Method comparison for Japanese encephalitis virus detection in samples collected from the Indo-Pacific region

Abstract

IntroductionJapanese encephalitis virus (JEV) is a mosquito-borne viral pathogen, which is becoming a growing public health concern throughout the Indo-Pacific. Five genotypes of JEV have been identified. Current vaccines are based on genotype III and provide a high degree of protection for four of the five known genotypes.MethodsRT-PCR, Magpix, Twist Biosciences Comprehensive Viral Research Panel (CVRP), and SISPA methods were used to detect JEV from mosquito samples collected in South Korea during 2021. These methods were compared to determine which method would be most effective for biosurveillance in the Indo-Pacific region.ResultsOur data showed that RT-PCR, Twist CVRP, and SISPA methods were all able to detect JEV genotype I, however, the proprietary Magpix panel was only able to detect JEV genotype III. Use of minION sequencing for pathogen detection in arthropod samples will require further method development.ConclusionBiosurveillance of vectorborne pathogens remains an area of concern throughout the Indo-Pacific. RT-PCR was the most cost effective method used in the study, but TWIST CVRP allows for the identification of over 3,100 viral genomes. Further research and comparisons will be conducted to ensure optimal methods are used for large scale biosurveillance.

Direct genome sequencing of respiratory viruses from low viral load clinical specimens using target capture sequencing technology

“…Here, we have demonstrated the effectiveness of target capture sequencing directly from respiratory clinical specimens with low viral load. The capture panel, the Twist Comprehensive Viral Research Panel, was selected for its ability to capture a very wide range of viral pathogens; to our knowledge, this is the first evaluation using human respiratory clinical specimens with low viral load although the panel has been used on human cerebrospinal fluid (26), saliva, blood and feces from wild bats (27) and mosquito samples (28). Target capture sequencing with this panel yielded approximately 180- and 2000-fold higher read counts of SARS-CoV-2 or influenza A virus, respectively, than metagenomic sequencing when RNA extracts from specimens containing 59.3 or 544 RNA copies/µL of SARS-CoV-2 or influenza A virus were used, respectively.…”

Comparison of the performance of two targeted metagenomic virus capture probe-based methods using synthetic viral sequences and clinical samples

“…In 17 cases, AAV2 was detected using targeted sequencing, while in seven of these pediatric cases AAV2 was missed by untargeted metagenomic sequencing, illustrating the significance of the use of enrichment by hybridization. With regard to costefficiency, a recent study compared PCR, sequence-independent single primer amplification (SISPA), and the Twist Comprehensive Viral Research Panel for the detection of Japanese encephalitis 44 . The authors concluded that the PCR panels were not able to detect all genotypes, whereas broader surveillance of vector-borne pathogens would be more effective though costly 44 .…”

“…With regard to costefficiency, a recent study compared PCR, sequence-independent single primer amplification (SISPA), and the Twist Comprehensive Viral Research Panel for the detection of Japanese encephalitis 44 . The authors concluded that the PCR panels were not able to detect all genotypes, whereas broader surveillance of vector-borne pathogens would be more effective though costly 44 . Hybridization capture has been approved by the FDA for SARS-CoV-2 variant monitoring, illustrating the acknowledged significance of this type of enrichment.…”

Genomic characterization of a reemerging Chikungunya outbreak in Kedougou, Southeastern Senegal, 2023

“…Samples from confirmed cases with acute infection were processed to obtain the whole genome by a target enrichment standard hybridization workflow using the Twist Biosciences Comprehensive Viral Research Panel (CVRP), comprising approximately one million 120-base pair probes designed to target 15,488 distinct viral strains that infect both humans and animals, as previously described [ 14 ]. Briefly, extracted RNA samples were used as a template for a reverse transcription step with the SuperScript IV Reverse Transcriptase kit (Invitrogen, Thermo Fisher, USA) followed by a DNA fragmentation, telomere repair, dA-Tailing and a ligation with Universal Twist adapters before a final libraries amplification.…”