Human cytomegalovirus (HCMV) infection is the most common congenital infection globally and an important cause of disease among immunocompromised patients. HCMV genomic characterization is complicated by its genetic diversity, intra-host variability and complex genome structure. We compared the accuracy of Illumina and Oxford Nanopore Technologies (ONT) sequencing platforms to analyze full-length HCMV genomes of laboratory CMV strains enriched by hybridization capture. Both platforms achieved high per-base accuracy with DNA from high viral load (106 international units, IU) samples: 99.97% (95% CI: 0.999–1.000) with Illumina and 99.71% (95% CI: 0.994–1.000) for ONT. Low viral load (103 IU) samples, however, yielded insufficient data for downstream analysis (0.17% genome coverage, 0.01 × mean depth) using Illumina while ONT achieved 99.91% (95% CI: 0.997–1.000) per-base accuracy. Short-fragment DNA enrichment using biotinylated RNA baits produced ONT reads of only 372 ± 28 bp, inadequate for spanning complex genomic regions. To address this, we modified the enrichment method to increase fragment lengths and evaluated its performance using full-length HCMV genomes of CMV laboratory strains. The modified enrichment method substantially improved ONT performance: read lengths increased to 945 ± 363.3 bp, recovering 89% of the genome versus 83% with Illumina at ≥ 5x depth. The modified method also improved assembly contiguity from mean N50 of 150 bp to 1692 bp. Validation of the modified protocol with synthetic DNA confirmed that 5 × depth was adequate for variant calling, with consistent performance metrics across 5–500 × depth. Long-fragment DNA enrichment with ONT sequencing appears to be optimally suited for comprehensive HCMV genomic characterization.
Probabilistic methods for phylogenetic inference rely on substitution models of molecular evolution. However, at the protein level, the availability of empirical substitution models remains limited, particularly for viruses. Here, we present empirical substitution models of protein evolution for several Crimean-Congo hemorrhagic fever virus (CCHFV) proteins, including the nucleoprotein, glycoprotein precursor, and RNA-dependent RNA polymerase. For each protein, we estimated the exchangeability matrix and amino acid frequencies at equilibrium from hundreds of sequences using maximum likelihood, and we evaluated the models using independent data. The inferred CCHFV models revealed distinct evolutionary patterns among viral proteins, including patterns in polar and glycosylation-associated regions, while generally preserving common physicochemical features such as amino acid charge, polarity, and hydrophobic packing shaped by selective pressures. Remarkably, the CCHFV protein substitution models consistently outperformed all currently available empirical substitution models, including those inferred from other viral proteins, in terms of phylogenetic likelihood with independent CCHFV protein sequences. These findings support the use of these substitution models to improve the accuracy of phylogenetic reconstructions of CCHFV proteins, thereby enabling more reliable inference of evolutionary trajectories that may be useful for therapeutic design.
During a field study about porcine reproductive and respiratory syndrome virus (PRRSV) in Danish pig herds, it was discovered that the quantitative real-time RT-PCR (RT-qPCR) assay used in the study was unable to detect PRRSV-2 in two herds (Herds A and B) exhibiting clinical signs consistent with PRRS. Two additional herds (Herd C, identified through clinical suspicion, and Herd D, identified during the field study) showed reduced assay sensitivity. Sequence analysis of viruses from all four herds demonstrated that the reduced sensitivity was caused by mutations within the primer and probe binding sites. Full genome analysis of Danish PRRSV-2 sequences revealed the existence of two phylogenetically separate mutated variants with a high degree of identity to previously identified Danish variants, suggesting that they were not newly introduced but likely arose separately in Denmark.
Hantaan virus (HTNV) and Seoul virus (SEOV) are the primary causative agents of hemorrhagic fever with renal syndrome (HFRS), presenting a persistent challenge to global public health. Prompt and precise diagnostic tools are essential for clinical investigation and the containment of HFRS outbreaks. Leveraging the programmable precision of CRISPR/Cas12b system, which has significantly advanced the field of molecular diagnostics, we developed a rapid, high-sensitivity assay for the dual detection of HTNV and SEOV. Each virus was detected in a separate, target-specific single-tube reaction. The assay combines recombinase polymerase amplification (RPA) with CRISPR/Cas12b-mediated fluorescence detection and uses virus-specific sgRNAs and RPA primers targeting conserved genomic regions. Using recombinant plasmid standards, both targets were detected at 1 & times; 101 copies/ test in three technical replicates; this is a preliminary analytical detection threshold and does not incorporate extraction losses or matrix-associated inhibition. No cross-reactivity was observed within the evaluated four-organism non-target panel. In a preliminary feasibility assessment, all nine RT-qPCR-positive rodent samples (seven HTNV and two SEOV) were positive by the RPA-CRISPR/Cas12b assay, indicating complete positive agreement in this all-positive sample set. Because no negative samples were included, negative agreement, diagnostic specificity, false-positive rate, and overall clinical diagnostic accuracy could not be assessed. These results support further development of the assay as a potential screening tool; larger studies using well-characterized positive and negative clinical or field specimens are required.
In Atlantic salmon hatcheries, Salmon gill poxvirus (SGPV) can cause parachute, high mortality that can be alleviated by immediate countermeasures. However, this is hampered by a lack of good rapid diagnostics for SGPV. In this study, we developed and evaluated two rapid non-invasive isothermal amplification assays, loopmediated isothermal amplification (LAMP) and recombinase polymerase amplification (RPA), each coupled with a lateral flow device (LFD) for detection of SGPV in gill mucus or lysates from SGPV-infected salmon. Both assays demonstrated high analytical specificity and strong agreement with the reference real-time PCR assay when purified DNA was tested. The RPA-LFD assay achieved sensitivity comparable to real-time PCR but was consistently inhibited by crude gill mucus, limiting its suitability for direct field testing. In contrast, the LAMPLFD assay maintained high sensitivity and reproducibility using crude gill mucus without prior nucleic acid extraction, detecting SGPV in 14 of 15 field samples with excellent agreement with real-time PCR. Although analytical sensitivity of LAMP was approximately fourfold lower than real-time PCR, this did not compromise SGPV detection in relevant samples and a final mucus concentration of approximately 4% was optimal for SGPV amplification. Both assays showed no cross-reactivity with common salmon gill pathogens, including Candidatus Branchiomonas cysticola, Desmozoon lepeophtherii, and Paramoeba perurans. The LAMP-LFD workflow provides visual results within 45 min, representing a viable, rapid, and non-invasive diagnostic platform for field surveillance. These findings demonstrate that LAMP-LFD is well suited for SGPV detection and has strong potential to improve early outbreak detection and enhance the management of SGPV in salmon aquaculture.
