H5 Highly Pathogenic Avian Influenza Viruses Research Articles

Selection of antigenic variants of an H5N1 highly pathogenic avian influenza virus in vaccinated chickens

Vaccination-primed immunity in poultry has been suggested for selection of antigenically drifted highly pathogenic avian influenza viruses (HPAIVs). In this study, we performed two consecutive passage studies of an H5N1 HPAIV in vaccinated chickens, namely, study-I and study-II, to select antigenic variants under immune pressure from the vaccination. In study-I, nine consecutive passages of a wild-type H5N1 HPAIV were carried out in chickens vaccinated with the homologous challenge strain. Antigenically drifted variants with mutations at position 179 in the hemagglutinin (HA) were selected after three passages. Similarly, in study-II, a vaccination-mediated antigenic variant isolated in study-I was used as the vaccine and challenge strain to confirm further antigenic drift after updating the vaccine; after the third passage, additional antigenic variants with a mutation at position 256 in the HA were selected. Thus, our study demonstrated the contribution of vaccination in the selection of antigenic variants of H5 HPAIVs in chickens.

Avian Influenza Virus RNA in Groundwater Wells Supplying Poultry Farms Affected by the 2015 Influenza Outbreak

During the 2015 outbreak of highly pathogenic avian influenza virus (HPAI) on poultry farms in the midwestern United States, concern was raised about the potential for HPAI to contaminate groundwater. Our study objective was to evaluate the occurrence of HPAI in the groundwater supply wells on 13 outbreak-affected poultry farms in Iowa and Wisconsin. We sampled 20 wells, six waste-storage lagoons, and one pond. Three wells and one lagoon were positive for the matrix gene indicative of influenza A virus. Using a semi-nested qPCR assay specific to the H5 HPAI outbreak strain, one well was H5-positive, matching the outbreak virus hemagglutinin gene. Matrix gene-positive samples analyzed for avian influenza virus (AIV) by cell culture and embryonating egg culture were negative. Seven wells were positive by PCR for a poultry-specific parvovirus, thus providing corroborating evidence of virus transport pathways between poultry fecal wastes and groundwater. Our data suggest it is possible for AIV to be transport...

THE PATHOGENESIS OF CLADE 2.3.4.4 H5 HIGHLY PATHOGENIC AVIAN INFLUENZA VIRUSES IN RUDDY DUCK (OXYURA JAMAICENSIS) AND LESSER SCAUP (AYTHYA AFFINIS).

Waterfowl are the natural hosts of avian influenza virus (AIV) and disseminate the virus worldwide through migration. Historically, surveillance and research efforts for AIV in waterfowl have focused on dabbling ducks. The role of diving ducks in AIV ecology has not been well characterized. In this study, we examined the relative susceptibility and pathogenicity of clade 2.3.4.4 H5 highly pathogenic AIV (HPAIV) in two species of diving ducks. Juvenile and adult Ruddy Duck (Oxyura jamaicensis) and juvenile Lesser Scaup (Aythya affinis) were intranasally inoculated with A/Northern Pintail/WA/40964/2014 H5N2 HPAIV. Additional groups of juvenile Lesser Scaups were inoculated with A/Gyrfalcon/WA/41088/2014 H5N8 HPAIV. The approximate 50% bird infectious doses (BID50) of the H5N2 isolate for adult Ruddy Ducks was <102 50% egg infectious doses (EID50) and for the juvenile Lesser Scaups it was <104 EID50. There were insufficient juvenile Ruddy Ducks to calculate the BID50. The BID50 for the juvenile Lesser Scaups inoculated with the H5N8 isolate was 103 EID50. Clinical disease was not observed in any group; however, mortality occurred in the juvenile Ruddy Ducks inoculated with the H5N2 virus (three of five ducks), and staining for AIV antigen was observed in numerous tissues from these ducks. One adult Ruddy Duck also died and although it was infected with AIV (the duck was positive for virus shedding and AIV antigen was detected in tissues), it was also infected with coccidiosis. The proportion of ducks shedding virus was related to the dose administered, but the titers were similar among dose groups. The group with the fewest ducks shedding virus was the adult Ruddy Ducks. There was a trend for the Lesser Scaups to shed higher titers of virus than the Ruddy Ducks. No virus shedding was detected after 7 d postinoculation in any group. Similar to dabbling ducks, Lesser Scaups and Ruddy Ducks are susceptible to infection with this H5 HPAIV lineage, although they excrete lower titers of virus.

Antigenic diversity of H5 highly pathogenic avian influenza viruses of clade 2.3.4.4 isolated in Asia.

H5 highly pathogenic avian influenza viruses (HPAIV) have spread in both poultry and wild birds since late 2003. Continued circulation of HPAIV in poultry in several regions of the world has led to antigenic drift. In the present study, we analyzed the antigenic properties of H5 HPAIV isolated in Asia using four neutralizing mAbs recognizing hemagglutinin, which were established using A/chicken/Kumamoto/1-7/2014 (H5N8), belonging to clade 2.3.4.4 and also using polyclonal antibodies. Viruses of clades 1.1, 2.3.2.1, 2.3.4, and 2.3.4.4 had different reactivity patterns to the panel of mAbs, thereby indicating that the antigenicity of the viruses of clade 2.3.4.4 were similar but differed from the other clades. In particular, the antigenicity of the viruses of clade 2.3.4.4 differed from those of the viruses of clades 2.3.4 and 2.3.2.1, which suggests that the recent H5 HPAIV have further evolved antigenically divergent. In addition, reactivity of antiserum suggests that the antigenicity of viruses of clade 2.3.4.4 differed slightly among groups A, B, and C. Vaccines are still used in poultry in endemic countries, so the antigenicity of H5 HPAIV should be monitored continually to facilitate control of avian influenza. The panel of mAbs established in the present study will be useful for detecting antigenic drift in the H5 viruses that emerge from the current strains.

Isolation of an H5N8 Highly Pathogenic Avian Influenza Virus Strain from Wild Birds in Seoul, a Highly Urbanized Area in South Korea.

Asian-lineage H5 highly pathogenic avian influenza viruses (HPAIV) have caused recurrent outbreaks in poultry and wild birds. In January 2014, H5N8 HPAIV caused outbreaks in South Korea and subsequently spread to East Asia, Europe, and North America. We report the isolation of an H5N8 HPAIV strain from wild birds in Seoul, the most-developed city in South Korea. We analyzed the complete genome sequence of this isolate and estimated its origin using a phylogenetic analysis. The Seoul H5N8 isolate clustered phylogenetically with strains isolated from migratory wild birds but was distinct from Korean poultry isolates. This H5N8 virus was likely introduced into the urbanized city by migratory wild birds. Therefore, wild bird habitats in urbanized areas should be carefully monitored for HPAIV.

Novel real-time PCR-based patho- and phylotyping of potentially zoonotic avian influenza A subtype H5 viruses at risk of incursion into Europe in 2017.

Since November 2016, Europe witnesses another wave of incursion of highly pathogenic avian influenza (HPAI) A(H5) viruses of the Asian origin goose/Guangdong (gs/GD) lineage. Infections with H5 viruses of clade 2.3.4.4b affect wild bird and poultry populations. H5 viruses of clades 2.2, 2.3.1.2c and 2.3.4.4a were detected previously in Europe in 2006, 2010 and 2014. Clades 2.2.1.2 and 2.3.2.1.c are endemic in Egypt and Western Africa, respectively and have caused human fatalities. Evidence exists of their co-circulation in the Middle East. Subtype H5 viruses of low pathogenicity (LPAI) are endemic in migratory wild bird populations. They potentially mutate into highly pathogenic phenotypes following transmission into poultry holdings. However, to date only the gs/GD H5 lineage had an impact on human health. Rapid and specific diagnosis marks the cornerstone for control and eradication of HPAI virus incursions. We present the development and validation of five real-time RT-PCR assays (RT-qPCR) that allow sequencing-independent pathotype and clade-specific distinction of major gs/GD HPAI H5 virus clades and of Eurasian LPAI viruses currently circulating. Together with an influenza A virus-generic RT-qPCR, the assays significantly speed up time-to-diagnosis and reduce reaction times in a OneHealth approach of curbing the spread of gs/GD HPAI viruses.

Recent outbreaks of highly pathogenic avian influenza viruses in South Korea

Outbreaks of H5 highly pathogenic avian influenza viruses (HPAIVs) have caused economic loss for the poultry industry and posed a threat to public health. In South Korea, novel reassortants of HPAIVs such as H5N6 and H5N8 had been circulating in poultry. Here, we will discuss the identity of recent novel reassortants of Korean H5 HPAIVs and the recent advances in vaccine development, which will be useful for controlling HPAIV transmission in poultry and for effectively preventing future epidemics and pandemics.

Evolution, global spread, and pathogenicity of highly pathogenic avian influenza H5Nx clade 2.3.4.4.

Novel subtypes of Asian-origin (Goose/Guangdong lineage) H5 highly pathogenic avian influenza (HPAI) viruses belonging to clade 2.3.4, such as H5N2, H5N5, H5N6, and H5N8, have been identified in China since 2008 and have since evolved into four genetically distinct clade 2.3.4.4 groups (A–D). Since 2014, HPAI clade 2.3.4.4 viruses have spread rapidly via migratory wild aquatic birds and have evolved through reassortment with prevailing local low pathogenicity avian influenza viruses. Group A H5N8 viruses and its reassortant viruses caused outbreaks in wide geographic regions (Asia, Europe, and North America) during 2014–2015. Novel reassortant Group B H5N8 viruses caused outbreaks in Asia, Europe, and Africa during 2016–2017. Novel reassortant Group C H5N6 viruses caused outbreaks in Korea and Japan during the 2016–2017 winter season. Group D H5N6 viruses caused outbreaks in China and Vietnam. A wide range of avian species, including wild and domestic waterfowl, domestic poultry, and even zoo birds, seem to be permissive for infection by and/or transmission of clade 2.3.4.4 HPAI viruses. Further, compared to previous H5N1 HPAI viruses, these reassortant viruses show altered pathogenicity in birds. In this review, we discuss the evolution, global spread, and pathogenicity of H5 clade 2.3.4.4 HPAI viruses.

Pathogenicity and Transmission of H5 and H7 Highly Pathogenic Avian Influenza Viruses in Mallards.

The spread of H5 subtype highly pathogenic avian influenza (HPAI) viruses of the Gs/GD lineage by migratory waterfowl is a serious concern for animal and public health. H5 and H7 HPAI viruses are considered to be adapted to gallinaceous species (chickens, turkeys, quail, etc.) and less likely to infect and transmit in wild ducks. In order to understand why this is different with certain Gs/GD lineage H5 HPAI viruses, we compared the pathogenicity and transmission of several H5 and H7 HPAI viruses from previous poultry outbreaks to Gs/GD lineage H5 viruses, including H5N1 (clade 2.2), H5N8 and H5N2 (clade 2.3.4.4) viruses, in mallards as a representative wild duck species. Surprisingly, most HPAI viruses examined in this study replicated well and transmitted among mallards; however, the three Gs/GD lineage H5 HPAI viruses replicated to higher titers, which could explain the transmission of these viruses in susceptible wild duck populations.

Lessons from the Largest Epidemic of Avian Influenza Viruses in Taiwan, 2015.

The largest epidemic of avian influenza (AI) in history attacked poultry and wild birds throughout Taiwan starting January 6, 2015. This study analyzed surveillance results, epidemiologic characteristics, and viral sequences by using government-released information, with the intention to provide recommendations to minimize future pandemic influenza. The H5 clade 2.3.4.4 highly pathogenic AI viruses (HPAIVs) had not been detected in Taiwan before 2015. During this epidemic, four types of etiologic agents were identified: the three novel subtypes H5N2, H5N8, and H5N3 clade 2.3.4.4 HPAIVs and one endemic chicken H5N2 subtype (Mexican-like lineage) of low pathogenic AI viruses. Cocirculation of mixed subtypes also occurred, with H5N2 clade 2.3.4.4 HPAIVs accompanied by the H5N8 and H5N3 subtypes or old H5N2 viruses in the same farm. More than 90% of domestic geese died from this AI epidemic; geese were affected the most at the early outbreaks. The epidemic peaked in mid-January for all three novel H5 subtypes. Spatial epidemiology found that most affected areas were located in southwestern coastal areas. In terrestrial poultry (mostly chickens), different geographic distributions of AI virus subtypes were detected, with hot spots of H5N2 clade 2.3.4.4 vs. past-endemic old H5N2 viruses in Changhwa (P = 0.03) and Yunlin (P = 0.007) counties, respectively, of central Taiwan. Phylogenetic and sequence analyses of all the early 10 Taiwan H5 clade 2.3.4.4 isolates covering the three subtypes showed that they were very different from the HA of the past local H5 viruses from domestic ducks (75%-80%) and chickens (70%-75%). However, they had the highest sequence identity percentages (99.53%-100%), with the HA of A/crane/Kagoshima/KU13/2014(H5N8) isolated on December 7, 2014, in Japan being higher than those of recent American and Korean H5 HPAIVs [A/Northern pintail/Washington/40964/2014 (H5N2) and A/gyrfalcon/Washington/41088-6/2014 (H5N8): 99.02%-99.54% and A/Baikal teal/Korea/Donglim3/2014 (H5N8): 98.61%-99.08%], implying a likely common ancestor of these H5 clade 2.3.4.4 viruses. The multiple subtypes of H5 clade 2.3.4.4 HPAIVs imply high viral reassortment. We recommend establishing an integrated surveillance system, involving clinical, virologic, and serologic surveillance in poultry and wild birds, swine and other mammals prevalent on multiple-animal mixed-type traditional farms, and high-risk human populations, as a crucially important step to minimize future pandemic influenza.

Lack of chicken adaptation of newly emergent Eurasian H5N8 and reassortant H5N2 high pathogenicity avian influenza viruses in the U.S. is consistent with restricted poultry outbreaks in the Pacific flyway during 2014–2015

In 2014–2015, the U.S. experienced an unprecedented outbreak of Eurasian clade 2.3.4.4 H5 highly pathogenic avian influenza (HPAI) virus, initially affecting mainly wild birds and few backyard and commercial poultry premises. To better model the outbreak, the pathogenesis and transmission dynamics of representative Eurasian H5N8 and reassortant H5N2 clade 2.3.4.4 HPAI viruses detected early in the North American outbreak were investigated in chickens. High mean chicken infectious doses and lack of seroconversion in survivors indicated the viruses were poorly chicken adapted. Pathobiological features were consistent with HPAI virus infection, although the delayed appearance of lesions, longer mean death times, and reduced replication in endothelial cells differed from features of most other Eurasian H5N1 HPAI viruses. Although these initial U.S. H5 HPAI viruses had reduced adaptation and transmissibility in chickens, multi-generational passage in poultry could generate poultry adapted viruses with higher infectivity and transmissibility.

Application of Diagnostic Microarray Technique in Subtyping and Pathotyping of Avian Influenza Viruses Isolated in Mongolia

Asian-lineage H5 highly pathogenic avian influenza (HPAI) viruses have caused continuous outbreaks in poultry and wild birds. Development of rapid and accurate diagnostic methods is needed for preventing further spread of the virus and reducing the time required for eradication of the virus. We developed a low-density microarray for the rapid detection and identification of avian influenza virus subtypes H5, H7, and H9 and their pathotypes in a previous study. In the present study, we evaluated previously developed diagnostic microarray using avian influenza viruses isolated in Mongolia, including H5 HPAI viruses. All H5 HPAI viruses isolated in Mongolia were shown as H5-specific and highly pathogenic pattern in the microarray. H2, H3 and H12 viruses isolated in Mongolia used in this study did not show any H5, H7 and H9 patterns. These results indicated that this diagnostic microarray has enormous potential for the rapid subtyping and pathotyping of influenza viruses, including viruses isolated in Mongolia.

Supplemented vaccination with tandem repeat M2e virus-like particles enhances protection against homologous and heterologous HPAI H5 viruses in chickens

Highly pathogenic avian influenza (HPAI) H5 viruses derived from A/Goose/Guangdong/1/96 have been continuously circulating globally, severely affecting the public health and poultry industries. The matrix 2 protein ectodomain (M2e) is considered a promising candidate for a universal cross-protective influenza vaccine that provides more effective control over HPAI H5 viruses harboring variant hemagglutinin (HA)-antigens. Here, we evaluated the protective efficacy of a tandem repeat construct of heterologous M2e presented on virus-like particles (M2e5x VLPs) either alone or as a supplement against HPAI H5 viruses in a chicken model. Chickens immunized with M2e5x VLPs alone induced M2e-specific antibodies but were not protected against HPAI H5. The homo- and cross-protective efficacy of M2e5x VLP-supplemented vaccination of chickens was also examined. Importantly, supplementation with M2e5x VLPs induced significantly higher levels of antibodies specific for M2e and different viruses as well as provided improved protection against homologous and heterologous HPAI H5 viruses. Considering the limited efficacy of inactivated vaccines, supplement vaccination with M2e5x VLPs may be an effective measure for preventing outbreaks of HPAI viruses that have the ability to constantly change their antigenic properties in poultry.

Infection Risk for Persons Exposed to Highly Pathogenic Avian Influenza A H5 Virus-Infected Birds, United States, December 2014-March 2015.

Newly emerged highly pathogenic avian influenza (HPAI) A H5 viruses have caused outbreaks among birds in the United States. These viruses differ genetically from HPAI H5 viruses that previously caused human illness, most notably in Asia and Africa. To assess the risk for animal-to-human HPAI H5 virus transmission in the United States, we determined the number of persons with self-reported exposure to infected birds, the number with an acute respiratory infection (ARI) during a 10-day postexposure period, and the number with ARI who tested positive for influenza by real-time reverse transcription PCR or serologic testing for each outbreak during December 15, 2014-March 31, 2015. During 60 outbreaks in 13 states, a total of 164 persons were exposed to infected birds. ARI developed in 5 of these persons within 10 days of exposure. H5 influenza virus infection was not identified in any persons with ARI, suggesting a low risk for animal-to-human HPAI H5 virus transmission.

Outbreaks of highly pathogenic avian influenza H5N1 clade 2.3.2.1c in hunting falcons and kept wild birds in Dubai implicate intercontinental virus spread.

Highly pathogenic avian influenza viruses (HPAIVs) of subtype H5N1 have continued to perpetuate with divergent genetic variants in poultry within Asia since 2003. Further dissemination of Asian-derived H5 HPAIVs to Europe, Africa and, most recently, to the North American continent has occurred. We report an outbreak of HPAIV H5N1 among falcons kept for hunting and other wild bird species bred as falcon prey in Dubai, United Arab Emirates, during the autumn of 2014. The causative agent was identified as avian influenza virus subtype H5N1, clade 2.3.2.1c, by genetic and phylogenetic analyses. High mortality in infected birds was in accordance with systemic pathomorphological and histological alterations in affected falcons. Genetic analysis showed the HPAIV H5N1 of clade 2.3.2.1c is a reassortant in which the PB2 segment was derived from an Asian-origin H9N2 virus lineage. The Dubai H5N1 viruses were closely related to contemporary H5N1 HPAIVs from Nigeria, Burkina-Faso, Romania and Bulgaria. Median-joining network analysis of 2.3.2.1c viruses revealed that the Dubai outbreak was an episode of a westward spread of these viruses on a larger scale from unidentified Asian sources. The incursion into Dubai, possibly via infected captive hunting falcons returning from hunting trips to central Asian countries, preceded outbreaks in Nigeria and other West African countries. The alarmingly enhanced geographical mobility of clade 2.3.2.1.c and clade 2.3.4.4 viruses may represent another wave of transcontinental dissemination of Asian-origin HPAIV H5 viruses, such as the outbreak at Qinghai Lake caused by clade 2.2 (‘Qinghai’ lineage) in 2005.

Intracontinental and intercontinental dissemination of Asian H5 highly pathogenic avian influenza virus (clade 2.3.4.4) in the winter of 2014-2015.

Asian H5 highly pathogenic avian influenza viruses (HPAIVs) that possess the clade 2.3.4.4 HA gene have been identified in wild birds and poultry since late 2014 in both Europe and North America (N. America). Clade 2.3.4.4 H5 HPAIVs of the H5N8 subtype have been isolated in both regions, whereas reassortment viruses with NA N1 and N2 subtypes of the North American (N. American). avian lineage have only been identified in N. America. The HA genes of those isolates were closely related to genes of the HPAIVs that have caused massive outbreaks in poultry in Korea since January 2014. The outbreaks caused by those viruses and the genetic relatedness of their HA and NA genes are reviewed in this study. Although the illegal movement of poultry and poultry products cannot be ruled out as a cause of intercontinental and intracontinental dissemination of clade 2.3.4.4 H5 HPAIVs during the winter of 2014-2015, transmission of the viruses by infected migratory birds appears to be a more plausible mechanism for their dissemination. In particular, the involvement of migratory birds in HPAIV transmission between Asia and N. America is highly likely because of the reassortments between H5N8 HPAIV and the N. American lineage avian influenza viruses.

Protective efficacy of stockpiled vaccine against H5N8 highly pathogenic avian influenza virus isolated from a chicken in Kumamoto prefecture, Japan, in 2014.

H5 highly pathogenic avian influenza (HPAI) viruses have spread worldwide, and antigenic variants of different clades have been selected. In this study, the national stockpiled vaccine prepared from A/duck/Hokkaido/Vac-1/2004 (H5N1) strain was evaluated for the protective efficacy against H5N8 HPAI virus isolated in Kumamoto prefecture, Japan, in April 2014. In the challenge test, all of the vaccinated chickens survived without showing any clinical signs and reduced virus shedding. It was concluded that the present stockpiled vaccine was effective against the H5N8 HPAI virus.

Characterization of an H5N8 influenza A virus isolated from chickens during an outbreak of severe avian influenza in Japan in April 2014.

A highly pathogenic avian influenza virus (HPAIV) of subtype H5N8, A/chicken/Kumamoto/1-7/2014, was isolated from a Japanese chicken farm during an outbreak in April 2014. Phylogenetic analysis revealed that this virus belonged to HA clade 2.3.4.4. All eight genomic segments showed high sequence similarity to those of the H5N8 subtype HPAIVs A/broiler duck/Korea/Buan2/2014 and A/baikal teal/Korea/Donglim3/2014, which were isolated in Korea in January 2014. Intranasal experimental infection of chickens and ducks with A/chicken/Kumamoto/1-7/2014 was performed to assess the pathogenicity of the virus in chickens and the potential for waterfowl to act as a virus reservoir and carrier. A high-titer virus challenge (10(6) EID50 per animal) was lethal in chickens, but they were unaffected by lower virus doses (10(2) EID50 or 10(4) EID50 per animal). Virus challenge at all doses examined was found to result in asymptomatic infection of ducks. An HI assay revealed that A/chicken/Kumamoto/1-7/2014 possessed relatively low cross-reactivity with H5 viruses belonging to clades other than clade 2.3.4.4. These results suggest that waterfowl may be able to spread the virus even if they possess antibodies resulting from a previous infection with H5 HPAIV that was antigenically distinguishable from viruses belonging to clade 2.3.4.4.

Establishment of MDCK Stable Cell Lines Expressing TMPRSS2 and MSPL and Their Applications in Propagating Influenza Vaccine Viruses in Absence of Exogenous Trypsin.

We established two Madin-Darby canine kidney (MDCK) cell lines stably expressing human airway transmembrane protease: transmembrane protease, serine 2 (TMPRSS2) and mosaic serine protease large form (MSPL) which support multicycle growth of two H5 highly pathogenic avian influenza viruses (HPAIV) recombinant vaccines (Re-5 and Re-6) and an H9 avian influenza virus (AIV) recombinant vaccine (Re-9) in the absence of trypsin. Data showed that the cell lines stably expressed TMPRSS2 and MSPL after 20 serial passages. Both MDCK-TMPRSS2 and MDCK-MSPL could proteolytically cleave the HA of Re-5, Re-6, and Re-9 and supported high-titer growth of the vaccine without exogenous trypsin. Re-5, Re-6, and Re-9 efficiently infected and replicated within MDCK-TMPRSS2 and MDCK-MSPL cells and viral titer were comparable to the virus grown in MDCK cells with TPCK-trypsin. Thus, our results indicate a potential application for these cell lines in cell-based influenza vaccine production and may serve as a useful tool for HA proteolytic cleavage-related studies.

The NS segment of H5N1 avian influenza viruses (AIV) enhances the virulence of an H7N1 AIV in chickens

Some outbreaks involving highly pathogenic avian influenza viruses (HPAIV) of subtypes H5 and H7 were caused by avian-to-human transmissions. In nature, different influenza A viruses can reassort leading to new viruses with new characteristics. We decided to investigate the impact that the NS-segment of H5 HPAIV would have on viral pathogenicity of a classical avian H7 HPAIV in poultry, a natural host. We focussed this study based on our previous work that demonstrated that single reassortment of the NS-segment from an H5 HPAIV into an H7 HPAIV changes the ability of the virus to replicate in mammalian hosts. Our present data show that two different H7-viruses containing an NS-segment from H5–types (FPV NS GD or FPV NS VN) show an overall highly pathogenic phenotype compared with the wild type H7–virus (FPV), as characterized by higher viral shedding and earlier manifestation of clinical signs. Correlating with the latter, higher amounts of IFN-β mRNA were detected in the blood of NS-reassortant infected birds, 48 h post-infection (pi). Although lymphopenia was detected in chickens from all AIV-infected groups, also 48 h pi those animals challenged with NS-reassortant viruses showed an increase of peripheral monocyte/macrophage-like cells expressing high levels of IL-1β, as determined by flow cytometry. Taken together, these findings highlight the importance of the NS-segment in viral pathogenicity which is directly involved in triggering antiviral and pro-inflammatory cytokines found during HPAIV pathogenesis in chickens.