Characterization of avipoxviruses from chickens and domestic pigeons in Tanzania

Abstract

Avipoxviruses (APVs) are double stranded DNA viruses belonging to the family Poxviridae, sub-family Chordopoxvirinae and genus Avipoxvirus. APVs cause pox in birds; in chickens the disease is known as fowl pox, caused by Fowlpox virus (FWPV). In the recent years the incidence and prevalence of fowl pox in chickens has increased in Tanzania, characterized by high mortalities of chicks and growers. The main research question among researchers has been “are there variant strains of FWPV or other APVs circulating in domestic birds that pose a threat to chickens in Tanzania?’’ The general objective of this study was to investigate the characteristics of APVs from chickens and domestic pigeons in Tanzania, focusing on development of appropriate fowl pox vaccine. The specific objectives were to determine genetic characteristics of APV isolates from chickens and domestic pigeons in Tanzania, to investigate integration of Reticuloendotheliosis virus (REV) genes in the Tanzanian FWPV field isolates and the imported commercial FWPV vaccines currently used in the country, and to evaluate virulence characteristics of Tanzanian strains of FWPV and pigeonpox virus (PGPV) in chickens. Samples of cutaneous nodular lesions were collected from chickens (n = 154) and pigeons (n = 17) suspected to have pox, followed by virus isolation. Genetic characterization involved DNA extraction; polymerase chain reaction (PCR) amplification of FWPV and PGPV P4b gene, REV envelope (env) gene and REV 5' long terminal repeat (LTR); gel electrophoresis of PCR products; purification of PCR products; sequencing of purified PCR products and analysis of the sequence data using standard procedures. Biological characteristics, particularly virulence characteristics of Tanzanian strain(s) of FWPV and PGPV in chickens, were investigated by inoculating 10-day old embryonated chicken eggs and susceptible chickens with either REV-integrated Tanzanian FWPV strain, REV-free Tanzanian FWPV strain, or Tanzanian PGPV strain. Sixty six (66) out of the 154 samples (42.86%) analyzed for the presence of FWPV were found to contain FWPV, indicating that the 66 chickens from which the samples were collected had fowl pox as a result of FWPV infection. Analysis of sequences of the P4b gene (open reading frame [ORF] 167) revealed that the Tanzanian FWPV isolates were 99.65 – 100% identical to each other and 99 – 100% identical to several published sequences of FWPV isolates from various countries in different continents of the world, including Europe and Asia. Phylogenetic analysis revealed that all Tanzanian FWPV isolates belong to clade A subclade A1. This implies that based on sequences of the P4b gene the FWPVs currently prevalent in Tanzania are genetically and phylogenetically closely related. However, analysis of selected FWPV isolates in other genomic regions (between ORFs 201 and 203) revealed integration of various genomic fragments of REV in the genome of FWPV. Out of 55 field isolates of FWPV analyzed for integration of REV env gene and REV 5'LTR, 96.36% (n = 53) were found to have genomic fragments of REV. Most of them, 69.09% (n = 38) contained REV env gene and REV 5'LTR fragments, 18.18% (n = 10) contained fragments of REV env gene only, and 9.09% (n = 5) contained fragments of REV 5'LTR only. Two isolates (3.64%) were found not to be integrated with either REV env gene or REV 5'LTR. None of the screened vaccine strains from the imported commercial fowlpox virus vaccines was found to be integrated with REV env gene and/or REV 5'LTR. Analysis of sequences of a PCR product with fragment size 807 bp showed 95 – 100% identity to sequences of several REV env gene obtained in the GenBank and 100% identity to sequence of env gene of REV provirus integrated in a FWPV isolate. The sequence of a PCR product with fragment size 370 bp showed 88 – 99% identity to sequences of several REV LTR obtained in the GenBank, and 99 – 100% identity to sequences of LTR of REV provirus integrated in several FWPV isolates from other countries. Of the 17 samples of cutaneous nodular lesions collected from pigeons, two (both from Morogoro region) were found to contain PGPV. Sequence analysis revealed that the Tanzanian PGPV isolate derived from this study was 90 - 99% identical to several APV isolates from birds belonging to different species from several countries. The Tanzanian PGPV isolate showed 91% identity to each of the Tanzanian FWPV isolates, also derived from the present study, and 99% identity to all three PGPV isolates obtained in the GenBank. Phylogenetic analysis revealed that the Tanzanian PGPV isolate belongs to clade A, subclade A2, sharing a recent common ancestor with APVs belonging to subclade A3. Biological characterization revealed that unlike a PGPV isolate from a Norwegian wild pigeon, Palumbus palumbus, that could infect and cause pox in chickens; the Tanzanian strain of PGPV can infect but does not cause pox in chickens. The study also revealed high mortality rate (57%) in chickens inoculated with REV-integrated Tanzanian FWPV strains as compared to zero mortality observed in chickens inoculated with REV-free Tanzanian FWPV strains, or a Tanzanian PGPV strain currently circulating in Morogoro region, and chickens in the control group. Based on the findings from this study the following conclusions are drawn: (a) Currently there is a heterogeneous population of FWPV in Tanzania comprising of REV-integrated FWPV strains and REV-free FWPV strains. (b) REV-integrated FWPV strains are more virulent in susceptible chickens than REV-free FWPV strains. (c) The increased incidences and prevalence of fowl pox currently experienced in Tanzania, characterized with high mortality rates of chicks and growers, could be attributed to the emergence of variant strains of FWPV which are REV-integrated. (d) The imported commercial fowl pox vaccines currently used in Tanzania are not contaminated with REV, therefore the vaccines can safely continue to be used in the country. (e) Integration of genomic fragments of REV in the genome of field strains of FWPV currently prevalent in Tanzania is not attributed to imported commercial fowl pox virus vaccines currently used in the country. It could be attributed to recombination of field strains of REV and field strains of FWPV. (f) As opposed to a PGPV isolate from a Norwegian wild pigeon, Palumbus palumbus, that could infect and cause pox in chickens; this study has revealed that the Tanzanian strain of PGPV currently circulating in Morogoro region is not pathogenic in chickens, therefore it does not pose a threat to chickens in the country. On basis of the findings from this study the following future studies are recommended: (a) More studies aiming at detection and characterization of PGPV isolates from other regions and geographical locations of Tanzania should be conducted in order to establish genetic and antigenic characteristics of PGPV currently circulating in the country. This recommendation is based on the fact that in the present study only two isolates of PGPV could be obtained from pigeons in Morogoro region, Eastern Tanzania. (b) Studies to determine pathogenicity and lethality of PGPV and FWPV in different host systems are required. (c) Epidemiological features and risk factors for FWPV and PGPV transmission ability and spread should be investigated. (d) Evolutionary characteristics of FWPV, PGPV and REV should be systematically studied to unravel possible factors that could be linked with their genetic and antigenic diversity.