Molecular epidemiology of Avian influenza and Newcastle disease using samples transported without a cold chain

Limited infrastructure in developing countries makes rapid and accurate diagnosis of Avian Influenza (AI) and Newcastle disease (ND) difficult. This project has examined a method for collecting and transporting samples that eliminates the problems associated with previous ways of managing viral outbreaks. This method, verified using Real time (quantitative) reverse transcriptase polymerase chain reaction (RT-qPCR), also overcomes quarantine restrictions between countries. An epidemiological study was then carried out using the collection and transportation method and the results of this study can be used to support further epidemiological studies of AI and ND and to standardise assays between countries. Diagnostic assays for AI and ND based on RT-qPCR have been developed and published. These assays use the deoxyribonucleic acid (DNA) binding dye, SYBR Green, or dual labelled TaqMan probes, (van Elden et al., 2001; Spackman et al., 2002; Tan et al., 2004; Ward et al., 2004; Pham et al., 2005; Payungporn et al., 2006b; Ong et al., 2007). However, SYBR Green and TaqMan probes have limitations. SYBR Green can inhibit PCR reactions because it degrades PCR products, while TaqMan probes have less sensitivity due to the specificity of the probe sequences. Therefore, a SYTO 9 RT-qPCR was developed for the AI and ND diagnosis. SYTO 9 RT-qPCR for AI virus (AIV) and ND virus (NDV) assays were successfully used. A series of new primers were evaluated and developed in these assays such as M+4100 forward and M-4220 reverse for ND detection of Class II NDVs (chicken) (Wise et al., 2004) and NDV MGB1 for ND detection of Class I NDVs (wild birds). For the AI screening test, a few pairs of primers based on the M gene were also evaluated (Ward et al., 2004; Heine et al., 2005). Subsequently, these diagnostic assays were used to evaluate sample collection and transportation without a cold chain. Further studies were also performed to evaluate the sequences of NDVs. This sequencing was intended to show the viral pathotyping and phylogenetic relationships of NDV. Primers that targeted a 343 bp Fusion sequence were designed and successfully used to amplify six Australian NDV isolates, both NDV Class I and Class II. Based on the amino acid sequence in the Fusion cleavage site, the six isolates were confirmed as low virulence of NDV. The motif of the cleavage sites were S-G-G-E-RQ- E-R-L-V, S-G-G-E-Q-Q-G-R-L-I and S-G-G-G-K-G-R-L-I. Evaluations of suitable buffers showed that Solid Tissue Digest Buffer (STDB) and Lysis Buffer (LB) were effective buffers for sample collection and transportation. Further studies showed that there was no significant difference between these two buffers. However, viability experiments showed that the LB deactivated both AI and NDV while STDB failed to fully inactivate NDV. Therefore, the lysis buffer was chosen for further sample collection and transportation without a cold chain. A protocol for the extraction of samples collected without cold chain was evaluated. This modified protocol was successfully integrated into the nucleic purification system using a robotic device and ribonucleic acid (RNA) extraction protocol (Corbett Research, Brisbane). However, extracted and purified RNA may be degraded by ribonuclease contamination. Therefore, this project also evaluated buffers for RNA storage after extraction. Evaluation of two modified buffers, the Tris EDTA (TE) carrier buffer (TCB) and modified RNA safe buffer (mRSB), showed that both buffers can prevent RNA degradation with no significant difference in cycle threshold (Ct)-Value detected by RT-qPCR. The method of collection and transportation samples without a cold chain was applied by collecting live viruses and also viral antigens in Indonesia and transporting to Australia. Due to the time limitation, viral isolates were collected only from AI viruses representing four different regions in Indonesia (Timika, Ambon, Tanggerang, and Sukabumi). For ND commercial antigens (Balitvet and Pusvetma) were purchased. The AI and ND samples were processed using a lysis buffer in the Quarantine Agency Laboratory of Indonesia and transported to James Cook University (JCU) in Townsville, Australia. Transported AI and ND viral RNA were successfully amplified using SYTO 9 RT-qPCR. This study confirmed that not only viral RNA but also inactivated viral RNA can be preserved, transported and detected after a month of processing. This transportation also verified that viral RNA can be recovered and detected after chemical inactivation. SYTO 9 RT-qPCR and TaqMan dual labelled probed RT-qPCR assays were carried out to screen the presence of AI viruses in the transport buffer. These assays used published protocols (Ward et al., 2004; Heine et al., 2005). The assays successfully detected viral RNA. However, the TaqMan dual labelled probe assay for H5 (Heine et al., 2005) failed to detect viral RNA in the Sukabumi isolates. Further studies of Indonesian AI isolates using High Resolution Melt assay recognised at least two populations from four different regions. Normalization data demonstrated that Timika and Ambon isolates were very similar while Tanggerang and Sukabumi isolates represented different populations. These results suggest that there is continued evolution of AI viruses in Indonesia. The ability of lysis buffer to preserve AI viral RNA was confirmed by amplification of 590bp and 1370 bp products from the Haemagglutinin (HA) gene. These products were successfully sequenced demonstrating that the viral RNA transported using lysis buffer was not fragmented. As a result, a molecular epidemiology study of AI and ND isolates was successfully carried out using the samples transported without a cold chain. However, at this stage, the method of collection and transportation of samples without a cold chain has not been applied to field and clinical samples. The value of the technique would be enhanced if it could be demonstrated that it works equally well for swabs and tissues collected in the field.