Serotype and genomic diversity of dengue virus during the 2023 outbreak in Pakistan reveals the circulation of genotype III of DENV-1 and cosmopolitan genotype of DENV-2.

Dengue, a mosquito-borne viral disease, poses a significant public health challenge in Pakistan, with a significant outbreak in 2023, prompting our investigation into the serotype and genomic diversity of the dengue virus (DENV). NS-1 positive blood samples from 153 patients were referred to the National Institute of Health, Pakistan, between July and October 2023. Among these, 98 (64.1%) tested positive using multiplex real-time PCR, with higher prevalence among males (65.8%) and individuals aged 31-40. Serotyping revealed DENV-1 as the predominant serotype (84.7%), followed by DENV-2 (15.3%). Whole-genome sequencing of 18 samples (DENV-1 = 17, DENV-2 = 01) showed that DENV-1 (genotype III) samples were closely related (>99%) to Pakistan outbreak samples (2022), and approx. > 98% with USA (2022), Singapore and China (2016), Bangladesh (2017), and Pakistan (2019). The DENV-2 sequence (cosmopolitan genotype; clade IVA) shared genetic similarity with Pakistan outbreak sequences (2022), approx. > 99% with China and Singapore (2018-2019) and showed divergence from Pakistan sequences (2008-2013). No coinfection with dengue serotypes or other viruses were observed. Comparisons with previous DENV-1 sequences highlighted genetic variations affecting viral replication efficiency (NS2B:K55R) and infectivity (E:M272T). These findings contribute to dengue epidemiology understanding and underscore the importance of ongoing genomic surveillance for future outbreak responses in Pakistan.

Recovery of complete genome sequences of Crimean-Congo haemorrhagic fever virus (CCHFV) directly from clinical samples: A comparative study between targeted enrichment and metagenomic approaches.

Crimean-Congo haemorrhagic fever (CCHF) is the most prevalent human tick-borne viral disease, endemic to the Balkans, Africa, Middle East and Asia. There are currently no licensed vaccines or effective antivirals against CCHF. CCHF virus (CCHFV) has a negative sense segmented tripartite RNA genome consisting of the small (S), medium (M) and large (L) segments. Depending on the segment utilised for genetic affiliation, there are up to 7 circulating lineages of CCHFV. The current lack of geographical representation of CCHFV sequences in various repositories highlights a requirement for increased CCHFV sequencing capabilities in endemic regions. We have optimised and established a multiplex PCR tiling methodology for the targeted enrichment of complete genomes of Europe 1 CCHFV lineage directly from clinical samples and compared its performance to a non-targeted enrichment approach on both short-read and long-read sequencing platforms. We have found a statistically significant increase in mapped viral sequencing reads produced with our targeted enrichment approach. This has allowed us to recover near complete S segment sequences and above 90% of the M and L segment sequences for samples with Ct values as high as 31.3. This study demonstrates the superiority of a targeted enrichment approach for recovery of CCHFV genomic sequences from samples with low virus titre. CCHFV is an important vector-borne human pathogen with wide geographical distribution. The validated methodology reported here adds value to front-line public health laboratories employing genomic sequencing for CCHFV Europe 1 lineage surveillance, particularly in the Balkan and Middle Eastern territories currently monitoring the spread of the pathogen. Tracking the genomic evolution of the virus across regions improves risk assessment and directly informs the development of diagnostics, therapeutics, and vaccines.

Investing in health preparedness, response and resilience: a genomics costing tool focused on next generation sequencing.

The world has seen unprecedented gains in the global genomic surveillance capacities for pathogens with pandemic and epidemic potential within the last 4 years. To strengthen and sustain the gains made, WHO is working with countries and partners to implement the Global Genomic Surveillance Strategy for Pathogens with Pandemic and Epidemic Potential 2022-2032. A key technical product developed through these multi-agency collaborative efforts is a genomics costing tool (GCT), as sought by many countries. This tool was developed by five institutions - Association of Public Health Laboratories, FIND, The Global Fund to Fight AIDS, Tuberculosis and Malaria, UK Health Security Agency, and the World Health Organization. These institutions developed the GCT to support financial planning and budgeting for SARS-CoV-2 next-generation sequencing activities, including bioinformatic analysis. The tool costs infrastructure, consumables and reagents, human resources, facility and quality management. It is being used by countries to (1) obtain costs of routine sequencing and bioinformatics activities, (2) optimize available resources, and (3) build an investment case for the scale-up or establishment of sequencing and bioinformatics activities. The tool has been validated and is available in English and Russian at https://www.who.int/publications/i/item/9789240090866. This paper aims to highlight the rationale for developing the tool, describe the process of the collaborative effort in developing the tool, and describe the utility of the tool to countries.