Characterization of changes in the hemagglutinin that accompanied the emergence of H3N2/1968 pandemic influenza viruses.

The hemagglutinin (HA) of A/H3N2 pandemic influenza viruses (IAVs) of 1968 differed from its inferred avian precursor by eight amino acid substitutions. To determine their phenotypic effects, we studied recombinant variants of A/Hong Kong/1/1968 virus containing either human-type or avian-type amino acids in the corresponding positions of HA. The precursor HA displayed receptor binding profile and high conformational stability typical for duck IAVs. Substitutions Q226L and G228S, in addition to their known effects on receptor specificity and replication, marginally decreased HA stability. Substitutions R62I, D63N, D81N and N193S reduced HA binding avidity. Substitutions R62I, D81N and A144G promoted viral replication in human airway epithelial cultures. Analysis of HA sequences revealed that substitutions D63N and D81N accompanied by the addition of N-glycans represent common markers of avian H3 HA adaptation to mammals. Our results advance understanding of genotypic and phenotypic changes in IAV HA required for avian-to-human adaptation and pandemic emergence.

Avian influenza H9N2 subtype in Ghana: virus characterization and evidence of co-infection.

Between November 2017 and February 2018, Ghanaian poultry producers reported to animal health authorities a dramatic increase in mortality rate and a relevant drop in egg production in several layer hen farms. Laboratory investigations revealed that the farms had been infected by the H9N2 influenza subtype. Virological and molecular characterization of the viruses identified in Ghana is described here for the first time. Whole genome analysis showed that the viruses belong to the G1-lineage and cluster with viruses identified in North and West Africa. The low pathogenicity of the virus was confirmed by the intravenous pathogenicity index assay. Further investigations revealed co-infection with infectious bronchitis virus of the GI-19 lineage, which very likely explained the severity of the disease observed during the outbreaks. The H9N2 outbreaks in Ghana highlight the importance of performing a differential diagnosis and an in-depth characterization of emerging viruses. In addition, the detection of a potentially zoonotic subtype, such as the H9N2, in a region where highly pathogenic avian influenza H5Nx is currently circulating highlights the urgency of implementing enhanced monitoring strategies and supporting improved investments in regional diagnostic technologies. RESEARCH HIGHLIGHTS Influenza A H9N2 subtype was detected in layer hens in Ghana in 2017-2018 Whole genome characterization of seven H9N2 viruses was performed Phylogenetic trees revealed that the H9N2 viruses belong to the G1 lineage The HA protein possesses the amino acid mutations 226L and 155T Co-infection with infectious bronchitis virus of the GI-19 lineage was identified.

Influenza A(H9N2) Virus, Burkina Faso.

We identified influenza A(H9N2) virus G1 lineage in poultry in Burkina Faso. Urgent actions are needed to raise awareness about the risk associated with spread of this zoonotic virus subtype in the area and to construct a strategy for effective prevention and control of influenza caused by this virus.

Unexpected Interfarm Transmission Dynamics during a Highly Pathogenic Avian Influenza Epidemic.

UNLABELLED: Next-generation sequencing technology is now being increasingly applied to study the within- and between-host population dynamics of viruses. However, information on avian influenza virus evolution and transmission during a naturally occurring epidemic is still limited. Here, we use deep-sequencing data obtained from clinical samples collected from five industrial holdings and a backyard farm infected during the 2013 highly pathogenic avian influenza (HPAI) H7N7 epidemic in Italy to unravel (i) the epidemic virus population diversity, (ii) the evolution of virus pathogenicity, and (iii) the pathways of viral transmission between different holdings and sheds. We show a high level of genetic diversity of the HPAI H7N7 viruses within a single farm as a consequence of separate bottlenecks and founder effects. In particular, we identified the cocirculation in the index case of two viral strains showing a different insertion at the hemagglutinin cleavage site, as well as nine nucleotide differences at the consensus level and 92 minority variants. To assess interfarm transmission, we combined epidemiological and genetic data and identified the index case as the major source of the virus, suggesting the spread of different viral haplotypes from the index farm to the other industrial holdings, probably at different time points. Our results revealed interfarm transmission dynamics that the epidemiological data alone could not unravel and demonstrated that delay in the disease detection and stamping out was the major cause of the emergence and the spread of the HPAI strain. IMPORTANCE: The within- and between-host evolutionary dynamics of a highly pathogenic avian influenza (HPAI) strain during a naturally occurring epidemic is currently poorly understood. Here, we perform for the first time an in-depth sequence analysis of all the samples collected during a HPAI epidemic and demonstrate the importance to complement outbreak investigations with genetic data to reconstruct the transmission dynamics of the viruses and to evaluate the within- and between-farm genetic diversity of the viral population. We show that the evolutionary transition from the low pathogenic form to the highly pathogenic form occurred within the first infected flock, where we identified haplotypes with hemagglutinin cleavage site of different lengths. We also identify the index case as the major source of virus, indicating that prompt application of depopulation measures is essential to limit virus spread to other farms.

Reassortant avian influenza A(H5N1) viruses with H9N2-PB1 gene in poultry, Bangladesh.

Bangladesh has reported a high number of outbreaks of highly pathogenic avian influenza (HPAI) (H5N1) in poultry. We identified a natural reassortant HPAI (H5N1) virus containing a H9N2-PB1 gene in poultry in Bangladesh. Our findings highlight the risks for prolonged co-circulation of avian influenza viruses and the need to monitor their evolution.

Genomic Analysis of Infectious Bursal Disease Virus in Nigeria: Identification of Unique Mutations of Yet Unknown Biological Functions in Both Segments A and B.

Infectious bursal disease (IBD) is a viral poultry disease known worldwide for impacting the economy and food security. The disease is endemic in Nigeria, with reported outbreaks in vaccinated poultry flocks. To gain insight into the dynamics of infectious bursal disease virus (IBDV) evolution in Nigeria, near-complete genomes of four IBDVs were evaluated. Amino acid sequences in the hypervariable region of the VP2 revealed conserved markers (222A, 242I, 256I, 294I and 299S) associated with very virulent (vv) IBDV, including the serine-rich heptapeptide motif (SWSASGS). Based on the newly proposed classification for segments A and B, the IBDVs clustered in the A3B5 group (where A3 are IBDVs with vvIBDV-like segment A, and where B5 are from non-vvIBDV-like segment B) form a monophyletic subcluster. Unique amino acid mutations with yet-to-be-determined biological functions have been observed in both segments. Amino acid sequences of the Nigerian IBDVs showed that they are reassortant viruses. Circulation of reassortant IBDVs may be responsible for the vaccination failures observed in the Nigerian poultry population. Close monitoring of changes in the IBDV genome is recommended to nip deleterious changes in the bud through the identification and introduction of the most appropriate vaccine candidates and advocacy/extension programs for properly implementing disease control.

Phylogeography and evolutionary history of reassortant H9N2 viruses with potential human health implications.

Avian influenza viruses of the H9N2 subtype have seriously affected the poultry industry of the Far and Middle East since the mid-1990s and are considered one of the most likely candidates to cause a new influenza pandemic in humans. To understand the genesis and epidemiology of these viruses, we investigated the spatial and evolutionary dynamics of complete genome sequences of H9N2 viruses circulating in nine Middle Eastern and Central Asian countries from 1998 to 2010. We identified four distinct and cocirculating groups (A, B, C, and D), each of which has undergone widespread inter- and intrasubtype reassortments, leading to the generation of viruses with unknown biological properties. Our analysis also suggested that eastern Asia served as the major source for H9N2 gene segments in the Middle East and Central Asia and that in this geographic region within-country evolution played a more important role in shaping viral genetic diversity than migration between countries. The genetic variability identified among the H9N2 viruses was associated with specific amino acid substitutions that are believed to result in increased transmissibility in mammals, as well as resistance to antiviral drugs. Our study highlights the need to constantly monitor the evolution of H9N2 viruses in poultry to better understand the potential risk to human health posed by these viruses.

Genetic characterization of astroviruses detected in guinea fowl (Numida meleagris) reveals a distinct genotype and suggests cross-species transmission between turkey and guinea fowl.

Astroviruses can infect mammalian and avian species and are often responsible for gastroenteric disease symptoms. In this study, the complete open reading frame (ORF) 2, the 3' end of ORF1b and the corresponding intergenic region of astroviruses identified in farmed guinea fowl (Numida meleagris) were sequenced and genetically analysed. Overall, the genetic sequence of guinea fowl astroviruses was related to turkey astrovirus type 2 (TastV2), although a marked genetic distance was revealed based on ORF2, which might indicate the circulation of a distinct virus genotype and serotype in guinea fowl. Furthermore, the genetic data presented herein suggest that either recombination between different astroviruses infecting distinct hosts or adaptation of a given astrovirus to a new host had occurred. In either case, direct or indirect interspecies transmission of astroviruses is likely to have occurred between turkey and guinea fowl, indicating the ability of viruses belonging to the family Astroviridae to cross species barriers.

Experimental infection of poults and guinea fowl with genetically distinct avian astroviruses.

Avian astroviruses, of the genus Avastrovirus, are recognized as being the cause of enteritis in different bird species worldwide. In particular, turkeys are very susceptible and can be severely affected by this viral agent. More recently, astroviruses were detected in diseased guinea fowl in Italy but whether or not they were the causative agents of the clinical disease was not established. Despite the distribution and relevance of Avastrovirus infection, very little information on pathogenesis or factors influencing the pathogenicity of astroviruses is available. To increase available data on the pathogenesis of these viruses and to test the hypothesis of possible interspecies transmission, experimental infections were carried out in turkeys and guinea fowl with two genetically distinct avian astroviruses, namely TK-6363 and GF-5497, originating respectively from diseased turkey poults and guinea fowl. Data obtained in our study show that both of the viruses selected were able to infect young birds of the species in which they were originally detected. Additionally, these viruses were able to infect young birds of different species causing clinical signs, thus providing experimental evidence for the infection of distinct avian astroviruses in different avian species.

Evolutionary dynamics of multiple sublineages of H5N1 influenza viruses in Nigeria from 2006 to 2008.

Highly pathogenic A/H5N1 avian influenza (HPAI H5N1) viruses have seriously affected the Nigerian poultry industry since early 2006. Previous studies have identified multiple introductions of the virus into Nigeria and several reassortment events between cocirculating lineages. To determine the spatial, evolutionary, and population dynamics of the multiple H5N1 lineages cocirculating in Nigeria, we conducted a phylogenetic analysis of whole-genome sequences from 106 HPAI H5N1 viruses isolated between 2006 and 2008 and representing all 25 Nigerian states and the Federal Capital Territory (FCT) reporting outbreaks. We identified a major new subclade in Nigeria that is phylogenetically distinguishable from all previously identified sublineages, as well as two novel reassortment events. A detailed analysis of viral phylogeography identified two major source populations for the HPAI H5N1 virus in Nigeria, one in a major commercial poultry area (southwest region) and one in northern Nigeria, where contact between wild birds and backyard poultry is frequent. These findings suggested that migratory birds from Eastern Europe or Russia may serve an important role in the introduction of HPAI H5N1 viruses into Nigeria, although virus spread through the movement of poultry and poultry products cannot be excluded. Our study provides new insight into the genesis and evolution of H5N1 influenza viruses in Nigeria and has important implications for targeting surveillance efforts to rapidly identify the spread of the virus into and within Nigeria.

Evolutionary Dynamics of H5 Highly Pathogenic Avian Influenza Viruses (Clade 2.3.4.4B) Circulating in Bulgaria in 2019-2021.

The first detection of a Highly Pathogenic Avian Influenza (HPAI) H5N8 virus in Bulgaria dates back to December 2016. Since then, many outbreaks caused by HPAI H5 viruses from clade 2.3.4.4B have been reported in both domestic and wild birds in different regions of the country. In this study, we characterized the complete genome of sixteen H5 viruses collected in Bulgaria between 2019 and 2021. Phylogenetic analyses revealed a persistent circulation of the H5N8 strain for four consecutive years (December 2016-June 2020) and the emergence in 2020 of a novel reassortant H5N2 subtype, likely in a duck farm. Estimation of the time to the most recent common ancestor indicates that this reassortment event may have occurred between May 2019 and January 2020. At the beginning of 2021, Bulgaria experienced a new virus introduction in the poultry sector, namely a HPAI H5N8 that had been circulating in Europe since October 2020. The periodical identification in domestic birds of H5 viruses related to the 2016 epidemic as well as a reassortant strain might indicate undetected circulation of the virus in resident wild birds or in the poultry sector. To avoid the concealed circulation and evolution of viruses, and the risk of emergence of strains with pandemic potential, the implementation of control measures is of utmost importance, particularly in duck farms where birds display no clinical signs.

Gene segment reassortment between Eurasian and American clades of avian influenza virus in Italy.

All genes of avian influenza A viruses are phylogenetically distinguished into two large clades, namely the American and Eurasian clade. Reassortments among the gene segments of influenza viruses belonging to the two distinct clades are rare events and have never been described in poultry in Europe and Asia before. This study presents the genetic characterization of two influenza viruses isolated from domestic mallards in Italy in 2004 and 2005. Phylogenetic analysis of the entire genome showed that these viruses contain mixed gene segments belonging to the American and Eurasian clades.

Full-length genome sequencing of the Polish HPAI H5N1 viruses suggests separate introductions in 2006 and 2007.

This paper describes the results of the molecular and phylogenetic analysis of seven highly pathogenic avian influenza (HPAI) H5N1 strains isolated in 2006 (n = 5) and 2007 (n = 2) from wild birds and poultry in Poland. The whole genome sequence of these isolates was determined. All of the isolates possessed the hemagglutinin (HA) cleavage site sequence PQGERRRKKR*GLF typical of HPAI. Molecular markers associated with increased adaptation and virulence in mammals, as well as susceptibility to neuraminidase inhibitors, were revealed in the HA, neuraminidase (NA), and PB2 proteins. Based on the sequencing results related to the HA and NA genomic segments, H5N1 viruses circulating in Poland all belong to lineage 2.2. However, isolates isolated in 2006 were genetically distinct from those isolated in 2007 and grouped in different sublineages. H5N1 viruses isolated from wild birds in 2006 are almost identical to each other (99.9% HA; 99.6%-100% NA), and they are grouped within a cluster of viruses isolated in Germany from wild and domestic birds and mammals in 2006. Isolates from 2007 are also closely related to each other (nucleotide homologies 99.9% and 100% for HA and NA, respectively), and they are grouped together with isolates from wild and domestic birds collected in Eastern and Central Europe (Romania, Germany), and the Middle East (Kuwait, Saudi Arabia). Phylogenetic analysis of the sequences related to the internal proteins confirmed the results obtained for the HA and NA genes. Overall, the results indicate that HPAI H5N1 in Poland in 2006-07 was caused by at least two separate incursions of genetically distinct viruses.

Introduction into Nigeria of a distinct genotype of avian influenza virus (H5N1).

Genetic characterization of highly pathogenic avian influenza viruses (H5N1) isolated in July 2008 in Nigeria indicates that a distinct genotype, never before detected in Africa, reached the continent. Phylogenetic analysis showed that the viruses are genetically closely related to European and Middle Eastern influenza A (H5N1) isolates detected in 2007.

Isolation and identification of highly pathogenic avian influenza H5N1 virus from Houbara bustards (Chlamydotis undulata macqueenii) and contact falcons.

Highly pathogenic influenza virus (HPAIV) H5N1 has caused mortality and morbidity in many species of domestic and wild bird. The Houbara bustard (Chlamydotis undulata macqueenii) is a solitary bird that inhabits semi-desert regions. It is known to be susceptible to avianpox, avian paramyxovirus type 1, and low-pathogenicity avian influenza H9N2. We report an outbreak of H5N1 HPAIV in Houbara bustards, which were introduced into the Kingdom of Saudi Arabia for falconry purposes. Ninety-three per cent mortality (38 out of 41 birds) in the infected Houbara bustard flock and about 62.5% mortality (10 out of 16 birds) in falcons that came in contact with these birds were observed. Pooled cloacal and tracheal swabs from Houbara bustards as well as visceral organ homogenates collected in Houbara bustards and falcons were tested by real-time reverse transcriptase-polymerase chain reaction, and virus isolation was attempted in specific pathogen free hens' eggs. The viruses isolated were characterized as HPAIV H5N1. Phylogenetic analysis of the haemagglutinating and Neuraminidase (NA) genes revealed that the viruses isolated from Houbara bustards and falcons were closely related to each other and to Kuwaiti H5N1 strains isolated in 2007. Interestingly, they were genetically distinguishable from the co-circulating A/H5N1 viruses in Kingdom of Saudi Arabia causing outbreaks in domestic birds. This case emphasizes the need for surveillance of this endangered species in its natural habitat.

Pathogenicity and Full Genome Sequencing of the Avian Influenza H9N2 Moroccan Isolate 2016.

In Morocco in early 2016, a low pathogenic avian influenza virus serotype H9N2 caused large economic losses to the poultry industry, with specific clinical symptoms and high mortality rates on infected farms. Subsequent to the H9N2 outbreak, the causal agent was successfully isolated from chicken flocks with high morbidity and mortality rates, propagated on embryonated eggs, and fully sequenced. The phylogenetic analysis suggested that the Moroccan isolate could have derived from the Middle East isolate A/chicken/Dubai/D2506.A/2015. This study was designed to assess the pathogenicity of the Moroccan isolate H9N2 in experimentally infected broiler and specific-pathogen-free (SPF) chickens. At 22 days of age, one broiler and two SPF chicken groups were inoculated by dropping 0.2 ml of the H9N2 isolate (107.5 EID50/ml) in both nostrils and eyes. Clinically inoculated chickens with H9N2 displayed mild lesions, low mortality rates, and an absence of clinical signs. The H9N2 virus was more pathogenic in broiler chickens and produced more severe tissue lesions compared to SPF chickens. The viral shedding was detected up to 6 days postinoculation (pi) in oropharyngeal and cloacal swabs in infected birds with a maximum shedding in the oropharynges of the broiler group. All experimental chickens seroconverted and registered high hemagglutination inhibition titers as early as day 7 pi. The present study indicates that the H9N2 virus isolated from a natural outbreak was of low pathogenicity under experimental conditions. However, under field conditions infection with other pathogens might have aggravated the disease.

Estudio de patogenicidad y secuenciación del genoma completo del aislamiento de virus de la influenza aviar H9N2 de Marruecos del año 2016. En Marruecos, a principios de año 2016, el serotipo H9N2 del virus de la influenza aviar de baja patogenicidad (LPAIV) causó grandes pérdidas económicas en la industria avícola, con signos clínicos específicos y altas tasas de mortalidad en las granjas infectadas. Posterior al brote de H9N2, el agente causal se aisló con éxito de parvadas de pollos con altas tasas de morbilidad y mortalidad, se propagó en huevos embrionados y se secuenció completamente. El análisis filogenético sugirió que el aislado marroquí podría haberse derivado del aislamiento de Medio Oriente (A/pollo/Dubai/D2506.A/2015). Este estudio se diseñó para evaluar la patogenicidad del aislado marroquí H9N2 en pollos de engorde infectados experimentalmente y en pollos libres de patógenos específicos (SPF). A los 22 días de edad, un grupo de pollos de engorde y dos grupos de aves libres de patógenos específicos se inocularon mediante la instilación de 0.2 ml del aislamiento H9N2 (107.5 dosis infectantes de embrión de pollo 50% [EID50] por ml) en ambas fosas nasales y en los ojos. Los pollos clínicamente inoculados con el virus subtipo H9N2 mostraron lesiones leves, bajas tasas de mortalidad y ausencia de signos clínicos. El virus H9N2 fue más patógeno en los pollos de engorde y produjo lesiones tisulares más graves en comparación con las aves libres de patógenos específicos. La excreción viral se detectó hasta seis días después de la inoculación en frotis orofaríngeos y cloacales de aves infectadas con una excreción máxima en la orofarínge del grupo de pollos de engorde. Todos los pollos experimentales seroconvirtieron y registraron altos títulos de inhibición de hemaglutinación tan pronto como en el día siete después de la inoculación. El presente estudio indicó que el aislamiento viral H9N2 de un brote natural fue de baja patogenicidad en condiciones experimentales. Sin embargo, en condiciones de campo, la infección con otros patógenos pudo haber agravado la enfermedad.

Development and validation of a one-step real-time PCR assay for simultaneous detection of subtype H5, H7, and H9 avian influenza viruses.

Among the different hemagglutinin (HA) subtypes of avian influenza (AI) viruses, H5, H7, and H9 are of major interest because of the serious consequences for the poultry industry and the increasing frequency of direct transmission of these viruses to humans. The availability of new tools to rapidly detect and subtype the influenza viruses can enable the immediate application of measures to prevent the widespread transmission of the infection. In this study, a novel one-step real-time reverse transcription-PCR (RRT-PCR) was developed to detect simultaneously the H5, H7, and H9 subtypes of AI viruses from clinical samples of avian origin. The sensitivity of the RRT-PCR assay was determined by using in vitro-transcribed RNA and 10-fold serial dilutions of titrated AI viruses. High sensitivity levels were obtained, with limits of detection ranging from 10(1) to 10(3) RNA copies and from 10(1) 50% egg infectious dose (EID(50))/100 microl to 10(2.74) EID(50)/100 microl with titrated viruses. Excellent results were achieved in the intra- and interassay variability tests. The comparison of the results with those obtained from the analysis of 725 avian samples by means of the reference method (virus isolation [VI]) showed a high level of agreement. To date, this is the first real-time PCR protocol available for the simultaneous detection of AI viruses belonging to subtypes H5, H7, and H9, and the results obtained indicate that this method is suitable as a routine laboratory test for the rapid detection and differentiation of the three most-important AI virus subtypes in samples of avian origin.

Conventional inactivated bivalent H5/H7 vaccine prevents viral localization in muscles of turkeys infected experimentally with low pathogenic avian influenza and highly pathogenic avian influenza H7N1 isolates.

Highly pathogenic avian influenza (HPAI) viruses cause viraemia and systemic infections with virus replication in internal organs and muscles; in contrast, low pathogenicity avian influenza (LPAI) viruses produce mild infections with low mortality rates and local virus replication. There is little available information on the ability of LPAI viruses to cause viraemia or on the presence of avian influenza viruses in general in the muscles of infected turkeys. The aim of the present study was to determine the ability of LPAI and HPAI H7N1 viruses to reach muscle tissues following experimental infection and to determine the efficacy of vaccination in preventing viraemia and meat localization. The potential of infective muscle tissue to act as a source of infection for susceptible turkeys by mimicking the practice of swill-feeding was also investigated. The HPAI virus was isolated from blood and muscle tissues of all unvaccinated turkeys; LPAI could be isolated only from blood of one bird and could be detected only by reverse transcriptase-polymerase chain reaction in muscles. In contrast, no viable virus or viral RNA could be detected in muscles of vaccinated/challenged turkeys, indicating that viral localization in muscle tissue is prevented in vaccinated birds.

Complete Genome Sequence of Psittacine Adenovirus 1, Identified from Poicephalus senegalus in Italy.

Using a metagenomics approach, we were able to determine for the first time the full-genome sequence of a psittacine adenovirus 1 isolate that was recovered from the liver of a dead Senegal parrot (Poicephalus senegalus) in Italy. The results of the phylogenetic investigations revealed the existence of high genetic diversity among adenoviruses circulating in psittacine birds.

Influenza virus surveillance in wild birds in Italy: results of laboratory investigations in 2003-2005.

Following the avian influenza (AI) epidemics occurring in different areas of the world, a surveillance program funded by the Italian Ministry of Health was implemented. In the framework of this program, an investigation of wild birds was carried out to assess the circulation of AI viruses in their natural reservoir. More than 3000 samples, mainly cloacal swabs, were collected from migratory wild birds belonging to the orders Anseriformes and Charadriiformes. Samples were screened by means of a real-time reverse transcriptase polymerase chain reaction (RRT-PCR), then processed for attempted virus isolation in embryonated fowl's specific pathogen-free eggs. Approximately 5% of the samples were positive for type A influenza viruses by RRT-PCR, and from 14 of those samples AI viruses were isolated and fully characterized. The isolates, belonging to 8 different avian influenza virus subtype combinations (H10N4, H1N1, H4N6, H7N7, H7N4, H5N1, H5N2, and H5N3), were obtained from migratory Anseriformes.