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.

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.

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.

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.

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.

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 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.

Vaccine immune pressure influences viral population complexity of avian influenza virus during infection.

Vaccines are useful tools to control influenza A virus infection in poultry, but they need to be periodically reformulated to guarantee appropriate protection from infection and to limit viral replication and circulation, which could favour the emergence of new variants. In this study, a deep sequencing approach was used to characterize and follow the evolution of the hemagglutinin of the H5N1 highly pathogenic avian influenza viral population in infected animals vaccinated with two vaccines conferring different protection levels. Results from this preliminary investigation suggested that the evolution of the viral population, as well as the abundance and heterogeneity of minority variants could be influenced by the immune pressure conferred by vaccination.

Circulation of multiple genotypes of H1N2 viruses in a swine farm in Italy over a two-month period.

In August 2012 repeated respiratory outbreaks caused by swine influenza A virus (swIAV) were registered for a whole year in a breeding farm in northeast Italy that supplied piglets for fattening. The virus, initially characterized in the farm, was a reassortant Eurasian avian-like H1N1 (H1avN1) genotype, containing a haemagglutinin segment derived from the pandemic H1N1 (A(H1N1)pdm09) lineage. To control infection, a vaccination program using vaccines against the A(H1N1)pdm09, human-like H1N2 (H1huN2), human-like H3N2 (H3N2), and H1avN1 viruses was implemented in sows in November 2013. Vaccine efficacy was assessed by sampling nasal swabs for two months in 35-75 day-old piglets born from vaccinated sows. Complete genome sequencing of eight swIAV-positive nasal swabs collected longitudinally from piglets after the implementation of the vaccination program was conducted to investigate the virus characteristics. Over the two-month period, two different genotypes involving multiple reassortment events were detected. The unexpected circulation of multiple reassortant genotypes in such a short time highlights the complexity of the genetic diversity of swIAV and the need for a better surveillance plan, based on the combination of clinical signs, epidemiological data and whole genome characterization.

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.

Spillback transmission of European H1N1 avian-like swine influenza viruses to turkeys: A strain-dependent possibility?

In 1979, an avian influenza virus of the H1N1 subtype began to circulate in European swine herds, rapidly replacing classical swine H1N1 viruses. Spill-back transmissions to turkeys were recorded occasionally, but they might have been underreported due to the asymptomatic nature of the infection and the lack of specific surveillance. In our study, we evaluated the infectivity and transmissibility in turkeys of seven strains of H1N1 avian-like swine viruses isolated from 1979 to 2006, and compared them with their closest progenitor A/duck/Bavaria/1/77 (H1N1), to establish whether the adaptation to pigs has gradually decreased their fitness in turkeys. Our data indicate that the circulation of European H1N1 in pigs might have impaired the possibility of infecting turkeys. Nevertheless, the two swine-origin strains, which showed the ability to replicate and transmit in turkeys, possess typical swine-like genetic traits, not different from the rest of the tested isolates, suggesting replication of avian-like swine H1N1 viruses in turkeys as a strain-dependent polygenic feature.

Evolutionary trajectories of two distinct avian influenza epidemics: Parallelisms and divergences.

Influenza A virus can quickly acquire genetic mutations that may be associated with increased virulence, host switching or antigenic changes. To provide new insights into the evolutionary dynamics and the adaptive strategies of distinct avian influenza lineages in response to environmental and host factors, we compared two distinct avian influenza epidemics caused by the H7N1 and H7N3 subtypes that circulated under similar epidemiological conditions, including the same domestic species reared in the same densely populated poultry area for similar periods of time. The two strains appear to have experienced largely divergent evolution: the H7N1 viruses evolved into a highly pathogenic form, while the H7N3 did not. However, a more detailed molecular and evolutionary analysis revealed several common features: (i) the independent acquisition of 32 identical mutations throughout the entire genome; (ii) the evolution and persistence of two sole genetic groups with similar genetic characteristics; (iii) a comparable pattern of amino acid variability of the HA proteins during the low pathogenic epidemics; and (iv) similar rates of nucleotide substitutions. These findings suggest that the evolutionary trajectories of viruses with the same virulence level circulating in analogous epidemiological conditions may be similar. In addition, our deep sequencing analysis of 15 samples revealed that 17 of the 32 parallel mutations were already present at the beginning of the two epidemics, suggesting that fixation of these mutations may occur with different mechanisms, which may depend on the fitness gain provided by each mutation. This highlighted the difficulties in predicting the acquisition of mutations that can be correlated to viral adaptation to specific epidemiological conditions or to changes in virus virulence.

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.

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.

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.

Occurrence and identification of risk areas of Ixodes ricinus-borne pathogens: a cost-effectiveness analysis in north-eastern Italy.

BACKGROUND: Ixodes ricinus, a competent vector of several pathogens, is the tick species most frequently reported to bite humans in Europe. The majority of human cases of Lyme borreliosis (LB) and tick-borne encephalitis (TBE) occur in the north-eastern region of Italy. The aims of this study were to detect the occurrence of endemic and emergent pathogens in north-eastern Italy using adult tick screening, and to identify areas at risk of pathogen transmission. Based on our results, different strategies for tick collection and pathogen screening and their relative costs were evaluated and discussed. METHODS: From 2006 to 2008 adult ticks were collected in 31 sites and molecularly screened for the detection of pathogens previously reported in the same area (i.e., LB agents, TBE virus, Anaplasma phagocytophilum, Rickettsia spp., Babesia spp., "Candidatus Neoehrlichia mikurensis"). Based on the results of this survey, three sampling strategies were evaluated a-posteriori, and the impact of each strategy on the final results and the overall cost reductions were analyzed. The strategies were as follows: tick collection throughout the year and testing of female ticks only (strategy A); collection from April to June and testing of all adult ticks (strategy B); collection from April to June and testing of female ticks only (strategy C). RESULTS: Eleven pathogens were detected in 77 out of 193 ticks collected in 14 sites. The most common microorganisms detected were Borrelia burgdorferi sensu lato (17.6%), Rickettsia helvetica (13.1%), and "Ca. N. mikurensis" (10.5%). Within the B. burgdorferi complex, four genotypes (i.e., B. valaisiana, B. garinii, B. afzelii, and B. burgdorferi sensu stricto) were found. Less prevalent pathogens included R. monacensis (3.7%), TBE virus (2.1%), A. phagocytophilum (1.5%), Bartonella spp. (1%), and Babesia EU1 (0.5%). Co-infections by more than one pathogen were diagnosed in 22% of infected ticks. The prevalences of infection assessed using the three alternative strategies were in accordance with the initial results, with 13, 11, and 10 out of 14 sites showing occurrence of at least one pathogen, respectively. The strategies A, B, and C proposed herein would allow to reduce the original costs of sampling and laboratory analyses by one third, half, and two thirds, respectively. Strategy B was demonstrated to represent the most cost-effective choice, offering a substantial reduction of costs, as well as reliable results. CONCLUSIONS: Monitoring of tick-borne diseases is expensive, particularly in areas where several zoonotic pathogens co-occur. Cost-effectiveness studies can support the choice of the best monitoring strategy, which should take into account the ecology of the area under investigation, as well as the available budget.

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.

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.

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.

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.