MOLECULAR AND ANTIGENIC CHARACTERISATION OF INFLUENZA VIRUSES ISOLATED FROM HORSES IN NIGERIA

Abstract

A nationwide equine influenza virus surveillance was carried out between January 1989 and January 1995 during which nasopharyngeal swabs, tracheal washes and respiratory tract tissue homogenates were obtained from symptomatic and asymptomatic equine species in various parts of Nigeria for virus isolation and characterisation. Three equine influenza viruses were independently isolated in 10-11 day old embryonated hen eggs and Madin Darby canine kidney monolayer cells with TPCK-trypsin incorporated into the culture medium. The virus isolates were characterized antigenically, immunogenically and genetically which made them the first equine influenza viruses isolated and characterized in tropical Africa. Antigenic analyses with mono-specific antisera raised against a number of respiratory disease viruses including Paramyxoviruses, Adenoviruses, Herpes viruses, Alphavirus, Rhinoviruses, Equine arteritis viruses, African horse sickness viruses and Influenza viruses showed that the three virus isolates were influenza A viruses while haemagglutination and neuraminidase inhibition assays using influenza virus specific Mabs and polyclonal antisera showed the three viruses to be antigenically H3N8, members of equine-2 subtype of influenza A viruses. The three isolates were respectively named A/Equine/Ibadan/4/91, A/Equine/Ibadan/6/91 and A/Equine/Ibadan/9/91 in accordance with the recommendations of the World Health Organisation and the International Committee on Virus Taxonomy. Their antigenic cross-reactivity with panels of Mabs and polyclonal antisera indicated that the three viruses were antigenically divergent although they were all H3N8 viruses and were representatives of a homogenous population. They were reactive with Mabs and antisera directed against H3 equine viruses isolated in 1963, 1976, 1979, 1981 and 1986. This broad reactivity suggested that these isolates were similar to but not still different from those isolated in Europe and USA between 1963 and 1987. Virus protein synthesis and cleavability of the HA polypeptide in tissue culture were investigated by pulse-chase experiments. The results showed some heterogeneity in the non-glycosylated polypeptides particularly those of the ribonucleoprotein (RNP) complex while the HA glycoproteins of the three viruses were not cleaved in any of the cell types used in contrast to equine H7 and pathogenic avian H5 and H7 HAs. The molecular weights of the polypeptides were within the range previously determined for influenza A viruses. The heterogeneity of the RNP complex and antigenic divergence of the viruses’ HAs were confirmed by sequence analysis carried out in molecular studies. Virus infectivity was investigated by plaque assay using chicken embryo fibroblast (CEF) monolayer cells and virus titration in embryonated hen eggs. The results showed that the viruses were infectious with plaguing efficiency being comparable to efficiency of virus infectivity in embryonated hen eggs. Infectivity, antigenicity and immunogenicity of the three viruses were confirmed in-vitro using peripheral blood lymphocytes in lymphoproliferation assays and in-vivo in an equine model in which the viruses induced haemagglutination inhibiting and protective neutralising antibodies following experimental infections. The proliferating cells were also characterised and the immunoglobulin isotypes produced were determined. Molecular characterisation and genetic analyses of the three viruses were accomplished to determine the origin of the genes encoding the virus non-glycosylated polypeptides as well as those of the surface HA glycoprotein. Reverse transcription (RT) results showed the eight RNA segments of the three isolates and confirmed them as influenza A virus RNAs. No subgenomic RNAs or defective interfering particles were observed in the RNA transcripts. Nucleotide sequence analyses were carried out using three sequencing strategies of cDNA, vRNA and plasmid DNA sequencing with the dideoxy chain termination procedure. Partial nucleotide sequences were obtained from cDNAs, vRNAs and plasmid DNA clones of RNA segments 1, 2, 3, 5, 7 and 8 with the method of multiplex RT/PCR and cycle sequencing using radiolabelled segment specific oigonucleotides of 18, 24 or 25 mers. Complete nucleotide sequences of segment 4 (HA genes) were also determined on the same PCR products (cDNA) and vRNA using end-labelled oligonucleotides of both plus and minus sense. The partial nucleotide sequence data were analysed using a programme for "best-local-homology- rapid-search" on a digital array processor while the complete HA nucleotide and deduced amino acid sequence analyses were carried out using the University of Wisconsin Genetics Computer Group (GCG) package of programmes. Phytogenetic analysis was done with the distances, neighbour joining and DNAPARS of the PHYLIP package. Analyses of the viruses’ gene sequences confirmed that their genomes were similar to each other and to those of other H3N8 influenza viruses isolated from equines and also revealed the origin, evolution and genetic relatedness of the genes. Comparison of the partial cDNA sequences with virus DNA sequences in the database (EMBL sequence library) showed that for segments 1, 2, 5, and 7, the closest related sequences were from equine H3 viruses isolated in 1986 in USA (Tennesse/5/86 for segments 2, 5 and 7, Kentucky/2/86 for segments 1 and 5. Segment 5 was equally related to both viruses). The nucleotide sequence for segment 3 was most closely related to an equine-1 virus isolated in U.K. in 1973 (London/1416/73, H7N7) probably due to genetic exchange while segment 8 sequence was most closely related to an equine H3N8 virus isolated in U.K. in 1976 (Newmarket/76). The complete nucleotide and deduced amino acid sequences as well as phytogenetic analysis of the HA genes (RNA segment 4) showed a closer relationship albeit with nucleotide and amino acid substitutions between the three Ibadan viruses and those that were isolated in Europe in 1989 and 1991, the prototypic European strain, Suffolk/89 and Arundel/12369/91 isolated in U.K., Taby/91 isolated in Sweden, Hong Kong/92 isolated in the Far East in 1992 and Laplata/1/93 isolated in South America. These findings group the Ibadan viruses with those predominating and contemporarily causing disease in the Western Hemisphere rather than with viruses previously isolated from the north and south of African continent. Variation was observed in the nucleotide sequences of the Ibadan viruses HA genes. Some of the base changes resulted in amino acid changes which mapped to antigenic sites or within signal sequence in the HA1 domain as a result of a process of antigenic or genetic drift. The Ibadan viruses also showed some variation from the prototypic European virus (Suffolk/89) and these base changes also resulted in amino acid changes resulting in antigenic drift. Phytogenetic analysis showed the evolutionary lineages in equine H3 viruses isolated since 1963 along two paths one of which included the Ibadan viruses as well as viruses isolated in Europe between 1989 and 1991 and the Far East in 1992 and S. America in 1993 which form the 1989/93 cluster while the other lineage included viruses isolated in South America in 1987 and 1988 (Brazil/87, Laplata/88) and in the Far East in 1971 (Tokyo/3/71) all of which are very close to the original prototype equine-2 virus (Miami/63). These results demonstrate a faster evolutionary rate for recent equine H3 HA genes away from the original prototype virus. Overall, the results of these studies have (i) confirmed the occurrence of equine-2 H3N8 influenza viruses of distinct lineages in Nigerian equine populations in a tropical environment (ii) indicated antigenic drift among equine H3N8 viruses as earlier reported and confirmed that drift strains can co-circulate in equine populations, (iii) showed the origin, evolution and genetic relatedness of the viruses genes as well as their biological characteristics, (iv) provided the hitherto unavailable information on the status of equine influenza virus in this part of the world and (v) served to re-emphasise the potential of influenza virus for rapid global spread and the need for better control strategies.