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PubMed2027

Nanopore Sequencing for Chikungunya Virus: Principles and Application.

Nanopore sequencing is transforming viral genomics through real-time, portable, long-read analysis of RNA and DNA. Unlike traditional short-read platforms, it detects nucleotide sequences by measuring ionic current changes as nucleic acids pass through nanoscale pores, enabling direct single-molecule sequencing and base modification detection. Its simplicity, flexibility, and capacity for ultra-long reads make it ideal for resolving complex genomic regions, structural variants, and full viral genomes. These advantages have accelerated its use in pathogen surveillance and outbreak response, especially in resource-limited settings. For chikungunya virus (CHIKV), nanopore sequencing allows rapid, culture-independent recovery of complete genomes from clinical and vector samples, enabling real-time tracking of viral diversity, evolution, and spread. Experiences from Ebola, Zika, and COVID-19 have demonstrated the power of portable sequencing, now applied to CHIKV monitoring. Advances in tools such as Guppy, Dorado, Minimap2, and Medaka enhance read quality, consensus accuracy, and downstream analyses. Despite challenges in basecalling and error correction, robust quality control pipelines ensure reliable results. Ongoing improvements in chemistry, flow cell design, and machine learning will further enhance fidelity and throughput, establishing nanopore sequencing as a cornerstone of CHIKV genomic surveillance and epidemic preparedness.

Chikungunya virus↗

PubMed2025

Oropouche Virus Importation in Southern Brazil and Emerging Concern Calling for Enhanced Public Health Surveillance.

Oropouche virus (OROV), an arthropod-borne virus transmitted by Culicoides paraensis, is an endemic arbovirus that historically circulates mostly in the Amazon basin. Between 2022 and 2024, it reemerged as a more widespread public health concern in South America. We conducted a pooled-sample molecular surveillance study to understand the prevalence of Oropouche fever in Brazil's southernmost state. Over 18 months, we analyzed 4060 samples to monitor the virus emergence in the Rio Grande do Sul state. We detected the first human case of OROV in the state, and our phylogenetic reconstruction indicated a travel-related introduction from the Amazon region into Rio Grande do Sul. Despite the absence of local transmission, the invasion of Culicoides paraensis and enzootic circulation of the OROV in Rio Grande do Sul highlight the risk of Oropouche fever outbreaks in the region. We demonstrated that pooled-sample surveillance effectively monitors virus introduction during periods of low endemic circulation, serving as an essential active surveillance tool for the timely detection of virus emergence and enhancing public health preparedness. The multiple introductions of distinct OROV lineages into southern Brazil underscore the importance of genomic surveillance and public health strategies to monitor and mitigate arbovirus spread in the region.

Brazil↗

PubMed2025

SMART-RNA-Metavirome: a practical RNA metavirome platform compatible with high-throughput sequencing of both short and long reads.

BACKGROUND: The RNA virosphere's extensive diversity and its role in emerging infectious diseases underscore the importance of non-targeted sequencing for identifying unknown or rare pathogens, including co-infections. However, enriching low-abundance viral sequences in RNA metaviromics, particularly in the preparation of cDNA libraries and their compatibility with next-generation sequencing (NGS) and third-generation sequencing (TGS), remains challenging. Therefore, our objective is to develop and systematically assess a practical RNA metavirome methodology specifically tailored for the enrichment of low-abundance viral sequences within samples. METHODS: We developed the SMART-RNA-Metavirome platform, integrating SMART-9n library preparation with NGS and TGS technologies. Total RNA was extracted from two field-collected wild Aedes albopictus pools, along with one laboratory-infected Ae. albopictus pool harboring dengue virus (DENV). This RNA was subjected to reverse transcription using both this optimized protocol and random primer-based methods, followed by high-throughput sequencing on Illumina, Oxford Nanopore, and QitanTech Nanopore technologies. Welch's t-test was employed for comparative analysis of the subsequent RNA metavirome data, specifically to evaluate differences in viral species composition and abundance of viral reads between experimental groups. Furthermore, the effectiveness of this platform was systematically validated via RT-qPCR and SMART-RNA-Metavirome-based Oxford Nanopore sequencing across multiple sample types, including mosquito specimens from DENV-infected Ae. albopictus, serum samples from dengue patients and viral isolates of Japanese encephalitis virus (JEV) and Zika virus (ZIKV). RESULTS: The SMART-RNA-Metavirome platform has been systematically validated to excel in enriching the composition and diversity of the RNA virome (P = 0.04), providing sufficient coverage for the complete reconstruction of viral genomes. When employed in the detection of DENV-infected Ae. albopictus, clinical serum samples, and viral isolates of JEV and ZIKV, this technique exhibits a robust correlation with RT-qPCR (r2 > 0.95). Notably, it demonstrates exceptional sensitivity, ensuring sufficient coverage even in samples of DENV-infected Ae. albopictus with a Ct-value of 35.3, attaining an impressive 99.88% genome coverage. Furthermore, this platform possesses the capability to identify virus species and determine their serotypes. CONCLUSIONS: In our study, the SMART-RNA-Metavirome platform outperforms traditional methods, enriching RNA virome composition and diversity, enabling practical compatibility with both NGS and TGS technologies. It demonstrates significant proficiency in detecting both known and unknown arboviruses, even in low-titer samples such as those from wild mosquitoes and clinical sera. This platform facilitates comprehensive monitoring, risk assessment, and early warning of RNA virus transmissions, enhancing our understanding of RNA virome diversity and ecological patterns.

High-Throughput Nucleotide Sequencing↗

PubMed1979

Seasonal abundance of Culex annulirostris and other mosquitoes at Kowanyama, north Queensland, and Charleville, south west Queensland.

At Kowanyama, 104,006 adult female mosquitoes of a probable 47 species were collected on twelve occasions from April, 1972, to April, 1976. At Charleville, 71,791 adult females of 20 taxa were taken during thirteen periods from February, 1974, to February, 1976. Culex annulirostris was predominant at Kowanyama and especially at Charleville where it comprised up to 99% of dry season collections. Culex fatigans, Ae normanensis, An annulipes and, at Kowanyama only, An bancroftii were the other major species collected. Several bait trapping methods were suitable for monitoring densities of Cx annulirostris and other species but mammalian baits were best. Because Cx annulirostris activity continued throughout winter even at Charleville, continuous cycles of arbovirus transmission are possible, although the potential of dormant Aedes eggs also bears investigation as a method of arbovirus survival.

Animals↗

PubMed2025

Clinical and laboratory profiles of Oropouche virus disease from the 2024 outbreak in Manaus, Brazilian Amazon.

BACKGROUND: The 2024 Oropouche virus (OROV) outbreak in Brazil raised public health concerns due to its unprecedented rapid spread, high incidence, and potential neurological complications. OROV symptoms overlap with locally endemic arbovirus diseases, like dengue virus (DENV), complicating diagnosis. The study aimed to compare clinical, laboratory, and immunological profiles in OROV and DENV cases, crucial for improving diagnosis and management. METHODS: This study analyzed 51 OROV and 78 of DENV cases consecutively enrolled in Manaus, Amazonas, Brazil, and monitored for 28 days. OROV diagnosis was performed by real-time PCR (RT-PCR) using serum and urine samples. OROV RT-PCR positive samples were genotyped. A paired Plaque Reduction Neutralization Test (PRNT) was conducted on samples collected at D1 and D28. Patients with a ≥ 4-fold increase in neutralizing antibody titer between D1 and D28 were considered OROV-positive. Clinical manifestations, hematology, biochemistry, and cytokine profiles were analyzed. Statistical analysis included comparison between OROV and DENV patients. RESULTS: Genome sequencing of OROV isolates confirmed presence of a previously reported novel reassortment event, consistent with ongoing localized transmission. Urine RT-PCR demonstrated low positivity compared to serum samples. The paired PRNT increased sensitivity in 45%. Clinically, OROV infection was associated with significantly higher frequencies of severe headache, myalgia, arthralgia, and rash compared to DENV infection (p < 0.001). Elevated alanine aminotransferase (ALT) levels were also observed in OROV patients (p < 0.001). Immunologically, OROV infection induced significantly increased levels of acute-phase CCL11 (eotaxin), CXCL10, IFN-γ, IL-1RA, and IL-10, which declined by day 28, while IL-5 increased during recovery. In contrast, DENV patients exhibited elevated levels of CCL2, G-CSF, and CCL3 in recovery phase. CONCLUSION: OROV symptoms overlap with DENV underscores the need for syndromic diagnostic approach in endemic regions. Continued genomic surveillance and expanded clinical studies are vital to assess long-term consequences. Given OROV's expanding geographic range, targeted public health measures are essential to mitigate future outbreaks and better understand its pathophysiology.

Humans↗

PubMed2027

Protocol for Detecting and Sequencing Chikungunya Virus from Field-Collected Mosquitoes.

Arboviral diseases represent a major public health challenge, especially in tropical regions where environmental conditions may favor the proliferation and spread of mosquito vectors. Thus, early and accurate detection of chikungunya virus (CHIKV) in mosquito populations can be a valuable tool for effective surveillance of circulating variants and for identifying new viral introductions. Given the challenges of detecting arboviruses in field-captured mosquitoes, we describe an integrated workflow for CHIKV molecular detection and whole-genome sequencing. This protocol includes mosquito homogenization using a bead-based mechanical disruptor, RNA extraction using TRIzol reagent with minor modifications, molecular screening using CHIKV-specific RT-qPCR, and whole-genome amplification followed by sequencing on Illumina platforms. Despite the protocol being optimized for individual mosquitoes, it results in high-quality RNA suitable for both entomological surveillance and genomic analysis. As this protocol allows recovery of complete CHIKV genomes from mosquito specimens, it can serve as a basis for genomic epidemiology studies, enabling monitoring of viral diversity and lineage dynamics, and facilitating early detection of emerging variants to support timely and targeted public health interventions in endemic and at-risk regions.

Animals↗

PubMed2026

Widespread circulation of Alongshan virus in Austria and serological evidence for infection in humans: a nationwide molecular and serological observational study.

BACKGROUND: Alongshan virus, a segmented RNA virus in the Jingmenvirus group of flaviviruses, was first identified in 2017 in China in patients with tick-borne encephalitis-like illness. Alongshan virus has since been detected in ticks and mammals in several European countries. In this study, we aimed to characterise the temporal and geographical distribution of Alongshan virus in Austria through nationwide tick surveillance combined with serological and molecular screening in individuals with suspected tick-borne encephalitis or tick exposure. METHODS: In this nationwide molecular and serological observational study, we conducted a PCR-based screening of ticks collected across Austria in 2024, using flagging, animal hosts, and citizen submissions. We also collated genomic data from stored nucleic acid extracts from ticks collected in Austria in 2005 and 2013 in previous surveillance studies and stored extracts of paired tick-human samples collected between 2015 and 2018. Alongshan virus-positive tick samples were subjected to whole-genome sequencing and phylogenetic analysis. In addition to tick samples, blood samples from Austrian patients with reported tick bite or suspected tick-borne encephalitis, submitted to the National Reference Center for Human Arbovirus Infections, Austria, were screened for Alongshan virus infection and subjected to serological and molecular testing. FINDINGS: 2952 ticks were collected between Feb 1, 2024, and Dec 6, 2024, from 29 (83%) of 35 NUTS-3 regions in Austria; the median detection rate for Alongshan virus was 1·2% (IQR 0·4-3·5). In addition, 1816 archived tick samples were analysed, with three testing positive for Alongshan virus. For the virus-positive samples, phylogenetic analysis showed that sequences from Austria grouped within the European clade, with Austrian sequences from the same region showing high sequence similarity. 1361 human serum samples collected between March 1, 2023, and May 16, 2025, were assessed for anti-Alongshan virus IgG antibodies. Two individuals had high antibody titres against Alongshan virus proteins VP1a and VP2. INTERPRETATION: Our study shows detection of Alongshan virus in archived tick samples dating back to 2005, representing the earliest documented occurrence of the virus to date and suggesting that Alongshan virus has been circulating in Austria for at least two decades. The detection of Alongshan virus-specific antibody titres in two individuals suggests past infection and previously unrecognised exposure. These results highlight the need for continued molecular surveillance of tick populations and serological monitoring in humans to define the epidemiology and public health relevance of Alongshan virus in Europe. FUNDING: One Health surveillance and Vector monitoring for cross-border pathogens (OH SURVector) and UNITED4Surveillance.

Adolescent↗

PubMed2026

Emergence and phylogeography of the dengue vector Aedes aegypti in Southeastern Iran.

BACKGROUND: Aedes (Stegomyia) aegypti (Linnaeus) is the primary vector of dengue, chikungunya, Zika, and yellow fever viruses. Its recent detection in southeastern Iran raises public health concerns about arbovirus spread to new regions. This study provides the first genetic and phylogeographic analysis of Ae. aegypti populations from Sistan and Baluchistan Province (SBP), Iran, to infer their origin and invasion pathways. METHODS: Mitochondrial COI and ND4 genes were analysed in newly collected Ae. aegypti specimens from border areas, ports, and urban centres of SBP. Haplotype network analyses were constructed using the TCS method in PopART, and phylogenetic analyses were conducted using global reference sequences. RESULTS: Iranian specimens comprised 7 COI haplotypes (n = 18) and 10 ND4 haplotypes (n = 17). COI phylogeny placed Iranian specimens into two main clades, while ND4 analysis distributed them across several derived clades, mostly clustering with lineages from Latin America (Brazil, Mexico) or Africa. One Iranian specimen showed a close relationship with a Saudi Arabian sequence (bootstrap: 98%) near the basal region. Combined COI + ND4 analysis revealed a monophyletic clade of Iranian specimens with a Sri Lankan specimen, distinct from other global lineages. The global COI network (n = 47) showed a star-like topology with a dominant haplotype 1 shared among 10 Iranian specimens. The ND4 network (n = 31) revealed a complex topology with 18 haplotypes, where a Saudi Arabian and one Iranian specimen (~30 mutational steps) possibly represented the peripheral root. CONCLUSIONS: Detection of diverse Ae. aegypti clades confirm establishment of this vector in southeastern Iran. Results support multiple introductions and genetic connectivity with Latin America, Africa, and South Asia, pointing to an emerging invasion corridor. Continued genomic surveillance and integrated vector monitoring are urgently needed to guide prevention strategies.

Animals↗