This study reported SARS-CoV-2 whole genome sequencing results from June 2021 to January 2022 from seven genome sequencing centers in Malaysia as part of the national surveillance program. COVID-19 samples that tested positive by reverse transcription polymerase chain reaction and with cycle threshold values <30 were obtained throughout Malaysia. Sequencing of SARS-CoV-2 complete genomes was performed using Illumina, Oxford Nanopore, or Ion Torrent platforms. A total of 6163 SARS-CoV-2 complete genome sequences were generated over the surveillance period. All sequences were submitted to the Global Initiative on Sharing All Influenza Data database. From June 2021 to January 2022, Malaysia experienced the fourth wave of COVID-19 dominated by the Delta variant of concern, including the original B.1.617.2 lineage and descendant AY lineages. The B.1.617.2 lineage was identified as the early dominant circulating strain throughout the country but over time, was displaced by AY.59 and AY.79 lineages in Peninsular (west) Malaysia, and the AY.23 lineage in east Malaysia. In December 2021, pilgrims returning from Saudi Arabia facilitated the introduction and spread of the BA.1 lineage (Omicron variant of concern) in the country. The changing trends of circulating SARS-CoV-2 lineages were identified, with differences observed between west and east Malaysia. This initiative highlighted the importance of leveraging research expertise in the country to facilitate pandemic response and preparedness.

The COVID-19 pandemic first emerged in Malaysia in Jan 2020. As of 12th Sept 2021, 1,979,698 COVID-19 cases that occurred over three major epidemic waves were confirmed. The virus contributing to the three epidemic waves has not been well-studied. We sequenced the genome of 22 SARS-CoV-2 strains detected in Malaysia during the second and the ongoing third wave of the COVID-19 epidemic. Detailed phylogenetic and genetic variation analyses of the SARS-CoV-2 isolate genomes were performed using these newly determined sequences and all other available sequences. Results from the analyses suggested multiple independent introductions of SARS-CoV-2 into Malaysia. A new B.1.524(G) lineage with S-D614G mutation was detected in Sabah, East Malaysia and Selangor, Peninsular Malaysia on 7th October 2020 and 14th October 2020, respectively. This new B.1.524(G) group was not the direct descendant of any of the previously detected lineages. The new B.1.524(G) carried a set of genetic variations, including A701V (position variant frequency = 0.0007) in Spike protein and a novel G114T mutation at the 5’UTR. The biological importance of the specific mutations remained unknown. The sequential appearance of the mutations, however, suggests that the spread of the new B.1.524(G) lineages likely begun in Sabah and then spread to Selangor. The findings presented here support the importance of SARS-CoV-2 full genome sequencing as a tool to establish an epidemiological link between cases or clusters of COVID-19 worldwide.

Background: Zika virus (ZIKV) and dengue virus (DENV) co-circulate in tropical regions and elicit cross-reactive immunity. However, differential neutralization of closely related ZIKV strains remains unclear. This study characterizes the neutralization profiles of dengue-immune sera against DENV and two Asian-lineage ZIKV strains, P6-740 and PRVABC59. Methods: Sera from suspected dengue infections and from healthy donors were screened for anti-DENV IgM and IgG antibodies and anti-ZIKV IgM and IgG antibodies by ELISA and grouped. Neutralization against DENV serotypes and two ZIKV strains was quantified using the foci reduction neutralization test (FRNT). Complete genome sequences of ZIKV P6-740 and PRVABC59 were compared to identify amino acid differences. Results: Sera containing anti-DENV IgG showed higher neutralizing activity against both DENV and ZIKV than IgG-negative sera. Neutralization was also detected in some ELISA-seronegative samples. Across all immunological groups, neutralization titers were higher against ZIKV P6-740 than ZIKV PRVABC59. Among healthy donors with evidence of previous dengue exposure, 58% exhibited a ≥4-fold difference between the two ZIKV strains, of which 80% favored ZIKV P6-740. Among seronegative healthy blood donors, 45% exhibited a 4-fold difference, of which 89% favored P6-740. Four amino acid substitutions were identified in the envelope (E) protein. Conclusions: Dengue-induced immunity differentially neutralizes closely related ZIKV strains, with generally stronger activity against P6-740, suggesting that viral genetic variation may influence cross-neutralization outcomes.

Various methods have been developed for rapid and high throughput full genome sequencing of SARS-CoV-2. Here, we described a protocol for targeted multiplex full genome sequencing of SARS-CoV-2 genomic RNA directly extracted from human nasopharyngeal swabs using the Ion Personal Genome Machine (PGM). This protocol involves concomitant amplification of 237 gene fragments encompassing the SARS-CoV-2 genome to increase the abundance and yield of viral specific sequencing reads. Five complete and one near-complete genome sequences of SARS-CoV-2 were generated with a single Ion PGM sequencing run. The sequence coverage analysis revealed two amplicons (positions 13751-13965 and 23941-24106), which consistently gave low sequencing read coverage in all isolates except 4Apr20-64-Hu. We analyzed the potential primer binding sites within these low covered regions and noted that the 4Apr20-64-Hu possess C at positions 13730 and 23929, whereas the other isolates possess T at these positions. The genetic variations observed suggest that the naturally occurring genome variations present in the actively circulating SARS-CoV-2 strains affected the performance of the target enrichment panel of the Ion AmpliSeq™ SARS‑CoV‑2 Research Panel. The possible impact of other genetic sequence variations warrants further investigation, and an improved version of the Ion AmpliSeq™ SARS‑CoV‑2 Research Panel, hence, should be considered.