Molecular Characterisation and Expression of the H5 and N1 Genes of an Indonesian Avian Influenza Virus Isolate

Abstract The emergence of Asian lineage highly pathogenic avian influenza (HPAI) virus subtype H5N1 in Afro-Eurasia continents has had serious repercussions. The battle against the virus has become a global concern; however, each nation may engage a different approach to their disease control program. In several countries where the disease is endemic, bird vaccination may become the primary option to deal with the situation. An ideal vaccine that can reliably prevent virus infection is essential. Therefore, the objective of this study was to investigate the efficacy of herpesvirus of turkey (HVT) as a vector to express the genes of the immunogenic proteins of H5N1 virus. A series of field and laboratory work was undertaken in order to accomplish this agenda. The fieldwork was carried out to collect a currently circulating field strain of H5N1 virus. As a result of field sampling in Tangerang (Banten Province), Indonesia, an HPAI virus of subtype H5N1 was successfully isolated. Since the study targeted the hemagglutinin and neuraminidase genes, reverse transcriptase-PCR was employed for gene isolation. Furthermore, the genetic material was preserved by cloning into pGEM®-T Easy vector system and transferred to Brisbane (Australia) in a dried state using ethanol precipitation methods. Meanwhile, the laboratory work consisted two main projects, namely genetic characterisation of the H5N1 field isolate and development of a mutagenesis system for HVT. Molecular characterisation by sequencing of multiple clones generated a total of 1,707 bp and 1,350 bp of nucleotide sequence, comprising the full-length of hemagglutinin and neuraminidase genes, respectively. Several nucleotide differences between clones were observed, which may have occurred either from random error during the PCR process or from viral RNA polymerase error resulting in viral quasi-species. Phylogenetic analysis revealed the isolate belonged to the sub-clade 2.1.3 and retained typical sequence characteristics of currently circulating Indonesian viruses, such as the amino acid motifs associated with the H5 cleavage site, affinity to avian receptor and sensitivity to neuraminidase inhibitor. The in vitro expression of both genes was evaluated in three different cell cultures (CEF, RK13 & VERO) using two expression vectors, pEGFP-C1 and pcDNA™3.3-TOPO® TA Cloning vectors. The study revealed different expression levels of these genes. Whereas the H5 gene was expressed at only low levels, the N1 gene was expressed at higher levels. The study also recognised different expression levels between two different N1 clones with only a single nucleotide difference. Although the results of this study support utilising the native N1 gene in a recombinant vaccine system, the low level expression of the native H5 gene indicates that it requires advanced engineering to increase expression levels. This engineering could involve codon optimization in order to enhance its expression level, either using a synthetic gene or alternatively exploring the expression levels of native H5 gene sequence variants that are generated in the viral replication process as was identified for N1 in this study. Meanwhile, the trial of a development mutagenesis system for HVT generated no infectious clones that would be suitable for recombinant vaccine purposes. The mutagenesis experiments to develop an infectious clone of HVT-BAC with GETrec system only generated several defective clones despite repeated attempts. In conclusion, despite the fact that the ultimate goal of recombining the HVT genome with avian influenza genes was not achieved within the time of study, several findings in this research may be valuable for future recombinant HVT-AI studies. This study successfully isolated a HPAI virus subtype H5N1 from Tangerang, Indonesia, and characterised its H5 and N1 genes identifying differential expression levels which may have implications for future AIV vaccine development studies.