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 âunknownâ 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.
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:ufs/oai:etd.uovs.ac.za:etd-09182009-083352 |
| Date | 18 September 2009 |
| Creators | Lee, Ji-Yun |
| Contributors | Prof RR Bragg |
| Publisher | University of the Free State |
| Source Sets | South African National ETD Portal |
| Language | en-uk |
| Detected Language | English |
| Type | text |
| Format | application/pdf |
| Source | http://etd.uovs.ac.za//theses/available/etd-09182009-083352/restricted/ |
| Rights | unrestricted, I hereby certify that, if appropriate, I have obtained and attached hereto a written permission statement from the owner(s) of each third party copyrighted matter to be included in my thesis, dissertation, or project report, allowing distribution as specified below. I certify that the version I submitted is the same as that approved by my advisory committee. I hereby grant to University Free State or its agents the non-exclusive license to archive and make accessible, under the conditions specified below, my thesis, dissertation, or project report in whole or in part in all forms of media, now or hereafter known. I retain all other ownership rights to the copyright of the thesis, dissertation or project report. I also retain the right to use in future works (such as articles or books) all or part of this thesis, dissertation, or project report. |
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