An epidemiological survey of Newcastle disease virus in South Africa

[摘要] English:The primary objective in this study was the investigation of the molecular epidemiologyof NDV strains isolated during epizooties in South Africa in 1998. Isolates purported tobe NDV were collected from the Onderstepoort Veterinary Institute, the University ofPretoria, and EarlyBird Farms, Standerton. Four of the twenty-two isolates collectedwere identified as NDV, and successfully grouped into genotype VIIb, previouslydescribed by Herezeg et al., (1999).The isolates were propagated in 9-10 day-old embryonated SPF eggs. Embryo mortalitywas observed, allantoic fluid harvested, and genomic RNA extracted using TRIZOL ®reagent, containing guanidine thiocyanate.An RT-PCR based detection method was used to screen the isolates received (Ballagi-Pordány et al., 1996). The optimised method entailed initial reverse transcription in areaction mixture containing IIlM random hexamers p(dN)6, 200 U Moloney MurineLeukemia (M-ML V) RT and 25 U RNasin. Aliquots of 5 ul, of the cDNA mix wereused as template in subsequent PCR amplification reactions.A 1349bp region of the fusion protein gene was amplified using primers ONDVlaa andONDV 4aa. Fusion protein amplicons were obtained from field isolates, M89/98 (OstPool I), M89/98 (Av Pool 2), M57/98, and M308/98 obtained from OP. Restrictionenzyme profiles of the fusion protein amplicons using restriction enzymes HinfI, BstO I,and Rsa I displayed fragment patterns that allowed their grouping into genotype VIIb,described by Herezeg et al., (1999). Non-group specific bands observed upon digestionof the amplicon of strain M308/98 with Hinf I released fragments not consistent withthose observed in genotype VIIb isolates. These fragments suggest the presence ofmutations in this area of the genome.Amplification of a region comprising 88% of the matrix protein gene was facilitatedusing primers Ml and M2. These amplicons were subjected to RE analysis usingrestriction enzymes Mbt) I and Hinf I. Analysis of the restriction profiles producedrevealed that these four strains were not re-isolated forms of the commonly used livevaccine LaSota strain.A region of the fusion protein gene encoding the fusion protein cleavage activation sitewas amplified by means of a nested peR, and sequenced. Initial peR amplified a regionspanning from the M gene nt 778 to F gene nt 545 using primers Kl and K2. Theseamplicons were purified and used as template in a nested peR using primers MV1 (Mgene nt 1163) and B2 (F gene nt 470).Sequence analysis revealed that the amino acid sequence at the fusion protein cleavagesite of strains M89/98 (Ost Pool I), M57/98,and M308/98 was RRQKR -l-F, indicative ofvelogenie strains. Strain M89/98 (Av Pool 2) displayed a sequence at the fusion proteincleavage site that is characteristic of lentogenie strains GRQGR-l-L. This findingsuggests that genotype VIIb isolates are heterogeneous, composed of strains of varyingpathogenicity .Phylogenetic analysis based on a 378nt long region of the nested peR amplicon allowedthe grouping of all these isolates into genotype VIIb. A 1.5-1% divergence was observedbetween group VIIb isolates collected in 1993, and 1995, and those used in this study,collected in 1998. This genetic distance is consistent with a 0.5-1% divergence in strainsper year.The remaining field strains not detected by RT-peR, but that displayed embryo mortalityindicative of pathogenic agents were subjected to HAlHI tests. Strains M193/98 (OstPool 6), M183/98 (Ost Liv B), and M193/98 (Ost Pool 5) agglutinated chickenerythrocytes. Haemagglutination was not inhibited by anti-NDV serum. These resultsled to the suspicion of the presence of an unidentified pathogen, most likely avianinfluenza, as all three samples were collected from ostrich hosts, from which this virushas previously been isolated in South Africa.

[发布机构] University of the Free State