The identification of unknown poultry viruses through established methods
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.
Afrikaans: Die pluimvee bedryf lei elke jaar groot verliese weens bakteriese en virale infeksies. As gevolg van hierdie verliese word navorsing toegespits op die isolasie en identifikasie van verskillende mikro-organismes wat pluimvee met siektes affekteer. Die doeltreffendheid van die entstowwe teen hierdie siektes is slegs so doeltreffend soos die navorsing wat gedoen word op die siekte-veroorsakende bakterieë of virusse. Daar is wel gevalle waar onbekende viruses in die industrie verwoesting saai, dit is hoofsaaklik toe te skryf aan a) die virulente aard van die virus, b) gereeldheid van infeksie en c) die gebrek aan kennis oor die virus. Die onbekende virus kan dalk ’n ”nuwe” virus wees of ’n reeds bestaande virus wat gemuteer het tydens replikasie. Dus is dit belangrik om die prosedure van isolasie en identifikasie van pluimvee virusse te verfyn sodat uitbreke van onbekende virus infeksies vinnig en doeltreffend opgelos kan word. Vir hierdie studie is twee virusse ontvang wat as Newcastle disease virus (NDV) geïdentifiseer is, maar hierdie virusse het nie net ND-spesifieke PKR priemstukke vermeerder nie en het ook nie rooibloedselle gehemagglutineer nie. Hierdie monsters was twee van ’n groep virusse ontvang deur die Veeartsnykundige Biotegnologie laboratorium aan die Universiteit van die Vrystaat vanaf die Onderstepoort Veeartsnykundige Instituut. Die doel van die studie was om ondersoek in te stel na die identiteit van die twee onbekende virusmonsters, D1446/95 en 834/05, met behulp van verskeie identifikasie metodes asook die isolasie en kweking van hierdie onbekende virusse. Die prosedures wat op hierdie virusse uitgevoer is (aangeneem om NDV te wees) het ingesluit: kweking in embryo bevattende spesifieke patogeen vrye (SPV) eiers sowel as in primêre hoender embrio fibroblaste (HEF) selkulture, suiwering van die virus vir ultrastrukturele identifikasie met die gebruik van transmissie elektronmikroskopie (TEM), serologiese studies wat HA en gemiddelde dodingstyd (GDT) ingesluit het. Laastens was tru-transkriptase (TT)-PKR uitgevoer op die isolate. Die virale monsters is gekweek in ge-embrioneerde SPV eiers via die allantoïese roete. Embrio mortaliteit is waargeneem en die virus-bevattende allantoïese vloeistof is geoes. Hoender embrio fibroblaste primêre selkulture is voorberei en geïnnokuleer met die individuele virus monsters. Die virus monsters het sitopatiese effekte (SPE) postinnokulasie en inkubasie van selkulture getoon. Hierdie SPE toon dat die virus die HEF monolaag geïnfekteer het en dus op die HEF selkulture kon groei. Dit was verkies om die virusse te kweek in SPV eiers. Weens die feit dat die laboratorium nie ingerig is vir die gebruik en produksie van selkulture nie. Met gebruik van die HA toetse is bevind dat die virus monsters nie die hoender rooibloedselle kon agglutineer nie, dus het die moontlikheid ontstaan dat die virus monsters dalk nie klassieke NDV was nie, alhoewel dit wel moontlik is dat die virusse die rooibloedselle van ander soogdier of pluimvee spesies kon agglutineer. Dit is ook moontlik dat hierdie virusse gemuteer het vanaf klassieke NDV in byvoorbeeld Goose paramyxovirus. Die ultrastruktuur van virusmonster D1446/95 was waargeneem met TEM. Die virus was omhul met ’n deursnee van ongeveer 100 – 150 nm. Alhoewel die ultrastruktuur van die virus nie sonder twyfel die virus kan identifiseer nie, kan die virus wel gegroepeer word met ander bekende virusse wat soortgelyke ultrastrukture het, wat kan bydra tot identifikasie. Ondersoek in die virulensie van die virus monsters met gebruik van GDT het getoon dat die virus monster D1446/95 matig virulent was met ’n GDT van 71,75 ure en virus monster 834/05 was hoogs virulent met ’n GDT van 60 ure. Newcastle disease virus strek van hoogs virulent (velogeniese stamme) na lae virulensie (lentogeniese stamme). Die standaard GDT van NDV stipuleer dat velogeniese stamme minder as 60 ure neem om dood te veroorsaak, mesogeniese stamme tussen 60 en 90 ure en lentogeniese stamme meer as 90 ure om die SPV embrios se dood te veroorsaak. Na die ekstraksie van ribonukleïensuur (RNS) vanaf die geoesde virusmonster met behulp van TRIzol reagens, is die virale RNS van D1446/95 en 834/05 geamplifiseer met gebruik van TT-PKR, die priemstukke is gebaseer op die Goose paramyxovirus ZJ1 isolaat en die Access RT-PCR sisteem (Promega). Die TT-PKR produkte is gevisualiseer op ’n 1% agarose gel met behulp van ultra violet illuminasie. Die TT-PKR produkte van die twee virusmonsters het veelvuldige bandjies vir beide monsters getoon wat daarop gewys het dat hierdie monsters waarskynlik nie NDV is nie, maar moontlik NDV van ’n ander spesie of moontlik gans NDV. Hierdie resultate sal wel verder ondersoek moet word. Dus, met hierdie studie is bevind dat die ongeïdentifiseerde virusmonsters, D1446/95 en 834/05 waarskynlik nie klassieke NDV is nie, maar eerder ’n variasie van klassieke NDV wat moontlik kan vertak in paramyxovirus of ander pluimvee of soogdier spesies soos gevind is in die studie gedoen op Goose paramyxovirus.
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