Phylogenetic characterisation of the Palyam serogroup orbiviruses and development of a real-time RT-PCR

Abstract

The Palyam serogroup of the genus Orbivirus and family Reoviridae are arthropodborne viruses that have been isolated in Africa, Australia and Asia. They are associated with abortion and teratogenesis in cattle and other ruminants. There are currently 13 serotypes recognized by the International Committee on Taxonomy of Viruses (ICTV) including Palyam, Kasba, Vellore, Abadina, D’Aguilar, Nyabira, CSIRO Village, Marrakai, Gweru, Bunyip Creek, Petevo, Marondera and Kindia. Although Palyam viruses had been isolated previously, it was only after an outbreak of congenital abnormalities in cattle in Japan from November 1985 to April 1986 that their pathogenic importance began to be investigated. Of the 13 different serotypes that have been identified, the full genome sequence of only one, Kasba, has been published. Sequences for certain genome segments of the serotypes from Japan, Australia and Zimbabwe are available but not the complete genome data. The few molecular studies that have been done on the Palyam serogroup viruses, focused mainly on Kasba virus and little is known about the other serotypes. In general, not much is published on Palyam viruses, their occurrence or prevalence, and the impact of their epidemiology in South Africa or elsewhere is unknown. The objective of this project was to perform phylogenetic analysis of the different serotypes of the Palyam viruses to enable a better understanding of the genomic features of the Palyam serogroup of orbiviruses, their relation to each other as well as to other orbiviruses. The study is presented in two parts. The aim of the first part was to obtain the full genome sequences of the different Palyam serotypes and Apies River virus, as well as selected field isolates from Africa in order to perform phylogenetic analysis. The aim of the second part was to develop a rapid diagnostic test to detect the Palyam viruses. In the first part, the viruses were propagated and after full-length amplification of cDNA (FLAC) the amplicons were sequenced on an Illumina® Mi-Seq sequencer, using the Nextera XT DNA sample preparation kit and 300-bp paired-end V3 Illumina chemistry. Sequence data generated by Illumina sequencing were analyzed using the CLC Genomics Main workbench, version, 8.0.1. De novo assembly of sequence reads was performed and contig sequences prepared. Sequences were aligned and converted into nexus and phylips files. Data-display networks (neighbour-networks) were constructed with SplitsTree 4 and the phylips files were used to initiate model estimation via jModel test2, by using the online portal Cipres Science gateway. Bayesian analyses was performed in MrBayes version 3. During analysis of the amino acid sequences of the separate genes of the Palyam serogroup serotypes, the gene encoding Viral Protein (VP) 7 (Segment 7) was found to be the most conserved. The amino acid sequences for VP2 and VP5 showed the highest degree of variation, with VP2 being the most variable of the two. Phylogenetic analysis indicated that the Palyam virus group was most closely related to AHSV, and EEV showed the most distant evolutionary relationship to the Palyam viruses. When comparing the different serotypes within the Palyam serogroup viruses, a high degree of sequence identity was found for isolates from the same geographical region. The phylogenetic analysis revealed two clades, which were supported by strong bootstrap values of 100 and a posterior probability value of 1. The African serotypes were all very closely related in one clade, with identical sequences for several gene segments. The second clade contained the Australian and Asian serotypes and one African serotype, Petevo. The high percentage sequence identity (85.6% - 77,5%) that exists between the viruses from Australia and Asia may suggest that there has been some gene flow between the serotypes. It was clear from the sequence data that the geographical origin of Palyam serogroup viruses played an important role in the development of the different serotypes. In the second part of the study, the sequence data obtained in the phylogenetic analysis was used to develop primers and a probe to detect all the Palyam serogroup serotypes in a real-time RT-PCR. The same viruses used in the first part of the study as well as other orbiviruses were propagated and RNA was extracted and tested in a real-time RT-PCR. The real-time RT-PCR was able to detect all the Palyam serogroup serotypes, but further validation is necessary for it to be used as a diagnostic test. The sequence data generated during this study could enable further investigation into molecular evolution of the Palyam serogroup viruses such as reassortment, genetic drift and intragenic recombination. The developed real-time RT-PCR could be a valuable diagnostic tool for both the detection and exclusion of Palyam serogroup viruses during outbreaks involving relevant symptoms.