The identification of unknown poultry viruses through established methods

[摘要] English: The poultry industry suffers severely every year due to bacterial and viral infections. Hence, great effort has been incorporated into isolating and identifying the different microorganisms that afflict poultry with their disease. The efficiency of the vaccines provided against such infections is only as good as the research of the responsible bacterium or virus. There are cases, however, when 'unknown viruses create havoc within industry. This is mainly due to the a) virulent nature of the virus, b) frequency of infection and c) lack of knowledge about the virus. The unknown virus may be a truly novel virus or an existing virus that has mutated whilst replicating. It is thus important to refine the procedure of isolation and identification of the poultry viruses in order for outbreaks of an unknown virus to be dealt with quickly and efficiently. In this study, the isolation and passaging of two unknown virus samples as Newcastle disease virus (NDV) but failed to react with ND-specific PCR primers and did not haemagglutinate red blood cells were investigated These samples were two of a group of viruses submitted as NDV to the Veterinary Biotechnology Laboratory of the University of the Free State by the Onderstepoort Veterinary Institute. The aim of this study was to investigate into the identity of the two unknown virus samples, D1446/95 and 834/05, using various methods of identification. Procedures performed on this virus include:(presumed to be Newcastle propagation in embryonated SPF eggs and primary chicken embryo fibroblast (CEF) cell cultures; Disease Virus), purification of virus samples for ultrastructure identification (ultrastulu studies as by transmission electron microscopy (TEM) and negative staining;) serology work as per haemagglutination and mean death time (MDT) studies as well as restriction transcriptase polymerase chain reaction (RT-PCR) work. The virus samples were cultivated in embryonated SPF eggs via the allantoic cavity route. The embryo morality was observed and recorded, and the allantoic fluid containing the virus was successfully harvested. CEF primary cell cultures that were prepared in-house were inoculated with the individual samples separately. The virus samples showed cytopathic effects (CPE) after inoculation and incubation of the cell cultures. These CPE indicated that the virus samples infected the CEF monolayer and could be grown on CEF cell cultures as well. However, the cultivation of the virus samples in SPF eggs was preferred due to the difficulty that the primary cell cultures presented in a laboratory that is not directed at cell culture production and use. Through haemagglutination (HA) tests the virus samples were found to be unable to haemagglutinate chicken red blood cells (RBCs). It is speculated through this result that the virus samples are not classical NDV. However, it is entirely possible that the viruses agglutinate the RBCs of other mammalian or avian species. It is possible that these viruses may be evolved from classical NDV into, for example, Goose paramyxovirus The ultrastructure of the virus sample D1446/95 as observed through transmission electron microscopy, found an enveloped virus that measures about 100 nm to 150 nm in diameter. Although the ultrastructure of the virus does not conclusively identify the virus, it helps in preliminarily grouping it to other known viruses with similar ultrastructure and eliminating it from groups of viruses that have obvious differences in ultrastructure. Investigation into the virulence of the virus samples using the mean death time (MDT) indicated that the virus sample D1446/95 was moderately virulent with an MDT of 71,75 hours and the sample 834/05 was highly virulent with an MDT of 60 hours. As NDV ranges from being highly virulent (velogenic strains) to having low virulent (lentogenic strains) the MDT results were compared to the standard of the MDT of NDV. The standard of MDT for NDV states that velogenic strains take less than 60 hours to kill; mesogenic strains kill between 60 to 90 hours and lentogenic strains take longer than 90 hours to kill the SPF embryos. After the extraction of RNA from the harvested virus sample using TRIzol reagent, the viral RNA of D1446/95 and 834/05 were amplified using RT-PCR, primer sets designed based on Goose paramyxovirus ZJ1 and the Access RT-PCR System (Promega). The RT-PCR products were run and observed on 1% gels using gel electrophoresis. The RT-PCR products of the two virus samples resulted in multiple bands for the two virus samples that indicated that they are probably not classical NDV, but may possibly be NDV of other species perhaps even of goose NDV. However, this is still to be investigated further. Thus, through this study it was found that the unidentified virus samples D1446/95 and 834/05 are most likely not classical NDV, but rather could be a variation of classical NDV that may be branching off into paramyxovirus of other poultry or mammalian species as was found in the study of Goose paramyxovirus.

[发布机构] University of the Free State