An epidemiological survey of Newcastle disease virus in South Africa

English:The primary objective in this study was the investigation of the molecular epidemiology of NDV strains isolated during epizooties in South Africa in 1998. Isolates purported to be NDV were collected from the Onderstepoort Veterinary Institute, the University of Pretoria, and EarlyBird Farms, Standerton. Four of the twenty-two isolates collected were identified as NDV, and successfully grouped into genotype VIIb, previously described by Herezeg et al., (1999). The isolates were propagated in 9-10 day-old embryonated SPF eggs. Embryo mortality was 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 a reaction mixture containing IIlM random hexamers p(dN)6, 200 U Moloney Murine Leukemia (M-ML V) RT and 25 U RNasin. Aliquots of 5 ul, of the cDNA mix were used as template in subsequent PCR amplification reactions. A 1349bp region of the fusion protein gene was amplified using primers ONDVlaa and ONDV 4aa. Fusion protein amplicons were obtained from field isolates, M89/98 (Ost Pool I), M89/98 (Av Pool 2), M57/98, and M308/98 obtained from OP. Restriction enzyme 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 digestion of the amplicon of strain M308/98 with Hinf I released fragments not consistent with those observed in genotype VIIb isolates. These fragments suggest the presence of mutations in this area of the genome. Amplification of a region comprising 88% of the matrix protein gene was facilitated using primers Ml and M2. These amplicons were subjected to RE analysis using restriction enzymes Mbt) I and Hinf I. Analysis of the restriction profiles produced revealed that these four strains were not re-isolated forms of the commonly used live vaccine LaSota strain. A region of the fusion protein gene encoding the fusion protein cleavage activation site was amplified by means of a nested peR, and sequenced. Initial peR amplified a region spanning from the M gene nt 778 to F gene nt 545 using primers Kl and K2. These amplicons were purified and used as template in a nested peR using primers MV1 (M gene nt 1163) and B2 (F gene nt 470). Sequence analysis revealed that the amino acid sequence at the fusion protein cleavage site of strains M89/98 (Ost Pool I), M57/98,and M308/98 was RRQKR -l-F, indicative of velogenie strains. Strain M89/98 (Av Pool 2) displayed a sequence at the fusion protein cleavage site that is characteristic of lentogenie strains GRQGR-l-L. This finding suggests that genotype VIIb isolates are heterogeneous, composed of strains of varying pathogenicity . Phylogenetic analysis based on a 378nt long region of the nested peR amplicon allowed the grouping of all these isolates into genotype VIIb. A 1.5-1% divergence was observed between 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 strains per year. The remaining field strains not detected by RT-peR, but that displayed embryo mortality indicative of pathogenic agents were subjected to HAlHI tests. Strains M193/98 (Ost Pool 6), M183/98 (Ost Liv B), and M193/98 (Ost Pool 5) agglutinated chicken erythrocytes. Haemagglutination was not inhibited by anti-NDV serum. These results led to the suspicion of the presence of an unidentified pathogen, most likely avian influenza, as all three samples were collected from ostrich hosts, from which this virus has previously been isolated in South Africa.