Alphaviruses are mosquito-borne RNA viruses that cause devastating or debilitating diseases in both humans and livestock. SAGV and GETV are two members of the Alphavirus genus of the family Togaviridae. GETV is widely distributed in southeast Asia and northern Australia along the Pacific Ocean [20–24]. GETV has been isolated from various mosquito species of the genera Culex, Aedes, and Armigeres. It is conceivable that GETV may use mammals as primary hosts in its enzootic cycle, and through these biting vectors, the virus may be transmitted to various animal species, including pigs, chickens, humans, and other mammals, on rare occasion, the virus may jump the species barrier and infect a human or other animal . Although the virus has not been linked to illness in humans, many studies have suggested that the virus is a latent pathogen of humans causing a fever of unknown origin. GETV could cause illnesses in humans and livestock animals and, indeed, antibodies to GETV have been detected in many species of animals around the world [4–6].
Analysis of all sequences included in this study showed that the nsP3 non-structural protein gene and the capsid protein gene nucleotide sequence identity between YN08 isolates and other Chinese isolates (GETV_M1 , ALPV_M1, HB0234 and YN0540) ranged from 98.0 to 99.31% and 97.56 to 99.31%, respectively. Multiple alignments showed that the S_Korea isolate does not possess the 92 nt sequence from 11341–11433 in the virus genome and there was a low level of identity (92.19–93.75%) between S_Korea and other GETV strain at the 3’-UTR sequences. Despite possessing 3’-UTR sequences of different lengths, GETV isolates contain various numbers of an identical sequence element that could have originated from a large ancestral 3’-UTR [26, 27].
Phylogenetic trees constructed using viruses sequence data are the best indication of the evolutionary relationships between viruses and genetic changes associated with antigenic drift. To provide further insight into the evolutionary relationship of YN08 and other alphaviruses, phylogenic analysis was performed based on the capsid protein gene and the 3’-UTR sequence of YN08 and other 9 alphaviruses. These analyses showed that YN08 is a member of the GETV and was most closely related to HB0234 and S_Korea and then with YN0540 and GETV_LEIV_17741_MPR to form a distinguishable branch based on nsP3 and capsid protein genes. Thus, the phylogenetic analysis clearly showed that YN08 is more closely related to Hebei HB0234 strain than YN0540 strain and more genetically distant to the MM2021 Malaysia primitive strain.
Present methods rely on prior genetic knowledge but are not effective for the identification of unknown viruses. Thus, we developed the simple VIDISCR method based on the cDNA-RAPD technique [8, 9]. The RAPD technique is a type of PCR but random segments of DNA are amplified. Unlike traditional PCR analysis, RAPD does not require any specific knowledge of the DNA sequence of the target organism by the use of 10-mer primers for the amplification of DNA. However, the resolving power of the VIDISCR method is prone to interference from DNA or RNA from the lysed host tissues and cells (or bacteria). Since VIDISCR relies on a large, intact DNA template sequence, it has some limitations in the use of degraded DNA samples. Therefore, the intact DNA template sequence of virus genomes required and chromosomal DNA, mitochondrial DNA, and cellular RNA must be removed from the preparation to perform VIDISCR. In the current study, approximately 50% of PCR assays amplified DNA fragments in 20 VIDISCR selective PCR reactions (11/20; Table 1) and 13 of 21 fragments showed sequence similarity to virus genes. Therefore, VIDISCR is a suitable method for the identification of unknown viruses.
The current study indicated that the VIDISCR is an efficient procedure for the identification of known and unknown viruses with the removal of contaminating cellular nucleic acids, optimized nucleic acid amplification, large-scale sequencing, and bioinformatics. The VIDISCR technology is general, non-selective, and rapid, that does not require prior knowledge of the target sequence. This technique could be adapted to include a set of universal primers for virus genomic analysis in a wide variety of species. VIDISCR can identify a range of known and unknown pathogens that can be applicable to clinical samples including tissues or culture supernatants. Therefore, it is well suited for the rapid identification of an unknown or unexpected virus involved in a disease outbreak.