In turkeys, unlike chickens, the non-structural NS1 protein does not play a significant role in the replication and tissue tropism of the H7N1 avian influenza virus.

The economic losses caused by high pathogenicity (HP) avian influenza viruses (AIV) in the poultry industry worldwide are enormous. Although chickens and turkeys are closely related Galliformes, turkeys are thought to be a bridging host for the adaptation of AIV from wild birds to poultry because of their high susceptibility to AIV infections. HPAIV evolve from low pathogenicity (LP) AIV after circulation in poultry through mutations in different viral proteins, including the non-structural protein (NS1), a major interferon (IFN) antagonist of AIV. At present, it is largely unknown whether the virulence determinants of HPAIV are the same in turkeys and chickens. Previously, we showed that mutations in the NS1 of HPAIV H7N1 significantly reduced viral replication in chickens in vitro and in vivo. Here, we investigated the effect of NS1 on the replication and virulence of HPAIV H7N1 in turkeys after inoculation with recombinant H7N1 carrying a naturally truncated wild-type NS1 (with 224 amino-acid "aa" in length) or an extended NS1 with 230-aa similar to the LP H7N1 ancestor. There were no significant differences in multiple-cycle viral replication or in the efficiency of NS1 in blocking IFN induction in the cell culture. Similarly, all viruses were highly virulent in turkeys and replicated at similar levels in various organs and swabs collected from the inoculated turkeys. These results suggest that NS1 does not play a role in the virulence or replication of HPAIV H7N1 in turkeys and further indicate that the genetic determinants of HPAIV differ in these two closely related galliform species.

Evidence for Different Virulence Determinants and Host Response after Infection of Turkeys and Chickens with Highly Pathogenic H7N1 Avian Influenza Virus.

Wild birds are the reservoir for all avian influenza viruses (AIV). In poultry, the transition from low pathogenic (LP) AIV of H5 and H7 subtypes to highly pathogenic (HP) AIV is accompanied mainly by changing the hemagglutinin (HA) monobasic cleavage site (CS) to a polybasic motif (pCS). Galliformes, including turkeys and chickens, succumb with high morbidity and mortality to HPAIV infections, although turkeys appear more vulnerable than chickens. Surprisingly, the genetic determinants for virulence and pathogenesis of HPAIV in turkeys are largely unknown. Here, we determined the genetic markers for virulence and transmission of HPAIV H7N1 in turkeys, and we explored the host responses in this species compared to those of chickens. We found that recombinant LPAIV H7N1 carrying pCS was avirulent in chickens but exhibited high virulence in turkeys, indicating that virulence determinants vary in these two galliform species. A transcriptome analysis indicated that turkeys mount a different host response than do chickens, particularly from genes involved in RNA metabolism and the immune response. Furthermore, we found that the HA glycosylation at residue 123, acquired by LP viruses shortly after transmission from wild birds and preceding the transition from LP to HP, had a role in virus fitness and virulence in chickens, though it was not a prerequisite for high virulence in turkeys. Together, these findings indicate variable virulence determinants and host responses in two closely related galliformes, turkeys and chickens, after infection with HPAIV H7N1. These results could explain the higher vulnerability to HPAIV of turkeys compared to chickens. IMPORTANCE Infection with HPAIV in chickens and turkeys, two closely related galliform species, results in severe disease and death. Although the presence of a polybasic cleavage site (pCS) in the hemagglutinin of AIV is a major virulence determinant for the transition of LPAIV to HPAIV, there are knowledge gaps on the genetic determinants (including pCS) and the host responses in turkeys compared to chickens. Here, we found that the pCS alone was sufficient for the transformation of a LP H7N1 into a HPAIV in turkeys but not in chickens. We also noticed that turkeys exhibited a different host response to an HPAIV infection, namely, a widespread downregulation of host gene expression associated with protein synthesis and the immune response. These results are important for a better understanding of the evolution of HPAIV from LPAIV and of the different outcomes and the pathomechanisms of HPAIV infections in chickens and turkeys.

Experimental infection of ostriches with H7N1 low pathogenic and H5N8 clade 2.3.4.4B highly pathogenic influenza A viruses.

Infection dynamics data for influenza A virus in a species is important for understanding host-pathogen interactions and developing effective control strategies. Seven-week-old ostriches challenged with H7N1 low pathogenic viruses (LPAIV) or clade 2.3.4.4B H5N8 high pathogenic viruses (HPAIV) were co- housed with non-challenged contacts. Clinical signs, virus shed in the trachea, cloaca, and feather pulp, and antibody responses were quantified over 14 days. H7N1 LPAIV-infected ostriches remained generally healthy with some showing signs of mild conjunctivitis and rhinitis attributed to Mycoplasma co-infection. Mean tracheal virus shedding titres in contact birds peaked 3 days (106.2 EID50 equivalents / ml) and 9 days (105.28 EID50 equivalents / ml) after introduction, lasting for at least 13 days post infection. Cloacal shedding was substantially lower and ceased within 10 days of onset, and low virus levels were detected in wing feather pulp up until day 14. H5N8 HPAIV -infected ostriches showed various degrees of morbidity, with 2/3 mortalities in the in-contact group. Mean tracheal shedding in contact birds peaked 8 days after introduction (106.32 EID50 equivalents/ ml) and lasted beyond 14 days in survivors. Cloacal shedding and virus in feather pulp was generally higher and more consistently positive compared to H7N1 LPAIV, and was also detectable at least until 14 days post infection in survivors. Antibodies against H5N8 HPAIV and H7N1 LPAIV only appeared after day 7 post exposure, with higher titres induced by the HPAIV compared to the LPAIV, and neuraminidase treatment was essential to remove non-specific inhibitors from the H5N8-positive antisera.

H7N1 Low Pathogenicity Avian Influenza Viruses in Poultry in the United States During 2018.

Here, we report three detections of H7N1 low pathogenicity avian influenza viruses (LPAIV) from poultry in Missouri (n = 2) and Texas (n = 1) during February and March 2018. Complete genome sequencing and comparative phylogenetic analysis suggest that the H7 LPAIV precursor viruses were circulating in wild birds in North America during the fall and winter of 2017 and spilled over into domestic poultry in Texas and Missouri independently during the spring of 2018.

Nota de investigación­Virus de la influenza aviar de baja patogenicidad H7N1 en avicultura, Estados Unidos, 2018. En este artículo se reportan tres detecciones del virus de influenza aviar de baja patogenicidad H7N1 (LPAIV) en avicultura en Missouri (n = 2) y Texas (n = 1) durante febrero y marzo del 2018. La secuenciación completa del genoma y el análisis filogenético comparativo sugieren que precursores de este virus de influenza de baja patogenicidad H7 circulaban en aves silvestres en América del Norte durante el otoño y el invierno de 2017 y se propagaron a las aves comerciales en Texas y Missouri de forma independiente durante la primavera del 2018.

Substitution Arg140Gly in Hemagglutinin Reduced the Virulence of Highly Pathogenic Avian Influenza Virus H7N1.

The H7 subtype of avian influenza viruses (AIV) stands out among other AIV. The H7 viruses circulate in ducks, poultry and equines and have repeatedly caused outbreaks of disease in humans. The laboratory strain A/chicken/Rostock/R0p/1934 (H7N1) (R0p), which was previously derived from the highly pathogenic strain A/FPV/Rostock/1934 (H7N1), was studied in this work to ascertain its biological property, genome stability and virulent changing mechanism. Several virus variants were obtained by serial passages in the chicken lungs. After 10 passages of this virus through the chicken lungs we obtained a much more pathogenic variant than the starting R0p. The study of intermediate passages showed a sharp increase in pathogenicity between the fifth and sixth passage. By cloning these variants, a pair of strains (R5p and R6p) was obtained, and the complete genomes of these strains were sequenced. Single amino acid substitution was revealed, namely reversion Gly140Arg in HA1. This amino acid is located at the head part of the hemagglutinin, adjacent to the receptor-binding site. In addition to the increased pathogenicity in chicken and mice, R6p differs from R5p in the shape of foci in cell culture and an increased affinity for a negatively charged receptor analogue, while maintaining a pattern of receptor-binding specificity and the pH of conformational change of HA.

Infectivity and pathobiology of H7N1 and H5N8 high pathogenicity avian influenza viruses for pigeons (Columba livia var. domestica).

Avian influenza (AI) is one of the most important viral diseases in poultry, wildlife and humans. Available data indicate that pigeons play a minimum role in the epidemiology of AI. However, a degree of variation exists in the susceptibility of pigeons to highly pathogenic AI viruses (HPAIVs), especially since the emergence of the goose/Guangdong H5 lineage. Here, the pathogenesis of H5N8 HPAIV in comparison with a H7N1 HPAIV and the role of pigeons in the epidemiology of these viruses were evaluated. Local and urban pigeons (Columba livia var. domestica) were intranasally inoculated with 105 ELD50 of A/goose/Spain/IA17CR02699/2017 (H5N8) or A/Chicken/Italy/5093/1999 (H7N1) and monitored during 14 days. Several pigeons inoculated with H5N8 or H7N1 seroconverted. However, clinical signs, mortality, microscopic lesions and viral antigen were only detected in a local pigeon inoculated with H5N8 HPAIV. This pigeon presented prostration and neurological signs that correlated with the presence of large areas of necrosis and widespread AIV antigen in the central nervous system, indicating that the fatal outcome was associated with neurological dysfunction. Viral RNA in swabs was detected in some pigeons inoculated with H7N1 and H5N8, but it was inconsistent, short-term and at low titres. The present study demonstrates that the majority of pigeons were resistant to H5N8 and H7N1 HPAIVs, despite several pigeons developing asymptomatic infections. The limited viral shedding indicates a minimum role of pigeons as amplifiers of HPAIVs, regardless of the viral lineage, and suggests that this species may represent a low risk for environmental contamination. RESEARCH HIGHLIGHTS H7N1 and H5N8 HPAIVs can produce subclinical infections in pigeons. The mortality caused by H5N8 HPAIV in one pigeon was associated with neurological dysfunction. Pigeons represent a low risk for environmental contamination by HPAIVs.

Experimental infection of domestic geese (Anser anser var. domesticus) with H5N8 Gs/GD and H7N1 highly pathogenic avian influenza viruses.

Prior to the emergence of the Asian-origin H5 Goose/Guangdong/1/96 (Gs/GD) lineage, highly pathogenic avian influenza viruses (HPAIV) had rarely caused high mortalities in domestic geese. In 2016/2017 European epidemics, H5N8 Gs/GD clade 2.3.4.4 Group B produced an unprecedented number of outbreaks in waterfowl holdings. In this study, the pathogenesis of H5N8 HPAIV in comparison with H7N1 HPAIV, and the role of domestic geese in the epidemiology of these viruses, were evaluated. Local and commercial geese (Anser anser var. domesticus) were intranasally inoculated with 105 ELD50 of A/goose/Spain/IA17CR02699/2017 (H5N8) or A/Chicken/Italy/5093/1999 (H7N1) and monitored daily during 15 days. H5N8 was highly virulent to domestic geese, reaching 100% mortality by 10 days post-infection. Systemic microscopic necrotizing lesions associated with widespread AIV-antigen were detected by IHC techniques, the central nervous system being the most severely affected. High viral loads, measured by qRT-PCR, were present in all samples collected: oral and cloacal swabs, plasma tissues, and moderate levels in pool water. Domestic geese were also susceptible to H7N1 infection, as demonstrated by seroconversion and detection of viral RNA in tissues and plasma in some geese, but all lacked clinical signs. Viral shedding was confirmed in only some geese and was restricted to the oral route, but levels were high and still detected at the end of the study. Overall, H7N1 presents a lower lethality and shedding than H5N8 in geese; however, the viral shedding indicates that these species could play a role in the epidemiology of Gs/GD and other lineages of HPAIVs. RESEARCH HIGHLIGHTS H5N8 Gs/GD clade 2.3.4.4 Group B is highly virulent to domestic geese. The severity of H5N8 is associated with multisystemic replication. H7N1 can infect domestic geese but is avirulent to this species. Domestic geese could play a role in the epidemiology of Gs/GD HPAIVs.

Mammalian pathogenicity and transmissibility of low pathogenic avian influenza H7N1 and H7N3 viruses isolated from North America in 2018.

ABSTRACTLow pathogenic avian influenza (LPAI) H7 subtype viruses are infrequently, but persistently, associated with outbreaks in poultry in North America. These LPAI outbreaks provide opportunities for the virus to develop enhanced virulence and transmissibility in mammals and have previously resulted in both occasional acquisition of a highly pathogenic avian influenza (HPAI) phenotype in birds and sporadic cases of human infection. Two notable LPAI H7 subtype viruses caused outbreaks in 2018 in North America: LPAI H7N1 virus in chickens and turkeys, representing the first confirmed H7N1 infection in poultry farms in the United States, and LPAI H7N3 virus in turkeys, a virus subtype often associated with LPAI-to-HPAI phenotypes. Here, we investigated the replication capacity of representative viruses from these outbreaks in human respiratory tract cells and mammalian pathogenicity and transmissibility in the mouse and ferret models. We found that the LPAI H7 viruses replicated to high titre in human cells, reaching mean peak titres generally comparable to HPAI H7 viruses. Replication was efficient in both mammalian species, causing mild infection, with virus primarily limited to respiratory tract tissues. The H7 viruses demonstrated a capacity to transmit to naïve ferrets in a direct contact setting. These data support the need to perform routine risk assessments of LPAI H7 subtype viruses, even in the absence of confirmed human infection.

Virulence of three European highly pathogenic H7N1 and H7N7 avian influenza viruses in Pekin and Muscovy ducks.

BACKGROUND: There is paucity of data on the virulence of highly pathogenic (HP) avian influenza viruses (AIV) H7 in ducks compared to HPAIV H5. Here, the virulence of HPAIV H7N1 (designated H7N1-FPV34 and H7N1-It99) and H7N7 (designated H7N7-FPV27) was assessed in Pekin and/or Muscovy ducklings after intrachoanal (IC) or intramuscular (IM) infection. RESULTS: The morbidity rate ranged from 60 to 100% and mortality rate from 20 to 80% depending on the duck species, virus strain and/or challenge route. All Muscovy ducklings inoculated IC with H7N7-FPV27 or H7N1-FPV34 exhibited mild to severe clinical signs resulting in the death of 2/10 and 8/10 ducklings, respectively. Also, 2/10 and 6/9 of inoculated Muscovy ducklings died after IC or IM infection with H7N1-It99, respectively. Moreover, 5/10 Pekin ducklings inoculated IC or IM with H7N1-It99 died. The level of virus detected in the oropharyngeal swabs was higher than in the cloacal swabs. CONCLUSION: Taken together, HPAIV H7 cause mortality and morbidity in Muscovy and Pekin ducklings. The severity of disease in Muscovy ducklings depended on the virus strain and/or route of infection. Preferential replication of the virus in the respiratory tract compared to the gut merits further investigation.

Intercontinental spread of Asian-origin H7 avian influenza viruses by captive bird trade in 1990's.

Wild bird migration and illegal trade of infected poultry, eggs, and poultry products have been associated with the spread of avian influenza viruses (AIV). During 1992-1996, H7N1 and H7N8 low pathogenic AIV (LPAIV) were identified from captive wild birds; such as Pekin robin (Leiothrix lutea), magpie robin (Copsychus saularis), flycatcher sp. (genus Empidonax), a species of softbill and parakeet, sun conure (Aratinga solstitialis), painted conure (Pyrrhura picta), fairy bluebird (Irena puella), and common iora (Aegithina tiphia), kept in aviaries or quarantine stations in England, The Netherlands, Singapore and the United States (U.S.). In this study, we sequenced these H7 viruses isolated from quarantine facilities and aviaries using next-generation sequencing and conducted a comparative phylogenetic analysis of complete genome sequences to elucidate spread patterns. The complete genome sequencing and phylogenetic analysis suggested that H7 viruses originated from a common source, even though they were obtained from birds in distant geographical regions. All H7N1 and H7N8 viruses were LPAIV, except a H7N1 highly pathogenic AIV (HPAIV), A/Pekin robin/California/30412/1994(H7N1) virus. Our results support the continued need for regulation of the captive wild bird trade to reduce the risk of introduction and dissemination of both LPAIV and HPAIV throughout the world.

Mutation of Influenza A Virus PA-X Decreases Pathogenicity in Chicken Embryos and Can Increase the Yield of Reassortant Candidate Vaccine Viruses.

The PA-X protein of influenza A virus has roles in host cell shutoff and viral pathogenesis. While most strains are predicted to encode PA-X, strain-dependent variations in activity have been noted. We found that PA-X protein from the A/PR/8/34 (PR8) strain had significantly lower repressive activity against cellular gene expression than PA-X proteins from the avian strains A/turkey/England/50-92/91 (H5N1) (T/E) and A/chicken/Rostock/34 (H7N1). Loss of normal PA-X expression, either by mutation of the frameshift site or by truncating the X open reading frame (ORF), had little effect on the infectious virus titer of PR8 or PR8 7:1 reassortants with T/E segment 3 grown in embryonated hens' eggs. However, in both virus backgrounds, mutation of PA-X led to decreased embryo mortality and lower overall pathology, effects that were more pronounced in the PR8 strain than in the T/E reassortant, despite the low shutoff activity of the PR8 PA-X. Purified PA-X mutant virus particles displayed an increased ratio of hemagglutinin (HA) to nucleoprotein (NP) and M1 compared to values for their wild-type (WT) counterparts, suggesting altered virion composition. When the PA-X gene was mutated in the background of poorly growing PR8 6:2 vaccine reassortant analogues containing the HA and neuraminidase (NA) segments from H1N1 2009 pandemic viruses or from an avian H7N3 strain, HA yield increased up to 2-fold. This suggests that the PR8 PA-X protein may harbor a function unrelated to host cell shutoff and that disruption of the PA-X gene has the potential to improve the HA yield of vaccine viruses.IMPORTANCE Influenza A virus is a widespread pathogen that affects both humans and a variety of animal species, causing regular epidemics and sporadic pandemics, with major public health and economic consequences. A better understanding of virus biology is therefore important. The primary control measure is vaccination, which for humans mostly relies on antigens produced in eggs from PR8-based viruses bearing the glycoprotein genes of interest. However, not all reassortants replicate well enough to supply sufficient virus antigen for demand. The significance of our research lies in identifying that mutation of the PA-X gene in the PR8 strain of virus can improve antigen yield, potentially by decreasing the pathogenicity of the virus in embryonated eggs.

Replication and pathogenic potential of influenza A virus subtypes H3, H7, and H15 from free-range ducks in Bangladesh in mammals.

Surveillance of wild aquatic birds and free-range domestic ducks in the Tanguar Haor wetlands in Bangladesh has identified influenza virus subtypes H3N6, H7N1, H7N5, H7N9, and H15N9. Molecular characterization of these viruses indicates their contribution to the genesis of new genotypes of H5N1 influenza viruses from clade 2.3.2.1a that are dominant in poultry markets in Bangladesh as well as to the genesis of the highly pathogenic H5N8 virus currently causing disease outbreaks in domestic poultry in Europe and the Middle East. Therefore, we studied the antigenicity, replication, and pathogenicity of influenza viruses isolated from Tanguar Haor in the DBA/2J mouse model. All viruses replicated in the lung without prior mammalian adaptation, and H7N1 and H7N9 viruses caused 100% and 60% mortality, respectively. H7N5 viruses replicated only in the lungs, whereas H7N1 and H7N9 viruses also replicated in the heart, liver, and brain. Replication and transmission studies in mallard ducks showed that H7N1 and H7N9 viruses replicated in ducks without clinical signs of disease and shed at high titers from the cloaca of infected and contact ducks, which could facilitate virus transmission and spread. Our results indicate that H7 avian influenza viruses from free-range ducks can replicate in mammals, cause severe disease, and be efficiently transmitted to contact ducks. Our study highlights the role of free-range ducks in the spread of influenza viruses to other species in live poultry markets and the potential for these viruses to infect and cause disease in mammals.

Major contribution of the RNA-binding domain of NS1 in the pathogenicity and replication potential of an avian H7N1 influenza virus in chickens.

BACKGROUND: Non-structural protein NS1 of influenza A viruses harbours several determinants of pathogenicity and host-range. However it is still unclear to what extent each of its two structured domains (i.e. RNA-binding domain, RBD, and effector domain, ED) contribute to its various activities. METHODS: To evaluate the respective contributions of the two domains, we genetically engineered two variants of an H7N1 low pathogenicity avian influenza virus harbouring amino-acid substitutions that impair the functionality of either domain. The RBD- and ED-mutant viruses were compared to their wt- counterpart in vivo and in vitro, notably in chicken infection and avian cell culture models. RESULTS: The double substitution R38A-K41A in the RBD dramatically reduced the pathogenicity and replication potential of the virus, whereas the substitution A149V that was considered to abrogate the IFN-antagonistic activity of the effector domain entailed much less effects. While all three viruses initiated the viral life cycle in avian cells, replication of the R38A-K41A virus was severely impaired. This defect was associated with a delayed synthesis of nucleoprotein NP and a reduced accumulation of NS1, which was found to reach a concentration of about 30 micromol.L- 1 in wt-infected cells at 8 h post-infection. When overexpressed in avian lung epithelial cells, both the wt-NS1 and 3841AA-NS1, but not the A149V-NS1, reduced the poly(I:C)-induced activation of the IFN-sensitive chicken Mx promoter. Unexpectedly, the R38A-K41A substitution in the recombinant RBD did not alter its in vitro affinity for a model dsRNA. When overexpressed in avian cells, both the wt- and A149V-NS1s, as well as the individually expressed wt-RBD to a lesser extent, enhanced the activity of the reconstituted viral RNA-polymerase in a minireplicon assay. CONCLUSIONS: Collectively, our data emphasized the critical importance and essential role of the RNA-binding domain in essential steps of the virus replication cycle, notably expression and translation of viral mRNAs.

Similar transmissibility of the Italian H7N1 highly pathogenic avian influenza virus and its low pathogenic avian influenza virus predecessor.

The transmissibility of the H7N1 highly pathogenic avian influenza virus (HPAIV), which caused a large epidemic in commercial poultry in Italy in 1999-2000, was studied in chickens and compared with that of the low pathogenic precursor virus (LPAIV). Group transmission experiments using the HPAIV were executed to estimate the infectious period (IP), the transmission parameter (ß) and the basic reproduction number (R0). These estimates were then compared with those reported for the LPAIV. The estimated ß and R0 were similar for both viruses, whilst the IP of the LPAIV was longer than that of the HPAIV. These findings indicate that transmissibility from chicken-to-chicken alone does not appear to confer an advantage for this LPAIV to evolve to a HPAIV.

A DNA Vaccine That Targets Hemagglutinin to Antigen-Presenting Cells Protects Mice against H7 Influenza.

Zoonotic influenza H7 viral infections have a case fatality rate of about 40%. Currently, no or limited human to human spread has occurred, but we may be facing a severe pandemic threat if the virus acquires the ability to transmit between humans. Novel vaccines that can be rapidly produced for global distribution are urgently needed, and DNA vaccines may be the only type of vaccine that allows for the speed necessary to quench an emerging pandemic. Here, we constructed DNA vaccines encoding the hemagglutinin (HA) from influenza A/chicken/Italy/13474/99 (H7N1). In order to increase the efficacy of DNA vaccination, HA was targeted to either major histocompatibility complex class II molecules or chemokine receptors 1, 3, and 5 (CCR1/3/5) that are expressed on antigen-presenting cells (APC). A single DNA vaccination with APC-targeted HA significantly increased antibody levels in sera compared to nontargeted control vaccines. The antibodies were confirmed neutralizing in an H7 pseudotype-based neutralization assay. Furthermore, the APC-targeted vaccines increased the levels of antigen-specific cytotoxic T cells, and a single DNA vaccination could confer protection against a lethal challenge with influenza A/turkey/Italy/3889/1999 (H7N1) in mice. In conclusion, we have developed a vaccine that rapidly could contribute protection against a pandemic threat from avian influenza.IMPORTANCE Highly pathogenic avian influenza H7 constitute a pandemic threat that can cause severe illness and death in infected individuals. Vaccination is the main method of prophylaxis against influenza, but current vaccine strategies fall short in a pandemic situation due to a prolonged production time and insufficient production capabilities. In contrast, a DNA vaccine can be rapidly produced and deployed to prevent the potential escalation of a highly pathogenic influenza pandemic. We here demonstrate that a single DNA delivery of hemagglutinin from an H7 influenza could mediate full protection against a lethal challenge with H7N1 influenza in mice. Vaccine efficacy was contingent on targeting of the secreted vaccine protein to antigen-presenting cells.

Risk for Low Pathogenicity Avian Influenza Virus on Poultry Farms, the Netherlands, 2007-2013.

Using annual serologic surveillance data from all poultry farms in the Netherlands during 2007-2013, we quantified the risk for the introduction of low pathogenicity avian influenza virus (LPAIV) in different types of poultry production farms and putative spatial-environmental risk factors: distance from poultry farms to clay soil, waterways, and wild waterfowl areas. Outdoor-layer, turkey (meat and breeder), and duck (meat and breeder) farms had a significantly higher risk for LPAIV introduction than did indoor-layer farms. Except for outdoor-layer, all poultry types (i.e., broilers, chicken breeders, ducks, and turkeys) are kept indoors. For all production types, LPAIV risk decreased significantly with increasing distance to medium-sized waterways and with increasing distance to areas with defined wild waterfowl, but only for outdoor-layer and turkey farms. Future research should focus not only on production types but also on distance to waterways and wild bird areas. In addition, settlement of new poultry farms in high-risk areas should be discouraged.

Rapid detection of avian influenza A virus by immunochromatographic test using a novel fluorescent dye.

Sensitive and rapid diagnostic systems for avian influenza (AI) virus are required to screen large numbers of samples during a disease outbreak and to prevent the spread of infection. In this study, we employed a novel fluorescent dye for the rapid and sensitive recognition of AI virus. The styrylpyridine phosphor derivative was synthesized by adding allyl bromide as a stable linker and covalently immobilizing it on latex beads with antibodies generating the unique Red dye 53-based fluorescent probe. The performance of the innovative rapid fluorescent immnunochromatographic test (FICT) employing Red dye 53 in detecting the AI virus (A/H5N3) was 4-fold and 16-fold higher than that of Europium-based FICT and the rapid diagnostic test (RDT), respectively. In clinical studies, the presence of human nasopharyngeal specimens did not alter the performance of Red dye 53-linked FICT for the detection of H7N1 virus. Furthermore, in influenza A virus-infected human nasopharyngeal specimens, the sensitivity of the Red dye 53-based assay and RDT was 88.89% (8/9) and 55.56% (5/9) relative to rRT-PCR, respectively. The photostability of Red dye 53 was higher than that of fluorescein isothiocyanate (FITC), showing a stronger fluorescent signal persisting up to 8min under UV. The Red dye 53 could therefore be a potential probe for rapid fluorescent diagnostic systems that can recognize AI virus in clinical specimens.

Modelling the impact of co-circulating low pathogenic avian influenza viruses on epidemics of highly pathogenic avian influenza in poultry.

It is well known that highly pathogenic avian influenza (HPAI) viruses emerge through mutation of precursor low pathogenic avian influenza (LPAI) viruses in domestic poultry populations. The potential for immunological cross-protection between these pathogenic variants is recognised but the epidemiological impact during co-circulation is not well understood. Here we use mathematical models to investigate whether altered flock infection parameters consequent to primary LPAI infections can impact on the spread of HPAI at the population level. First we used mechanistic models reflecting the co-circulatory dynamics of LPAI and HPAI within a single commercial poultry flock. We found that primary infections with LPAI led to HPAI prevalence being maximised under a scenario of high but partial cross-protection. We then tested the population impact in spatially-explicit simulations motivated by a major avian influenza A(H7N1) epidemic that afflicted the Italian poultry industry in 1999-2001. We found that partial cross-protection can lead to a prolongation of HPAI epidemic duration. Our findings have implications for the control of HPAI in poultry particularly for settings in which LPAI and HPAI frequently co-circulate.

Comparison of pathogenicities of H7 avian influenza viruses via intranasal and conjunctival inoculation in cynomolgus macaques.

The outbreak of H7N9 low pathogenic avian influenza viruses in China has attracted attention to H7 influenza virus infection in humans. Since we have shown that the pathogenicity of H1N1 and H5N1 influenza viruses in macaques was almost the same as that in humans, we compared the pathogenicities of H7 avian influenza viruses in cynomolgus macaques via intranasal and conjunctival inoculation, which mimics natural infection in humans. H7N9 virus, as well as H7N7 highly pathogenic avian influenza virus, showed more efficient replication and higher pathogenicity in macaques than did H7N1 and H7N3 highly pathogenic avian influenza viruses. These results are different from pathogenicity in chickens as reported previously. Therefore, our results obtained in macaques help to estimate the pathogenicity of H7 avian influenza viruses in humans.

Prevalence of the C-terminal truncations of NS1 in avian influenza A viruses and effect on virulence and replication of a highly pathogenic H7N1 virus in chickens.

Highly pathogenic (HP) avian influenza viruses (AIV) evolve from low pathogenic (LP) precursors after circulation in poultry by reassortment and/or single mutations in different gene segments including that encoding NS1. The carboxyl terminal end (CTE) of NS1 exhibits deletions between amino acid 202 and 230 with still unknown impact on virulence of AIV in chickens. In this study, NS1 protein sequences of all AIV subtypes in birds from 1902 to 2015 were analyzed to study the prevalence and distribution of CTE truncation (ΔCTE). Thirteen different ΔCTE forms were observed in NS1 proteins from 11 HA and 8 NA subtypes with high prevalences in H9, H7, H6 and H10 and N9, N2, N6 and N1 subtypes particularly in chickens and minor poultry species. With 88% NS217 lacking amino acids 218-230 was the most common ΔCTE form followed by NS224 (3.6%). NS217 was found in 10 and 8 different HA and NA subtypes, respectively, whereas NS224 was detected exclusively in the Italian HPAIV H7N1 suggesting relevance for virulence. To test this assumption, 3 recombinant HPAIV H7N1 were constructed carrying wild-type HP NS1 (Hp-NS224), NS1 with extended CTE (Hp-NS230) or NS1 from LPAIV H7N1 (Hp-NSLp), and tested in-vitro and in-vivo. Extension of CTE in Hp NS1 significantly decreased virus replication in chicken embryo kidney cells. Truncation in the NS1 decreased the tropism of Hp-NS224 to the endothelium, central nervous system and respiratory tract epithelium without significant difference in virulence in chickens. This study described the variable forms of ΔCTE in NS1 and indicated that CTE is not an essential virulence determinant particularly for the Italian HPAIV H7N1 but may be a host-adaptation marker required for efficient virus replication.