Clement Meseko

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.

Avian influenza risk perception among poultry workers, Nigeria.

The Department of Production Animal Studies, Faculty of Veterinary Science, University of Pretoria and ARC-Onderstepoort Veterinary Institute provided funding for this research.

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)

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.

Genetically Different Highly Pathogenic Avian Influenza A(H5N1) Viruses in West Africa, 2015.

To trace the evolution of highly pathogenic influenza A(H5N1) virus in West Africa, we sequenced genomes of 43 viruses collected during 2015 from poultry and wild birds in 5 countries. We found 2 co-circulating genetic groups within clade 2.3.2.1c. Mutations that may increase adaptation to mammals raise concern over possible risk for humans.

Swine influenza in sub-Saharan Africa - current knowledge and emerging insights.

Pigs have been associated with several episodes of influenza outbreaks in the past and are considered to play a significant role in the ecology of influenza virus. The recent 2009 pandemic influenza A/H1N1 virus originated from swine and not only did it cause widespread infection in humans, but was also transmitted back to swine in Asia, Europe and America. What may be the prevailing situation in Africa, particularly in sub‐Saharan Africa, with respect to the circulation of classical swine or pandemic influenza? The ecology of influenza viruses, as well as the epidemiology of human or animal influenza, is poorly understood in the region. In particular, little is known about swine influenza in Africa despite the relevance of this production in the continent and the widespread pig husbandry operations in urban and rural areas. In this review, the gap in the knowledge of classical and pandemic swine influenza is attributed to negligence of disease surveillance, as well as to the economic and public health impact that the disease may cause in sub‐Saharan Africa. However, emerging serological and virological evidence of swine influenza virus in some countries in the region underscores the importance of integrated surveillance to better understand the circulation and epidemiology of swine influenza, a disease of global economic and public health importance.

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.

Evidence of exposure of domestic pigs to Highly Pathogenic Avian Influenza H5N1 in Nigeria

Avian influenza viruses (AIV) potentially transmit to swine as shown by experiments, where further reassortment may contribute to the generation of pandemic strains. Associated risks of AIV inter-species transmission are greater in countries like Nigeria with recurrent epidemics of highly pathogenic AI (HPAI) in poultry and significant pig population. Analysis of 129 tracheal swab specimens collected from apparently healthy pigs at slaughterhouse during presence of HPAI virus H5N1 in poultry in Nigeria for influenza A by RT-qPCR yielded 43 positive samples. Twenty-two could be determined by clade specific RT-qPCR as belonging to the H5N1 clade 2.3.2.1c and confirmed by partial hemagglutinin (HA) sequence analysis. In addition, 500 swine sera were screened for antibodies against influenza A virus nucleoprotein and H5 HA using competition ELISAs and hemagglutination inhibition (HI) tests. Serologically, 222 (44.4%) and 42 (8.4%) sera were positive for influenza A virus NP and H5 antibodies, respectively. Sera reacted to H5N1 and A/H1N1pdm09 strains by HI suggesting exposure of the Nigerian domestic pig population to these viruses. We report for the first time in Nigeria, exposure of domestic pigs to H5N1 virus. This poses potential public health and pandemic risk due to interspecies transmission of avian and human influenza viruses.

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.

Armillifer armillatus infestation in Human; public health scenario of a snake parasite: a report of three cases

We report cases of Armillifer Armillatus infestation in three Nigerian adults within two and half years in our health facility. The first patient was a 70 year old farmer and a regular consumer of snake meat for over 50 years. He presented in February, 2014 for follow-up visit as he was a known systemic hypertensive patient. He was incidentally discovered to have multiple comma-shaped calcific lesions in the lungs and liver on a chest radiograph. These were better demonstrated on abdominal ultrasound and computed tomographic scans. He was asymptomatic. The second patient was a 42 year old male civil servant who presented in December 2015 with dry cough and right loin pain for five and three days respectively. His past medical history revealed that he had been treated previously for pneumonia. He has never eaten snake meat but consumed Alligator (Amphibious reptile) for many years but stopped about 12 years ago. Similar calcific lesions were also noted in his liver and lung parenchyma on chest radiograph and abdominal ultrasound scan. The third patient was an 80 year old man who presented in April, 2014 with dizziness and diminished urine output of one day duration. He was a farmer who has been consuming snake meat for many years, and has been on management for systemic arterial hypertension and prostatic hypertrophy. Chest radiograph and abdomino-pelvic ultrasound incidentally revealed multiple comma-shaped calcific lesions in the lungs and liver. The liver function test parameters were all within normal limits but the electrolytes were deranged and he was anaemic with a Packed Cell Volume of 27%. A diagnosis of Armillifer Armillatus infestation was made in these patients, and they were conservatively managed with Mebendazole. The third case was catherized and the deranged electrolytes were corrected. The first patient was lost to follow-up, whiles the second and third had no remarkable symptoms on subsequent follow-up visits.