Ismaila Shittu

Evolutionary Dynamics of Multiple Sublineages of H5N1 Influenza Viruses in Nigeria from 2006 to 2008

ABSTRACT 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.

Field and laboratory findings of the first incursion of the Asian H5N1 highly pathogenic avian influenza virus in Africa

In mid-January 2006 an outbreak of avian influenza in domestic birds was recorded in Kaduna State, Nigeria. The virus responsible for the outbreak was characterized as a highly pathogenic avian influenza H5N1 (A/chicken/Nigeria/641/06), belonging to the Qinghai lineage. The index case occurred on a farm containing mixed avian species including free-range birds. Clinical signs and mortality were observed in chickens, geese and ostriches. The present paper describes the clinical, pathological and virological findings of this outbreak.

Highly Pathogenic Avian Influenza A(H5N1) Virus in Poultry, Nigeria, 2015.

To the Editor: In Nigeria, from February 2006 through July 2008, outbreaks of highly pathogenic avian influenza (HPAI) subtype H5N1 virus infection in poultry negatively affected animal and public health as well as the agricultural sector and trade. These outbreaks were caused by viruses belonging to genetic clades 2.2 and 2.2.1 (1). In January 2015, seven years after disappearance of the virus, clinical signs of HPAI (swollen head and wattles, hemorrhagic shank and feet) and increased mortality rates were observed among backyard poultry in Kano and in a live bird market in Lagos State, Nigeria. The virus was isolated from 2 samples independently collected from the poultry farm (parenchymatous tissues) and the market (tracheal swab), and H5 subtype virus was identified by reverse transcription PCR. The samples were adsorbed onto 2 Flinders Technology Associates cards (GE Healthcare Life Sciences, Little Chalfont, UK), which were sent to the World Organisation for Animal Health/Food and Agriculture Organization of the United Nations Reference Laboratory for Avian Influenza in Italy for subtype confirmation and genetic characterization. Influenza A(H5N1) virus was detected in both samples, and sequencing of the hemagglutinin (HA) gene showed that the viruses possessed the molecular markers for HPAI viruses with a multibasic amino acid cleavage site motif (PQRERRRKR*G). The complete genome of the virus from backyard poultry was successfully sequenced from the genetic material extracted from the Flinders Technology Associates cards by using an Illumina MiSeq platform (2) and was submitted to the Global Initiative on Sharing All Influenza Data database (http://platform.gisaid.org/) under accession nos. EPI556504 and EPI567299–EPI567305. Maximum-likelihood trees were estimated for all 8 gene segments by using the best-fit general time reversible plus invariant sites plus gamma 4 model of nucleotide substitution with PhyML (3). The topology of the phylogenetic tree of the HA gene demonstrated that the H5N1 virus from Nigeria (A/chicken/Nigeria/15VIR339-2/2015) falls within genetic clade 2.3.2.1c (Figure, panel A). In particular, the HA gene sequence clustered with H5 viruses collected in China in 2013 and with an H5N1 virus (A/Alberta/01/2014) isolated from a Canada resident who had returned from China (similarity 99.3%–99.5%) (4). The remaining 7 genes were closely related to genes of A/Alberta/01/2014(H5N1), although the 2 viruses differed by 32 aa (Technical Appendix). Just as for the virus from Canada (4), 7 of 8 gene segments of the virus from Nigeria clustered with HPAI A(H5N1) virus circulating in Vietnam and China, while the polymerase basic 2 gene segment (Figure, panel B) resulted from reassortment with viruses circulating in the same Asian countries but belonged to the H9N2 subtype. Differing from the strain from Canada (only 2 aa mutations compared with the 2.3.2.1c candidate vaccine strain; 5), the strain from Nigeria possesses 6 aa differences: 3 in HA1 and 3 in HA2 (Technical Appendix). The effect of these mutations on the antigenic relatedness of these strains should be further explored. Figure Maximum likelihood phylogenetic trees of the A) hemagglutinin and B) polymerase basic 2 gene segments of highly pathogenic avian influenza A(H5N1) virus from poultry in Nigeria, 2015 (in red). Bootstrap values (100 replicates) >60 are shown at ... Molecular characterization demonstrated that the polymerase basic 2 sequence contains glutamic acid at position 627, establishing the lack of a well-known mammalian adaptation motif (6). Mutations associated with increased virulence in mice have been observed in the nonstructural protein 1 (P42S, D87E, L98F, I101M, and the 80–84 deletion) and in the matrix 1 proteins (N30D, T215A). In addition, the substitutions D94N, S133A, S155N (H5 numbering) associated with increased binding to α-2,6 sialic acid have been identified in the HA protein. However, most of these substitutions are present in the H5N1 virus sequences from Asia included in our phylogenetic analyses, suggesting that they may be common among the HPAI H5 virus subtype. Mutations associated with resistance to antiviral drugs have not been detected (7). The results obtained from whole-genome analysis provide evidence that a novel clade of the A(H5N1) virus, specifically clade 2.3.2.1c, has reached Nigeria. Although ascertaining how and exactly when this has happened is difficult, it seems most likely that the virus entered the country in December 2014, as evidenced by unverified accounts of increased poultry deaths in some live bird markets in Lagos, after the birds had been moved from the north (Kano) to the south during the festive season. The identification of genetic clustering between the strains from Nigeria analyzed here and the HPAI A(H5N1) viruses originally identified in Asia suggests an unknown epidemiologic link between these regions, probably associated with human activities, migratory bird movements, or both. Considering that this virus is an intersubtype reassortant and has already caused infection in humans, we believe that complete characterization of the strain in terms of virulence and host range is of high priority. Furthermore, because the reemergence of subtype H5N1 virus was followed by epidemiologic amplification (≈265 outbreaks in 18 states as of February 2015; T. Joannis, pers. comm., 2015) for which virus genetic characterization is not yet available, local veterinary and public health services and international organizations should take necessary measures to identify critical control points and stop circulation of this virus. Technical Appendix. Amino acid comparison of avian influenza viruses and sequences from the Global Initiative on Sharing All Influenza Data database. Click here to view.(221K, pdf)

Complete Genome Sequence of a Genotype XVII Newcastle Disease Virus, Isolated from an Apparently Healthy Domestic Duck in Nigeria

ABSTRACT The first complete genome sequence of a strain of Newcastle disease virus (NDV) of genotype XVII is described here. A velogenic strain (duck/Nigeria/903/KUDU-113/1992) was isolated from an apparently healthy free-roaming domestic duck sampled in Kuru, Nigeria, in 1992. Phylogenetic analysis of the fusion protein gene and complete genome classified the isolate as a member of NDV class II, genotype XVII.

Rapid antigen detection in the diagnosis of highly pathogenic avian influenza (H5N1) virus in Nigeria.

Highly pathogenic avian influenza (AI) is an infectious disease of agroeconomic and public health importance. The outbreak that occurred in Nigeria (2006-2008) was devastating to the poultry industry and raised public health concerns. In the course of its control, rapid laboratory confirmation of suspected cases in poultry was essential for prompt mobilization of control logistics for depopulation and decontamination of affected premises. Commercial rapid test kit was evaluated in the diagnosis of highly pathogenic AI (HPAI) as a preliminary to virus isolation. Between 2006 and 2007, 382 cases were tested out of which 149 were positive by rapid antigen detection. Virus isolation yielded 171 positive cases. The relative diagnostic sensitivity of Anigen Rapid AIV Ag test was 84.3% (95% confidence interval [CI], 78.1-88.9%), whereas the relative diagnostic specificity was 97.7% (95% CI, 94.2-99.1%). Rapid antigen detection is a useful technique for prompt diagnosis of HPAI for early detection and containment.

Highly pathogenic avian influenza (H5N1) in Nigeria in 2015: evidence of widespread circulation of WA2 clade 2.3.2.1c

Genetic analysis of the complete haemagglutinin (HA) gene of fourteen Nigerian avian influenza isolates showed multiple basic amino acids at the cleavage site (321PQRERRRKdelR*GLF333), characteristic of highly pathogenic avian influenza (HPAI). Substitution of Gln to Lys at position 322 (H5-specific numbering) was identified in one isolate. In some isolates, amino acid substitutions were observed across the HA gene, however the receptor binding, antigenic and glycosylation sites were conserved in all. Phylogenetic analysis revealed two clusters of the HPAI H5N1 clade 2.3.2.1c. Cluster I has close genetic relatedness (97.8–99.8%) with viruses circulating in some West Africa countries. Cluster II shared close identity (98.9–100.0%) with isolates from Europe, Côte d’Ivoire and Niger and viruses from this cluster were detected in five of the eleven states investigated in Nigeria. In view of the continuous HPAI outbreaks being recorded in Nigerian poultry and the zoonotic potential of the virus, extensive and continued characterization of HPAI isolates is advocated.

Identification and Complete Genome Sequence Analysis of a Genotype XIV Newcastle Disease Virus from Nigeria

ABSTRACT The first complete genome sequence of a strain of Newcastle disease virus (NDV) from genotype XIV is reported here. Strain duck/Nigeria/NG-695/KG.LOM.11-16/2009 was isolated from an apparently healthy domestic duck from a live bird market in Kogi State, Nigeria, in 2009. This strain is classified as a member of subgenotype XIVb of class II.

A two-year monitoring period of the genetic properties of clade 2.3.2.1c H5N1 viruses in Nigeria reveals the emergence and co-circulation of distinct genotypes

Phylogenetic analyses of the complete genomes of the highly pathogenic avian influenza (HPAI) 2.3.2.1c H5N1 virus strains causing outbreaks in Nigerias poultry population from 2014 to 2016 showed evidence of distinct co-circulating genotypes and the emergence of reassortant viruses. One of these reassortants became the predominant strain by 2016, and the NA protein of this strain possessed the V96A substitution known to confer reduced susceptibility to neuraminidase inhibiting antiviral drugs. Our findings also demonstrated evolutionary relationships between Nigerian isolates and European and Middle Eastern strains of H5N1 which provides further evidence for the proposed role of migratory birds in spreading the virus, although the involvement of the live poultry trade cannot be excluded. Efforts must be directed towards improving biosecurity and gaining the cooperation of poultry farmers for more effective control of HPAI, in order to mitigate the emergence of HPAI strains in Nigeria with biological properties that are potentially even more dangerous to animals and humans.

Newcastle disease in Nigeria: epizootiology and current knowledge of circulating genotypes

Over the years, Newcastle disease (ND) has defied all available control measures. The disease has remained at the forefront of infectious diseases afflicting poultry production after avian influenza. Despite the continuous global use of million doses of ND vaccine annually, the causative pathogen, avian paramyxovirus type 1 also known as Newcastle disease virus (NDV) has continued to evolve causing, even more, a threat not only to the unvaccinated but the vaccinated flocks inclusive. The disease has been well studied in the developed countries where the virus is found in circulation. However, limited information exists on the epizootiology and circulating genotypes of the virus in developing countries where the majority of the flocks are raised on the extensive management system. Identification of virulent NDV in apparently healthy free-range ducks in this system calls for concern and pragmatic approach to investigate factor(s) that favour the virus inhabiting the ducks without clinical manifestation of the disease. Recently, novel genotypes (XIV, XVII, and XVIII) with peculiarity to West and Central African countries have been discovered and due to lack or poor surveillance system possibility of hitherto unreported genotypes are likely. This review elucidates and discusses available literature on the diversity of the circulating NDV genotypes across the West Africa countries and the epizootiology (molecular) of the disease in Nigeria with the view of identifying gaps in knowledge that can assist in the development of effective vaccines and control strategies to combat the peril of the disease.

Newcastle Disease Virus Infection in Quail

Newcastle disease (ND), caused by virulent strains of Newcastle disease virus (NDV), is a devastating disease of poultry worldwide. The pathogenesis of ND in quail is poorly documented. To characterize the ability of virulent NDV strains to replicate and cause disease in quail, groups of 14 two-week-old Japanese quail (Coturnix japonica) were experimentally inoculated with 108 EID50 (embryo infectious dose 50%) units of 1 of 4 virulent NDV strains: 2 isolated from quail (N2, N23) and 2 from chickens (Israel, Pakistan). At day 2 postinfection, noninfected quail (contact group) were added to each infection group to assess the efficacy of virus transmission. Tested NDV strains showed moderate pathogenicity, with highest mortality being 28% for the N2 strain and below 10% for the others. Two N2-inoculated birds showed neurological signs, such as head tremor and ataxia. Microscopic lesions were present in N2-, Israel-, and Pakistan-inoculated birds and consisted of nonsuppurative encephalitis. Contact birds showed no clinical signs or lesions. In both inoculated and contact birds, virus replication was moderate to minimal, respectively, as observed by immunohistochemistry in tissues and virus isolation from oropharyngeal and cloacal swabs. Strains originally isolated from quail resulted in higher numbers of birds shedding in the inoculation group; however, transmission appeared slightly more efficient with chicken-derived isolates. This study shows that virulent NDV strains have limited replicative potential and mild to moderate disease-inducing ability in Japanese quail.