Metagenomic identification of Newcastle disease virus sequences in a pediatric case of acute undifferentiated febrile illness at the Colombian–Venezuelan border

Epidemiology and Molecular Characterization of Human Metapneumovirus in Influenza-Like Illness: A Decade of Surveillance in Dibrugarh, Assam (2014-2025).

BACKGROUND Human metapneumovirus (hMPV) is a globally recognized cause of influenza-like illness (ILI), especially among children, but data from Northeast India are limited. This study analyzed epidemiological and molecular features of hMPV detected in Dibrugarh, Assam, over a decade-long surveillance period. METHODS Between May 2014 and November 2025, 3,597 ILI patients were screened for hMPV at a tertiary hospital in Dibrugarh, Assam. Clinical samples (nasopharyngeal/throat swabs) were tested using multiplex respiratory panels and TaqMan-based real-time RT-PCR. Partial sequencing of the G gene was performed by Sanger sequencing, followed by phylogenetic and glycosylation analyses using MEGA 11, NetNGlyc, and NetOGlyc tools. Statistical analysis was done using SPSS v26. RESULTS Overall, hMPV prevalence was 3.1% (112/3597), significantly higher among children aged <5 years than older individuals (3.9% vs. 1.3%; p = 0.003). hMPV activity exhibited marked winter seasonality, with significantly higher positivity during the colder months (5.6% vs. 0.8%; p < 0.001). Phylogenetic analysis demonstrated the circulation of A2.2.1 (A2b1), A2.2.2 (A2b2), and B2 lineages in Assam. The two 2025 A2.2.2 strains harbored the characteristic 111-nucleotide G-gene duplication, while comparative glycosylation analysis revealed lineage-specific variation in predicted N- and O-linked glycosylation profiles. CONCLUSION hMPV remains an important cause of pediatric influenza-like illness in Northeast India, with marked winter seasonality. The detection of A2.2.1, A2.2.2, and B2 lineages, including A2.2.2 strains with the characteristic 111-nucleotide G-gene duplication, underscores the importance of continued molecular surveillance.

First detection and molecular characterization of influenza D virus in livestock from Kazakhstan reveals circulation of the D/Yama2019 lineage

Influenza D virus (IDV) is an emerging Orthomyxovirus primarily associated with cattle and increasingly recognized as a pathogen of veterinary and zoonotic relevance. Despite growing evidence of its circulation across Asia, Europe, and North America, no data have previously been available from Kazakhstan or Central Asia. This study investigated the presence of IDV in domestic livestock samples collected during active surveillance between 2023 and 2026. A total of 867 biological samples were obtained from cattle (n = 611), camels (n = 175), Maral Deer (n = 51), and pigs (n = 30) across multiple regions of Kazakhstan. Viral RNA was screened by reverse transcription polymerase chain reaction (RT-PCR) targeting the HEF, PB1, PB2, and P3 genes using a newly designed primer panel. Positive amplicons were subjected to Sanger sequencing and phylogenetic analysis. IDV RNA was detected in 11 animals, corresponding to an overall prevalence of 1.27%. Positive samples were identified in several geographically distinct regions, indicating wider circulation than previously recognized. Complete HEF gene sequences were successfully obtained from two representative cattle strains, designated D/bovine/Zhetysu/KAZ/678/2025 and D/bovine/Zhetysu/KAZ/680/2025. Phylogenetic analysis demonstrated that both strains clustered within the D/Yama2019 lineage and shared >98.83% nucleotide identity with contemporary Asian strains. To our knowledge, this is the first report of IDV detection in Kazakhstan. These findings expand the known geographic range of IDV and suggest possible transboundary transmission routes across Central Asia. Continued molecular surveillance is warranted to clarify epidemiology, host range, and potential economic impact.

MOLECULAR – GENETIC CONFIRMATION OF INFLUENZA D VIRUS CIRCULATION IN LIVESTOCK OF KAZAKHSTAN

Influenza D virus (IDV) is an emerging respiratory pathogen with a broad host range and increasing recognition as a potential zoonotic agent. Despite growing evidence of IDV circulation across Asia, Europe, and North America, its epidemiology in Central Asia has remained unknown. Here, we report the first molecular detection and genetic characterization of IDV in Kazakhstan. Between 2023 and 2025, a total of 616 biological samples were collected from livestock in multiple regions of Kazakhstan, including cattle (n=387), Altai maral (n=51), camels (n=148), and pigs (n=30). Samples were screened using real-time reverse transcription PCR, and positive specimens were subjected to metagenomic sequencing and phylogenetic analysis targeting partial polymerase basic 1 (PB1) and hemagglutinin-esterase-fusion (HEF) gene regions. Influenza D virus RNA was detected in 9 of 616 samples (1.46%), with positive detections restricted to cattle (7/387; 1.81%) and Altai maral (2/51; 3.92%) from the Zhetysu and East Kazakhstan regions. No viral RNA was detected in camel or pig samples. Phylogenetic analyses revealed that the detected strains clustered within the D/Yama2019 lineage and exhibited high nucleotide identity (>97-98%) with contemporary Chinese bovine strains collected during 2021-2022. Metagenomic sequencing confirmed the presence of IDV-associated PB1 and HEF fragments without evidence of major structural variation in the analyzed genomic regions. The identification of genetically related strains across neighboring regions suggests regional dissemination potentially associated with transboundary livestock movement and animal trade. The detection of IDV in Altai maral expands current knowledge regarding the host spectrum of the virus and raises the possibility that wild or semi-domesticated ruminants may contribute to local transmission ecology. Collectively, these findings provide the first evidence of IDV circulation in Kazakhstan, extend the known geographic range of the virus to Central Asia, and highlight the need for integrated veterinary and zoonotic surveillance under a One Health framework. Вирус гриппа D (Influenza D virus, IDV) представляет собой эмерджентный респираторный патоген с широким спектром хозяев и все более признаваемым зоонозным потенциалом. Несмотря на накопление данных о циркуляции IDV в странах Азии, Европы и Северной Америки, его эпидемиология в Центральной Азии до настоящего времени оставалась неизвестной. В настоящем исследовании представлено первое молекулярное выявление и генетическая характеристика вируса гриппа D в Казахстане. В период 2023–2025 гг. в различных регионах Казахстана было собрано 616 биологических образцов от сельскохозяйственных животных, включая крупный рогатый скот (n=387), алтайского марала (n=51), верблюдов (n=148) и свиней (n=30). Скрининг образцов проводили методом обратной транскрипции с полимеразной цепной реакцией в реальном времени (RT-PCR), а положительные образцы подвергали метагеномному секвенированию и филогенетическому анализу, направленным на частичные участки генов polymerase basic 1 (PB1) и hemagglutinin-esterase-fusion(HEF). РНК вируса гриппа D была обнаружена в 9 из 616 образцов (1,46%), при этом положительные результаты выявлены исключительно у крупного рогатого скота (7/387; 1,81%) и алтайских маралов (2/51; 3,92%) из Жетысуской и Восточно-Казахстанской областей. В образцах верблюдов и свиней вирусная РНК не обнаружена. Филогенетический анализ показал, что выявленные штаммы относятся к линии D/Yama2019 и характеризуются высокой нуклеотидной идентичностью (>97–98%) с современными китайскими штаммами, выделенными от крупного рогатого скота в 2021–2022 гг. Метагеномное секвенирование подтвердило присутствие фрагментов генов PB1 и HEF, ассоциированных с IDV, без признаков крупных структурных вариаций в исследованных геномных регионах. Выявление генетически близких штаммов в соседних регионах указывает на региональное распространение вируса, потенциально связанное с трансграничным перемещением животных и торговлей скотом. Обнаружение IDV у алтайского марала (Cervus elaphus maral) расширяет современные представления о спектре хозяев данного вируса и указывает на возможную роль диких или полуодомашненных жвачных в локальной экологии передачи инфекции. В совокупности полученные результаты представляют собой первое доказательство циркуляции IDV в Казахстане, расширяют известный географический ареал вируса на территорию Центральной Азии и подчеркивают необходимость интегрированного ветеринарного и зоонозного надзора в рамках концепции One Health(«Единое здоровье»).

Metagenomic sequencing detects viruses and bacteria in a cross-sectional clinical cohort of undifferentiated febrile illness in Nigeria

ABSTRACT Molecular and microscopy-based diagnostic capacity is often insufficient or unavailable in places where infectious disease burdens are highest, such as in West Africa. Rapid diagnostic testing (RDT) can provide quick and affordable diagnoses of common infections but is an imperfect solution due to limitations around detecting and dealing with false-negative and false-positive results. An alternative to RDT is unbiased metagenomic sequencing for pathogen surveillance. Here, we present data from unbiased metagenomic sequencing used to identify causes of undiagnosed febrile illness in Jos, Plateau State, Nigeria. Proof of concept for this approach has been demonstrated by several groups who have identified epidemic and endemic viral diseases like Lassa fever, yellow fever, and chikungunya. We show that unbiased deep sequencing and metagenomic analysis can be used to identify RNA viruses in clinical samples. We sequenced RNA from sera of patients (n = 343), many of whom were acutely febrile (76%), in a survey of clinics in Jos. We detected five human-infecting viruses in 39 (11 %) specimens. Among these were hepatitis B virus, human pegivirus, and several anelloviruses. While most of the viruses identified are unlikely to cause clinical symptoms in the patients we sampled, their presence demonstrates the validity of our approach. Additionally, our sequencing data allowed us to identify genetic material from potentially pathogenic bacteria, another possible etiological agent of febrile illness. IMPORTANCE In low-resource areas, fevers due to infectious pathogens are a major source of illness, but tools for detecting and identifying such pathogens are often limited. Unbiased approaches for identifying genetic material from all potentially infectious organisms in a sample represent an opportunity for discovering sources of fever. Metagenomic sequencing can improve insight into pathogen landscapes in low-resource settings, potentially providing early detection of disease outbreaks. However, unbiased metagenomic sequencing (mNGS) is no panacea; it is susceptible to contamination and false positives. We used mNGS to evaluate serum from >300 Nigerian clinic-goers in Jos, Nigeria, most of whom (>70%) had fevers of unknown origin. Our goal was to understand arbovirus prevalence in Jos, Nigeria, and identify the sources of infection not routinely monitored for at clinics. We detected hepatitis B virus, as well as nonpathogenic anelloviruses. Our study provides insight into the utility and limitations of mNGS for pathogen surveillance. In low-resource areas, fevers due to infectious pathogens are a major source of illness, but tools for detecting and identifying such pathogens are often limited. Unbiased approaches for identifying genetic material from all potentially infectious organisms in a sample represent an opportunity for discovering sources of fever. Metagenomic sequencing can improve insight into pathogen landscapes in low-resource settings, potentially providing early detection of disease outbreaks. However, unbiased metagenomic sequencing (mNGS) is no panacea; it is susceptible to contamination and false positives. We used mNGS to evaluate serum from >300 Nigerian clinic-goers in Jos, Nigeria, most of whom (>70%) had fevers of unknown origin. Our goal was to understand arbovirus prevalence in Jos, Nigeria, and identify the sources of infection not routinely monitored for at clinics. We detected hepatitis B virus, as well as nonpathogenic anelloviruses. Our study provides insight into the utility and limitations of mNGS for pathogen surveillance.

Shotgun metagenomic sequencing and genetic characterization of four enteric viruses in a dairy farm suffering a diarrhea outbreak of undetermined cause in adult cattle

Molecular Epidemiology of Emerging Subvariants of the SARS-CoV-2 Omicron Lineage: A Cross-sectional Study from Himachal Pradesh, India

Tracking SARS-CoV-2 variants revealed the predominance of the JN.1 sublineage of Omicron with generally mild symptoms, highlighting the need for robust, ongoing genomic surveillance integrated with epidemiological data to enable early detection of emerging variants and strengthen control strategies.