Simultaneous co-circulation of two genotypes of dengue virus serotype 3 causing a large outbreak in Sri Lanka in year 2023.

As many other countries, Sri Lanka experienced a marked rise in the number of dengue cases in 2023, with an unusual pattern of disease epidemiology. This rise coincided with the emergence of dengue virus (DENV) serotype 3 in Sri Lanka as the predominant serotype after 2009. Interestingly, a discrepancy between NS1 rapid antigen test positivity and quantitative real time PCR positivity was observed, with 50% of NS1 positive samples being negative by molecular diagnostics. Following sequencing of the DENV-3 strains in 2023, we identified two DENV-3 genotypes (I and III) co-circulating. While DENV-3 genotype III was detected by the modified CDC DENV-3 primers, genotype I evaded detection due to key mutations at forward and reverse primer binding sites. The co-circulation of multiple genotypes associated with an increase in cases highlights the importance of continuous surveillance of DENVs to identify mutations resulting in non-detection by diagnostics and differences in virulence.

Molecular Epidemiology of AY.28 and AY.104 Delta Sub-lineages in Sri Lanka.

Background: The worst SARS-CoV-2 outbreak in Sri Lanka was due to the two Sri Lankan delta sub-lineages AY.28 and AY.104. We proceeded to further characterize the mutations and clinical disease severity of these two sub-lineages. Methods: 705 delta SARS-CoV-2 genomes sequenced by our laboratory from mid-May to November 2021 using Illumina and Oxford Nanopore were included in the analysis. The clinical disease severity of 440/705 individuals were further analyzed to determine if infection with either AY.28 or AY.104 was associated with more severe disease. Sub-genomic RNA (sg-RNA) expression was analyzed using periscope. Results: AY.28 was the dominant variant throughout the outbreak, accounting for 67.7% of infections during the peak of the outbreak. AY.28 had three lineage defining mutations in the spike protein: A222V (92.80%), A701S (88.06%), and A1078S (92.04%) and seven in the ORF1a: R24C, K634N, P1640L, A2994V, A3209V, V3718A, and T3750I. AY.104 was characterized by the high prevalence of T95I (90.81%) and T572L (65.01%) mutations in the spike protein and by the absence of P1640L (94.28%) in ORF1a with the presence of A1918V (98.58%) mutation. The mean sgRNA expression levels of ORF6 in AY.28 were significantly higher compared to AY.104 (p < 0.0001) and B.1.617.2 (p < 0.01). Also, ORF3a showed significantly higher sgRNA expression in AY.28 compared to AY.104 (p < 0.0001). There was no difference in the clinical disease severity or duration of hospitalization in individuals infected with these sub lineages. Conclusions: Therefore, AY.28 and AY.104 appear to have a fitness advantage over the parental delta variant (B.1.617.2), while AY.28 also had a higher expression of sg-RNA compared to other sub-lineages. The clinical implications of these should be further investigated.

Comparison of different sequencing techniques for identification of SARS-CoV-2 variants of concern with multiplex real-time PCR.

As different SARS-CoV-2 variants emerge and with the continuous evolvement of sub lineages of the delta variant, it is crucial that all countries carry out sequencing of at least >1% of their infections, in order to detect emergence of variants with higher transmissibility and with ability to evade immunity. However, due to limited resources as many resource poor countries are unable to sequence adequate number of viruses, we compared to usefulness of a two-step commercially available multiplex real-time PCR assay to detect important single nucleotide polymorphisms (SNPs) associated with the variants and compared the sensitivity, accuracy and cost effectiveness of the Illumina sequencing platform and the Oxford Nanopore Technologies' (ONT) platform. 138/143 (96.5%) identified as the alpha and 36/39 (92.3%) samples identified as the delta variants due to the presence of lineage defining SNPs by the multiplex real time PCR, were assigned to the same lineage by either of the two sequencing platforms. 34/37 of the samples sequenced by ONT had <5% ambiguous bases, while 21/37 samples sequenced using Illumina generated <5%. However, the mean PHRED scores averaged at 32.35 by Illumina reads but 10.78 in ONT. This difference results in a base error probability of 1 in 10 by the ONT and 1 in 1000 for Illumina sequencing platform. Sub-consensus single nucleotide variations (SNV) are highly correlated between both platforms (R2 = 0.79) while indels appear to have a weaker correlation (R2 = 0.13). Although the ONT had a slightly higher error rate compared to the Illumina technology, it achieved higher coverage with a lower number or reads, generated less ambiguous bases and was significantly less expensive than Illumina sequencing technology.

Regional Variation in Dengue Virus Serotypes in Sri Lanka and Its Clinical and Epidemiological Relevance.

Dengue is a significant health concern in Sri Lanka, but diagnosis of the infecting dengue virus (DENV) serotype has hitherto been largely restricted to the Colombo district in the western province. Salinity tolerant Aedes vectors are present in the island's northern Jaffna peninsula, which is undergoing rapid groundwater salinization. Virus serotypes were determined by RT-qPCR in 107 and 112 patients diagnosed by NS1 antigen positivity from the Jaffna district in 2018 and 2019, respectively, and related to clinical characteristics. DENV1 and DENV2 were the most common serotypes in both years. Infections with multiple serotypes were not detected. DENV1 was significantly more prevalent in 2019 than 2018, while DENV3 was significantly more prevalent in 2018 than 2019 among the Jaffna patients. Limited genomic sequencing identified DENV1 genotype-I and DENV3 genotype-I in Jaffna patients in 2018. Dengue was more prevalent in working age persons and males among the serotyped Jaffna patients. DENV1 and DENV2 were the predominant serotypes in 2019 in the Colombo district. However, DENV1 and DENV3 were significantly more prevalent in Colombo compared with Jaffna in 2019. The differences in the prevalence of DENV1 and DENV3 between the Jaffna and Colombo districts in 2019 have implications for dengue epidemiology and vaccination. Salinity-tolerant Aedes vector strains, widespread in the Jaffna peninsula, may have contributed to differences in serotype prevalence compared with the Colombo district in 2019. Significant associations were not identified between virus serotypes and clinical characteristics among Jaffna patients.

Genomic and Epidemiological Analysis of SARS-CoV-2 Viruses in Sri Lanka.

Background: In order to understand the molecular epidemiology of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) in Sri Lanka, since March 2020, we carried out genomic sequencing overlaid on available epidemiological data until April 2021. Methods: Whole genome sequencing was carried out on diagnostic sputum or nasopharyngeal swabs from 373 patients with COVID-19. Molecular clock phylogenetic analysis was undertaken to further explore dominant lineages. Results: The B.1.411 lineage was most prevalent, which was established in Sri Lanka and caused outbreaks throughout the country until March 2021. The estimated time of the most recent common ancestor (tMRCA) of this lineage was June 1, 2020 (with 95% lower and upper bounds March 30 to July 27) suggesting cryptic transmission may have occurred, prior to a large epidemic starting in October 2020. Returning travellers were identified with infections caused by lineage B.1.258, as well as the more transmissible B.1.1.7 lineage, which has replaced B.1.411 to fuel the ongoing large outbreak in the country. Conclusions: The large outbreak that started in early October, is due to spread of a single virus lineage, B.1.411 until the end of March 2021, when B.1.1.7 emerged and became the dominant lineage.

Genomic analysis of a novel Rhodococcus (Prescottella) equi isolate from a bovine host.

Rhodococcus (Prescottella) equi causes pneumonia-like infections in foals with high mortality rates and can also infect a number of other animals. R. equi is also emerging as an opportunistic human pathogen. In this study, we have sequenced the genome of a novel R. equi isolate, B0269, isolated from the faeces of a bovine host. Comparative genomic analyses with seven other published R. equi genomes, including those from equine or human sources, revealed a pangenome comprising of 6876 genes with 4141 genes in the core genome. Two hundred and 75 genes were specific to the bovine isolate, mostly encoding hypothetical proteins of unknown function. However, these genes include four copies of terA and five copies of terD genes that may be involved in responding to chemical stress. Virulence characteristics in R. equi are associated with the presence of large plasmids carrying a pathogenicity island, including genes from the vap multigene family. A BLAST search of the protein sequences from known virulence-associated plasmids (pVAPA, pVAPB and pVAPN) revealed a similar plasmid backbone on two contigs in bovine isolate B0269; however, no homologues of the main virulence-associated genes, vapA, vapB or vapN, were identified. In summary, this study confirms that R. equi genomes are highly conserved and reports the presence of an apparently novel plasmid in the bovine isolate B0269 that needs further characterisation to understand its potential involvement in virulence properties.