal strategy for viral eradication in poultry. Here, according to the emerging highly pathogenic H7N9 avian influenza virus (AIV), a DIVA vaccine strain, named rGD4HALo-mH3-TX, was successfully developed, based on a substituted 12 peptide of H3 virus located at HA2. In order to meet with the safety requirement of vaccine production, the multi-basic amino acid located at the HA cleavage site was modified. Meanwhile, six inner viral genes from a H9N2 AIV TX strainwere introduced for increasing viral production. The rGD4HALo-mH3-TX strain displayed a similar reproductive ability with rGD4 and low pathogenicity in chickens, suggesting a good productivity and safety. In immuned chickens, rGD4HALo-mH3-TX induced a similar antibody level with rGD4 and provided 100% clinical protection and 90% shedding protection against highly pathogenic virus challenge. rGD4HALo-mH3-TX strain also produced a good cross-protection against low pathogenic AIV JD/17. Moreover, serological DIVA characteristics were evaluated by a successfully established competitive inhibition ELISA based on a 3G10 monoclonal antibody, and the result showed a strong reactivity with antisera of chickens vaccinated with H7 subtype strains but not rGD4HALo-mH3-TX. Collectedly, rGD4HALo-mH3-TX is a promising DIVA vaccine candidate against both high and low pathogenic H7N9 subtype AIV.

The highly pathogenic avian influenza (HPAI) virus phenotype is restricted to influenza A viruses of the H5 and H7 hemagglutinin (HA) subtypes. To obtain more information on the apparent subtype-specific nature of the HPAI virus phenotype, a low-pathogenic avian influenza (LPAI) H6N1 virus was generated, containing an HPAI H5 RRRKKR [downward arrow] G multibasic cleavage site (MBCS) motif in HA (the downward arrow indicates the site of cleavage). This insertion converted the LPAI virus phenotype into an HPAI virus phenotype in vitro and in vivo. The H6N1 virus with an MBCS displayed in vitro characteristics similar to those of HPAI H5 viruses, such as cleavage of HA(0) (the HA protein of influenza A virus initially synthesized as a single polypeptide precursor) and virus replication in the absence of exogenous trypsin. Studies of chickens confirmed the HPAI phenotype of the H6N1 virus with an MBCS, with an intravenous pathogenicity index of 1.4 and systemic virus replication upon intranasal inoculation, the hallmarks of HPAI viruses. This study provides evidence that the subtype-specific nature of the emergence of HPAI viruses is not at the molecular, structural, or functional level, since the introduction of an MBCS resulted in a fully functional virus with an HPAI virus genotype and phenotype.

Laboratory-confirmed infections with H5N1 and H7N9 As per World Health Organization (WHO) reporting of human infections with H5 and H7 avian influenza viruses [2-4], Luczo and Spackman [5] reported laboratory-confirmed H7N9 (and H5N1) infections and the associated (laboratory-confirmed) case fatality rate. The cumulative number of human cases for H5N1 reported to WHO 2003-2025 also includes (laboratory-confirmed) reported detections in asymptomatic individuals. We have not misinterpreted the (laboratory-confirmed) H7N9 or H5N1 case fatality rate and have clearly defined that the metric reported is related to laboratory-confirmed infections. Notably, both the WHO and experts in the field continue to report this metric based on laboratory-confirmed infections. Pathogenicity of non-H5/H7 avian influenza viruses We don't believe that the statement “Experimental studies have shown that other subtypes like H2, H4, H6, H8, H9, and H14 can support a highly pathogenic phenotype” is at disharmony with non-H5/H7 subtypes having low pathogenic phenotypes in nature. Furthermore, in Luczo and Spackman [5], we describe that low pathogenicity avian influenza viruses generally cause non-clinical or mild disease compared to highly pathogenic avian influenza. Regardless, the pathobiology of low pathogenic avian influenza was not a primary focus of this work and has been reviewed elsewhere [6, 7]. However, to try to address Ghaemi et al. [1] concerns, first it is unclear whether Ghaemi et al. are referring to the World Organization for Animal Health (WOAH) regulatory definition of highly pathogenic avian influenza and low pathogenic avian influenza [8] or a more general clinical description of disease. Regardless, the severity of low pathogenic avian influenza pathogenesis is subjective and for poultry production even “mild” clinical disease [9] can result in substantial economic losses in the field due to decreased production. Low pathogenic avian influenza virus isolates of almost all subtypes have been reported to cause notable disease in the field [10-14] which has warranted the cost to implement control measures. Therefore, a broader view of the disease impact of low pathogenic avian influenza is not inappropriate regardless of how it is presented in Luczo and Spackman [5]. In the context of the WOAH definition, Luczo and Spackman [5] describe that highly pathogenic viruses have evolved naturally in the H5 an H7 subtypes only. However, there are several studies of interest with recombinant and wild type viruses that provide insights as to the complexity of influenza pathogenicity. For example, there is clear evidence that in experimental settings some non-H5/H7 subtypes have the potential to support a highly pathogenic phenotype [15-18]. Ghaemi et al. [1] also conclude that experimental changes can transform non-H5/H7 into highly pathogenic phenotype, making their argument on this a moot point. Of interest is why only H5 and H7 avian influenza viruses naturally convert to highly pathogenic forms – knowledge of this could be harnessed to mitigate the genesis of these highly pathogenic viruses. Additionally, it has been reported that a wild type H10 virus that has a low pathogenicity cleavage site has an intravenous pathogenicity index of 1.76, which meets the WOAH definition for a highly pathogenic avian influenza virus [19]. Finally, despite being classified as of low pathogenicity, H9 avian influenza viruses pose a significant threat to the poultry industry and it has been noted that infection of chickens with some H9N2 virus lineages cause severe disease and even death in poultry [20-25]. We also wish to clarify that H9 avian influenza viruses are not present in poultry globally – it is not enzootic in the Americas, Europe, nor Australia, for example. Collectively, whilst non-H5/H7 viruses are classified as low pathogenicity, there is clear evidence that some non-H5/H7 viruses do not follow this trend. Writing – original draft: J.M.L. Writing – review and editing, J.M.L. and E.S. The authors declare no conflicts of interest.

To the Editor: In 2005, a large population of wild migratory birds was infected with highly pathogenic avian influenza (HPAI) virus (H5N1) in the Qinghai Lake region of western People’s Republic of China, resulting in the death of ≈10,000 birds (1,2). On the basis of phylogenetic analysis of the hemagglutinin (HA) gene, the virus was classified as clade 2.2 according to the World Health Organization guidelines. Subsequently, viruses from this clade were found in Mongolia, Russia, Europe, and Africa along the migratory flyways of birds (3,4). This unique distribution of the same clade of HPAI virus (H5N1) through different migratory routes indicates that migratory birds might play a global role in virus dissemination (3,4). In 2006, viruses from the same clade were isolated in the Qinghai Lake region (3). Analysis of viral outbreaks along migratory flyways demonstrated a similar outbreak pattern for the past 4 years (2006–2009) (5). During that period, clade 2.2 avian influenza virus (H5N1) was isolated in China, Mongolia, Russia, Germany, Egypt, and Nigeria; all viruses were closely related to the Qinghai Lake virus. Despite the broad distribution of clade 2.2 viruses in migratory flyways, few isolates of clade 2.2 viruses in local domestic poultry were reported, especially in China (6). Outbreaks of these viruses were reported in poultry in Africa (7). The reason these viruses rarely cause outbreaks in poultry is unknown. During May–June 2009 and 2010, several dead migratory birds were found in the Qinghai Lake region. Nine HPAI viruses (H5N1) were isolated in 2009 and 2 were isolated in 2010 from great cormorants (Phalacrocorax carbo), brown-headed gulls (Chroicocephalus brunnicephalus), great black-headed gulls (Ichthyaetus ichthyaetus), great-crested grebes (Podiceps cristatus), and bar-headed geese (Anser indicus) and serotyped as described (3). HA genes from all 11 isolates were subsequently amplified by using reverse transcription–PCR and sequenced. Phylogenetic analysis of HA sequences and an additional HA gene sequence from the 2009 Qinghai Lake subtype H5N1 virus isolate from a great crested grebe (from the National Avian Influenza Virus Reference Laboratory, Harbin, China) (GenBank accession no. {type:entrez-nucleotide,attrs:{text:CY063318,term_id:301015462,term_text:CY063318}}CY063318) showed that HA genes from all 12 viruses clustered as clade 2.3.2 (Figure); none clustered with clade 2.2 viruses. Additionally, the HA cleavage site in the new isolates is PQRERRRKRG, which is identical to that of clade 2.3.2 viruses. In clade 2.2, the cleavage site is PQRERRRKKRG. Figure Bootstrapped (1,000×) maximum likelihood phylogenetic tree of hemagglutinin genes of avian influenza viruses (H5N1), People’s Republic of China, 2009–2010. Viruses isolated from the plateau pika near Qinghai Lake are indicated ... A bootstrap (1,000×) maximum likelihood tree (8) also demonstrated that Qinghai 2009 and 2010 virus isolates are closely related to those isolated in Mongolia and Uvs Nuur Lake in 2009, as reported by Sharshov et al. (5). Qinghai Lake and Uvs Nuur Lake, which are found along the migratory flyway in central Asia, are major lakes for bird migration and breeding. Many birds fly from Qinghai Lake to Uvs Nuur Lake in the spring. If one considers isolation date and bird species infected, viruses isolated in Mongolia and Russia and our isolates were likely transmitted between the 2 lake regions by bird migration. Moreover, HA sequences are closely related to viruses isolated from wild birds in Hong Kong and Japan during 2007–2008, which are the most recent isolates of clade 2.3.2 viruses before isolation of 2009 Qinghai Lake viruses. These results indicate that viruses in the Qinghai Lake region may be transmitted by wild birds along the migratory flyway in eastern Asia. However, there is no evidence that avian influenza virus (H5N1) is transmitted from eastern Asian (inner China or across the Himalayas) to the Qinghai Lake region. The 2009 and 2010 Qinghai Lake viruses are related to various viruses isolated from plateau pikas near Qinghai Lake (9). In 2007, clade 2.2 and clade 2.3.2 viruses were isolated from plateau pikas, but no clade 2.3.2 viruses were found in aquatic birds. Wild birds, pikas, and other animals near Qinghai Lake share the same environment, and viruses may be transmitted across species. However, surveillance data are limited for wild animals near Qinghai Lake. Therefore, further investigations need to be conducted to clarify relationships among birds, animals, and influenza viruses near Qinghai Lake. Our results and those of Sharshov et al. (5) show that in 2009 HPAI virus (H5N1) began infecting birds along the migratory route near Qinghai Lake and changed from clade 2.2 viruses to clade 2.3 viruses. New outbreaks of HPAI viruses (H5N1) along this migratory flyway should be investigated.

Homologous and heterologous antigenic matched vaccines containing different H5 hemagglutinins provide variable protection of chickens from the 2014 U.S. H5N8 and H5N2 clade 2.3.4.4 highly pathogenic avian influenza viruses.

Avian influenza virus (AIV) infections in cats are a new and not fully understood problem in Poland. These infections have drawn the attention of both veterinarians and human medical practitioners, mainly because of their zoonotic potential, i.e. possible spreading to humans and other mammals. In wild felids as well as in domestic cats, AIV can cause severe infections, often ending in death. Highly pathogenic avian influenza virus (HPAIV) and low pathogenic avian influenza virus (LPAIV) have been identified, with the recent H5N1 (2.3.4.4b clade) outbreak affecting poultry, wild birds and carnivores. Transmission likely occurs through contact with infected birds, their excretions or contaminated raw poultry, while cat-to-cat transmission remains unconfirmed. First reported in Thailand in 2003, H5N1 infections in cats have since occurred in multiple countries. In Poland, 25 confirmed cases were identified in June 2023, with genetic sequencing linking the virus to strains detected in local wild birds. The virus primarily replicates in the lower respiratory tract, spreading via viremia or nerve fibers, causing multi-organ failure. While avian influenza in cats is severe and often fatal, it should not yet be considered an epidemic. Further interdisciplinary research is essential to clarify transmission routes and assess the zoonotic risk. Additionally, differential diagnosis should include rabies, which presents similar neurological symptoms and remains a critical public health concern. This article presents the current knowledge of H5N1 virus infection in cats, especially the possible routes for its spreading, the current epizootic situation of the disease around the world, its pathogenesis, clinical course and methods of diagnosis.

BackgroundHighly-pathogenic avian influenza virus (HPAIV) and Newcastle disease virus (NDV) are the two most important poultry viruses in the world. Natural low-virulence NDV strains have been used as vaccines over the past 70 years with proven track records. We have previously developed a reverse genetics system to produce low-virulent NDV vaccine strain LaSota from cloned cDNA. This system allows us to use NDV as a vaccine vector for other avian pathogens.Methodology/Principal FindingHere, we constructed two recombinant NDVs (rNDVs) each of which expresses the hemagglutinin (HA) gene of HPAIV H5N1strain A/Vietnam/1203/2004 from an added gene. In one, rNDV (rNDV-HA), the open reading frame (ORF) of HA gene was expressed without modification. In the second, rNDV (rNDV-HAF), the ORF was modified so that the transmembrane and cytoplasmic domains of the encoded HA gene were replaced with those of the NDV F protein. The insertion of either version of the HA ORF did not increase the virulence of the rNDV vector. The HA protein was found to be incorporated into the envelopes of both rNDV-HA and rNDV-HAF. However, there was an enhanced incorporation of the HA protein in rNDV-HAF. Chickens immunized with a single dose of either rNDV-HA or rNDV-HAF induced a high titer of HPAIV H5-specific antibodies and were completely protected against challenge with NDV as well as lethal challenges of both homologous and heterologous HPAIV H5N1.Conclusion and SignificanceOur results suggest that these chimeric viruses have potential as safe and effective bivalent vaccines against NDV and. HPAIV. These vaccines will be convenient and affordable, which will be highly beneficial to the poultry industry. Furthermore, immunization with these vaccines will permit serological differentiation of vaccinated and avian influenza field virus infected animals.

The objective of the study was Investigation and isolation of the Low Pathogenic Avian Influenza virus in poultry farms (Broilers and layers) in different provinces in Iraq over the period from August 2020 to June 2021. A total of 241 samples, including (Larynx, trachea &amp; lunge) were collected from 31 poultry farms suspected to be infected with avian influenza and distributed in different provinces including Baghdad, Babylon, Wasit, Karbala &amp; Salahuddin provinces. Collected samples were prepared firstly then viral RNA was extracted by a special viral RNA extraction kit and amplified by using primers for M gene-specific for type A influenza virus and primers specific for H9 gene of LPAI in Real-time RT-PCR which showed that from (31) farms suspected to have low pathogenic avian influenza, six farms (19.35%) were positive H9 LPAI.

BackgroundAvian influenza virus (AIV) is classified into two pathotypes, low pathogenic (LP) and high pathogenic (HP), based on virulence in chickens.Differences in pathogenicity between HPAIV and LPAIV might eventually be related to specific characteristics of strains, tissue tropism and host responses.MethodsTo study differences in disease development between HPAIV and LPAIV, we examined the first appearance and eventual load of viral RNA in multiple organs as well as host responses in brain and intestine of chickens infected with two closely related H7N1 HPAIV or LPAIV strains.ResultsBoth H7N1 HPAIV and LPAIV spread systemically in chickens after a combined intranasal/intratracheal inoculation. In brain, large differences in viral RNA load and host gene expression were found between H7N1 HPAIV and LPAIV infected chickens. Chicken embryo brain cell culture studies revealed that both HPAIV and LPAIV could infect cultivated embryonic brain cells, but in accordance with the absence of the necessary proteases, replication of LPAIV was limited. Furthermore, TUNEL assay indicated apoptosis in brain of HPAIV infected chickens only. In intestine, where endoproteases that cleave HA of LPAIV are available, we found minimal differences in the amount of viral RNA and a large overlap in the transcriptional responses between HPAIV and LPAIV infected chickens. Interestingly, brain and ileum differed clearly in the cellular pathways that were regulated upon an AI infection.ConclusionsAlthough both H7N1 HPAIV and LPAIV RNA was detected in a broad range of tissues beyond the respiratory and gastrointestinal tract, our observations indicate that differences in pathogenicity and mortality between HPAIV and LPAIV could originate from differences in virus replication and the resulting host responses in vital organs like the brain.

Avian influenza (AVI) is being known for its pandemic potential and devastating effects on poultry and birds. The AVI outbreaks in domesticated birds are of concern because the Low pathogenic avian influenza virus (LPAI) tends to evolve into a High pathogenic avian influenza virus (HPAI) resulting in the rapid spread and significant outbreak in poultries. The containment should be rapid and stringent precautions should be taken in handling the infected poultry cases or infected materials. In general, AVI viruses do not replicate efficiently in humans, indicating that transmitting these viruses to humans directly is a very rare preference. However, the HPAI ability to the cross-species barrier and infect humans has been known for H5N1 and H7N9. Recently, the world's first human case of transmission of the H5N8 strain from the avian species to humans has been documented. In this recent scenario, it is worth discussing the strain variations, disease severity, economic loss, and effective controlling strategies for controlling avian influenza.

Highly Pathogenic Avian Influenza Viruses (HPAIVs) arise from low pathogenic precursors following spillover from wild waterfowl into poultry populations. The main virulence determinant of HPAIVs is the presence of a multi-basic cleavage site (MBCS) in the hemagglutinin (HA) glycoprotein. The MBCS allows for HA cleavage and, consequently, activation by ubiquitous proteases, which results in systemic dissemination in terrestrial poultry. Since 1959, 51 independent MBCS acquisition events have been documented, virtually all in HA from the H5 and H7 subtypes. In the present article, data from natural LPAIV to HPAIV conversions and experimental in vitro and in vivo studies were reviewed in order to compile recent advances in understanding HA cleavage efficiency, protease usage, and MBCS acquisition mechanisms. Finally, recent hypotheses that might explain the unique predisposition of the H5 and H7 HA sequences to obtain an MBCS in nature are discussed.