Rabies remains a formidable global public health challenge, attributable to viruses of the genus Lyssavirus. Control efforts have progressed markedly through the introduction of inactivated vaccines, rabies immunoglobulin (RIG), and monoclonal antibodies (mAbs), which collectively provide both pre- and post-exposure prophylaxis (PrEP and PEP). In low-resource environments, however, PEP implementation is hindered by the lack of highly sensitive diagnostic assays. At present, the gold-standard techniques endorsed by the World Organization for Animal Health (WOAH) and the World Health Organization (WHO) — namely the Rapid Fluorescent Focus Inhibition Test (RFFIT) and the Fluorescent Antibody Virus Neutralization (FAVN) assay — are employed to quantify rabies virus neutralizing antibody (VNA) titers. These assays, nonetheless, are labor-intensive, time-consuming, and necessitate handling of live virus. This review elucidates emerging methodologies for antibody quantification against rabies virus. Notably, the recent advancement of an Enzyme-Linked Immunosorbent Assay (ELISA) — characterized by rapid turnaround, high specificity and sensitivity, cost-effectiveness, and scalability has significantly enhanced detection of antibodies directed against the rabies virus glycoprotein. We also discuss the principle technical obstacles encountered during the assay development. Looking forward, these innovations are poised to improve both the accuracy and throughput of rabies serology, thereby streamlining efficacy evaluations and prophylaxis monitoring, and ultimately bolstering global rabies control and eradication initiatives.
Bean common mosaic virus (BCMV) is a serious viral pathogen of leguminous crops especially common beans. BCMV is transmitted through infected seeds and can also spread horizontally by aphid vectors in a non-persistent manner. It is highly stable within the embryo of seed that is the major factor contributing for its long-distance dissemination across continents. Therefore, there is an urgent need to develop a reliable, rapid and cost-effective diagnostic method that can facilitate the detection of the virus in seed certification programs and plant quarantine facilities, thereby prevents its further spread. The present study describes an efficient and accurate detection of BCMV by reverse transcriptase-recombinase polymerase amplification (RT-RPA) assay. The primers were designed from highly conserved region of coat protein region. The BCMV-specific primers successfully amplified fragment of the coat protein (CP) gene under isothermal conditions at 37 °C within 20 min. The limit of detection of the developed assay was determined using both purified RNA and crude plant sap extract. In both cases, the assay exhibited higher sensitivity than conventional RT-PCR, enabling the detection of BCMV at lower template concentrations. In addition, specificity of the assay was tested by cross reaction with closely related non-target viruses (bean common mosaic necrosis virus (BCMNV), clover yellow vein virus (ClYVV), soybean mosaic virus (SMV) and cucumber mosaic virus (CMV) BCMNV). The RT-RPA assay accurately distinguished BCMV from other non-target viruses showing comparable specificity with RT-PCR. To enhance the suitability of the assay for resource-limited settings and reduce the overall detection time, the assay was adapted to accurately detect BCMV directly from crude sap.
Monkeypox disease develops from the monkeypox virus, which contains double-stranded DNA and belongs to the orthopoxvirus genus found under the family Poxviridae, subfamily Chordopoxvirinae. The WHO director declared the multiregional monkeypox outbreak an international public health emergency. This review aims to summarise the pathogenesis, virology, epidemiology, clinical features, diagnostics, the available therapeutic and prophylactic measures, as well as future perspectives, research gaps, and global preparedness for monkeypox. Monkeypox virus has an architectural resemblance to the variola virus but has its own genetic and pathogenic characteristics. Monkeypox virus has evolved globally to spread from specific African regions to cause human-to-human transmission in other worldwide territories. Standard clinical signs, including fever and headache pain along with muscle pain, swollen lymph nodes, and rashes in specific body regions, manifest as the first step of diagnosis. Traditional polymerase chain reaction together with real-time polymerase chain reaction (RT-PCR) stands as the main diagnostic tool that healthcare providers use with imaging studies for complication assessment. Some antiviral drugs, including tecovirimat, cidofovir, and brincidofovir, are FDA-approved for the treatment of smallpox and are used for mpox under the Expanded Access Investigational New Drug (EA-IND) protocol or other authorised investigational/off-label settings for eligible patients. JYNNEOS (IMVANEX) is approved by the FDA and EMA for the prevention of both smallpox and mpox, whereas ACAM2000 is approved for smallpox and may be used for mpox in specific off-label or public health settings. The primary preventive strategy involves community-oriented preventive initiatives for infectious diseases, including future studies regarding gaps in vaccinations, the dynamics of transmission, and enhancing the preparedness of global health systems. The comprehensive review reveals Mpox disease history and current knowledge through a systematic evaluation of disease source, virology, disease transmission and clinical presentation, detection, management and prevention approaches and future plans to stop this infection.
Hepatitis E virus (HEV) genotypes A3 and A4 are predominantly associated with swine and have been reported in zoonotic infections. Differences in codon usage between these genotypes within a common swine reservoir remain poorly characterized. This study analyzed 114 complete coding sequences of swine-associated HEV-A3 (n = 62) and HEV-A4 (n = 52). Nucleotide composition, relative synonymous codon usage (RSCU), effective number of codons (ENC), neutrality, parity rule 2 (PR2), dinucleotide abundance, codon adaptation index (CAI), principal component analysis (PCA), PERMANOVA, and site-specific selection analyses were performed for the complete coding region and separately for ORF1, ORF2, and ORF3. Both genotypes were GC-rich, with GC3 values of 57.66
Infectious bronchitis virus (IBV) is a major respiratory pathogen in poultry, yet its impact on the tracheal microbiota under field conditions remains insufficiently understood. This study investigated the effects of IBV infection intensity on tracheal microbial diversity and composition in broiler chickens using 16 S rRNA gene sequencing. A total of 200 tracheal samples were collected from commercial farms and screened using RT-qPCR. Samples were stratified into high, low, and negative viral load groups based on cycle threshold (Ct) values, and a representative subset was selected for microbiome analysis. Alpha diversity analysis revealed a significant reduction in microbial richness (Observed species and Chao1 indices; q < 0.05) in the low-viral-load group compared with controls, while Shannon diversity remained unchanged, suggesting a selective loss of rare taxa. In contrast, no significant changes in alpha diversity were observed in the high viral load group. Beta-diversity analysis based on Bray–Curtis dissimilarity (PERMANOVA) revealed significant differences in microbial community composition among different viral load groups. Taxonomic analysis showed stability at the phylum level, with more pronounced shifts at the genus level. These findings indicate that IBV infection induces selective and load-dependent alterations in the tracheal microbiota. Notably, treating infection as a binary variable may obscure ecologically meaningful and biologically relevant microbial shifts. This preliminary field study provides evidence that IBV viral load is associated with differences in the respiratory microbiome of broiler chickens under field conditions.
Rabies, a fatal zoonotic encephalitis, causes approximately 60,000 deaths annually worldwide. The current gold standard for laboratory confirmation is the direct fluorescent antibody test (DFAT) on postmortem brain samples. To overcome the limitations of DFAT, real-time PCR for rabies viral RNA detection has emerged as a reliable alternative for both antemortem and postmortem samples. This study evaluates the diagnostic performance of the Quantiplus Rabies Virus Detection Kit, a commercially available real-time PCR kit for detecting rabies RNA. The study included 24 postmortem human brain samples, 14 antemortem human samples (cerebrospinal fluid, saliva, and nuchal skin), and 44 postmortem animal brain samples. DFAT and Quantiplus assays were performed on brain samples, while Real-time TaqMan RT-PCR and Quantiplus assays were used on antemortem human samples. Using DFAT as the reference standard, the Quantiplus assay demonstrated 100
Acute respiratory infections continue to be a major source of sickness and hospitalisation in all age groups, particularly in settings with limited access to molecular diagnostic data. To improve clinical management and public health initiatives, it is crucial to identify circulating respiratory viruses and their co-infection patterns. The State Virology Laboratory, Gandhi Medical College, Bhopal, India, conducted a short-term molecular study of ARIs from July to October 2025, and the results are reported in this article. A validated multiplex real-time PCR assay was used to examine swab samples from the nasopharynx and/or oropharynx of suspected cases. This assay can identify SARS-CoV-2, human adenovirus, respiratory syncytial virus, influenza A subtypes, and influenza B. Descriptive analysis was performed on clinical and demographic data, classifying cases as severe ARIs or ILI, and evaluating mono- and mixed-viral detection patterns. Multiple respiratory viruses were detected across all age categories, with influenza viruses and SARS-CoV-2 more common in adults and respiratory syncytial virus and adenovirus being the most prevalent, particularly in children. Mixed viral detections were observed across clinical categories and were more frequently recorded among SARI cases. To facilitate prompt clinical and public health interventions, these results highlight the utility of multiplex molecular diagnostics and the need for sustained respiratory virus surveillance.
Hepatitis B virus, a pararetrovirus and member of hepadnaviridae family, harbours a partially double-stranded DNA genome that is transcribed by the host RNA polymerase II to generate an intron-containing pregenomic RNA (pgRNA). Nuclear export of pgRNA utilizing the canonical host pathways remains central to successful virus replication since it is reverse transcribed by the viral P protein within the nucleocapsid in cytoplasm. We provide direct microscopic evidence of Polypyrimidine tract binding protein (PTB) mediated inhibition of nuclear export of a pgRNA based intron-containing pre-mRNA (CPS-PRE) with quantitative RNA-FISH. We also demonstrate through RNA immunoprecipitation experiments that CPS-PRE minigene transcripts stably interact with members of NXF1-NXT1 pathway and no crosslinking with karyopherin CRM1 could be seen. Our data reinforces a novel aspect of PTB in pre-mRNA nuclear export for active retention of intron-containing transcripts and directly shows the ability of pgRNA components to interact with NXF1-NXT1 heterodimer.
Despite the World Health Organization’s declaration in May 2023 of the end of the Public Health Emergency of International Concern (PHEIC), COVID-19 continues to circulate globally. As such, the continued transmission across the different parts of the globe resulted in the emergence of variants which could pose a constant challenge to public health. The isolation of circulating virus variants during these times is essential to assess the impact of the strains and establish effective counter-measures if necessary. This study focuses on the isolation and characterization of the SARS-CoV-2 recombinant Omicron variant XBB.1.16 from a clinical sample in Central India, collected during an early 2023 surge. Using a throat swab, confirmation of the variant by qRT-PCR and distinctive Cytopathic Effects (CPE) were observed on the Vero E6 cell line. The isolation by passages of the virus was found to be successful through positive qRT-PCR and next-generation sequencing (NGS). The isolate was found to be XBB.1.16, Clade GRA, a Variant of Interest (VOI) after sequencing. Detailed analysis of the spike gene indicated 53 mutations leading to 45 amino acid substitutions compared to the original Wuhan strain.
Sodium butyrate (NaB), a histone deacetylase inhibitor, can induce lytic replication of Epstein-Barr virus (EBV). This study aimed to investigate the potential role of EBV reactivation in tumorigenic mechanisms in EBV-associated gastric cancer (EBVaGC) by activating EBV lytic replication using NaB. AGS cells (EBV-negative) and AGS-EBV cells (EBV-positive) were treated with NaB for 12 and 24 h, followed by transcriptomic sequencing to obtain host gene expression profiles. Three groups of differentially expressed genes (DEGs) were identified: Control vs. NaB 12 h (813 DEGs), Control vs. NaB 24 h (1243 DEGs), and Control vs. NaB combining 12 h and 24 h (1528 DEGs). GO and KEGG analyses revealed that these DEGs were significantly enriched in EBV infection, immune-inflammatory pathways (such as TNF and NF-κB signaling), and cancer-related pathways, indicating that EBV lytic activation triggers these signaling events and induces a strong host immune response along with carcinogenic effects. A protein-protein interaction (PPI) network was constructed and imported into Cytoscape, and the cytoHubba plugin was used to screen for hub genes. Seven consensus hub genes (TLR2, IL1A, CSF1, PECAM1, CXCL10, CD4, CD34) were identified by intersecting the results from different algorithms, and their mRNA expression levels were validated by qRT-PCR. Notably, only CXCL10 expression was consistent with the transcriptomic sequencing results, suggesting that under the conditions of EBV infection and NaB treatment, CXCL10 may play a crucial role in the biological responses of host cells, providing important clues for exploring the mechanism of EBV infection and identifying the targets of NaB.
Crimean Congo haemorrhagic Fever remains a significant public health problem, especially in endemic areas, and is transmitted through Hyalomma ticks and direct contact with the blood or tissues of infected livestock. This study aimed to assess the association between household animals, tick bites, and contact with raw or fresh meat and the transmission of disease in Thi Qar Province, Iraq. A retrospective cross-sectional analytical study was conducted, including 806 suspected cases, of which 190 were positive for Crimean Congo haemorrhagic Fever (23.6
Foot-and-mouth disease (FMD) is a serious transboundary infectious disease that affects all cloven-hoofed animals of economic importance. Non-structural protein (NSP)-ELISA diagnostic assays are extensively used to detect FMD virus exposure in susceptible animals. The available NSP assays require different cutoffs for their implementation, and these cutoffs are determined through empirical and statistical (bootstrap and OptimalCutpoints) approaches. Further, the slightest changes in the cutoffs significantly affect the performance of these assays. Therefore, this study presents a cutoff independent machine learning-based computational model, FmdNspPred, for the prediction of FMD virus exposure of the susceptible animals. Here, logistic regression models were trained on absorbance data of 2B NSP-ELISA from the bovine serum samples (n = 1355) collected from various locations in India. The repeated 5-fold cross-validation accuracy and sensitivity of the FmdNspPred model were found to be highest (96 https://github.com/ICARNIFMD/FmdNspPred ). The source R-code is also provided for large-scale and offline predictions ( https://github.com/ICARNIFMD/FmdNspPred/tree/master/R ). The proposed FmdNspPred model is cutoff independent and expresses virus exposure in terms of probabilistic values, which is easy for epidemiological interpretations.
Broussonetia papyrifera (paper mulberry) is an economically important species in East Asia used for fiber production, traditional medicine, and ecological restoration. During a field survey, virus-like symptoms, including chlorotic mottling and mosaic were observed in B. papyrifera leaves. High-throughput sequencing of total RNA identified citrus leaf blotch virus (CLBV), a member of the genus Citrivirus within the family Betaflexiviridae. De novo assembly generated an 8698-nt contig showing 86% nt identity with CLBV isolate Ac-MX. The genome contained three open reading frames encoding a replication-associated polyprotein, a movement protein, and a coat protein. RT-PCR and Sanger sequencing confirmed the presence of CLBV. Rapid amplification of cDNA ends validated the genome termini and completed the viral genome sequence, which was deposited in GenBank under accession number PX939710. Phylogenetic analysis based on complete genome sequences placed the B. papyrifera isolate within a clade of CLBV isolates previously reported from Actinidia species. This study represents the first report of CLBV infecting B. papyrifera in South Korea.
Lichens represent complex symbiotic systems consisting of a fungal partner and photosynthetic microorganisms, and they could also harbor diverse associated microbiota including viruses. Despite increasing interest in mycoviruses from filamentous fungi, viruses infecting lichenized fungi remain poorly characterized. In this study, we identified and molecularly characterized two novel double-stranded RNA viruses infecting the lichenized fungus Calogaya decipiens (Arnold) Arup, Frödén Søchting. Viral sequences were detected from dsRNA-enriched extracts of a surface-sterilized lichen thallus using random PCR amplification and high-throughput sequencing. Genome analyses revealed that both viruses possess bipartite genomes typical of members of the family Partitiviridae, each consisting of two dsRNA segments encoding an RNA-dependent RNA polymerase (RdRp) and a capsid protein (CP). The genome segments of Calogaya decipiens partitivirus 1 (CdPV1) and Calogaya decipiens partitivirus 2 (CdPV2) range from 1861 to 2283 bp and contain single open reading frames. Phylogenetic analysis based on RdRp amino acid sequences placed CdPV1 within the genus Alphapartitivirus, whereas CdPV2 clustered with members of the genus Betapartitivirus. Conserved catalytic motifs characteristic of dsRNA virus polymerases were identified in both RdRps. Sequence identity comparisons with related viruses supported the classification of CdPV1 and CdPV2 as representatives of two novel partitivirus species. To our knowledge, this study represents the first report of mycoviruses infecting the lichenized fungus C. decipiens.
Okra enation leaf curl virus (OELCuV) is a monopartite begomovirus, frequently detected in mixed infections in okra, together with bhendi yellow vein mosaic virus (BYVMV) and/or its associated betasatellite. Such mixed infections often lead to synergistic relationships that can intensify disease severity and consequently reduce crop productivity. This study investigated the infectivity of OELCuV and its alphasatellite and evaluated their infection dynamics when co-inoculated with BYVMV and/or its betasatellite. Mixed infections resulted in higher virus accumulation compared to single infections, indicating potential synergism between the two okra-infecting begomoviruses. To identify RNA silencing suppressors of OELCuV, transient expression assays were carried out in 16c/GFPi transgenic line as well as using GFP as a reporter protein. The results revealed that multiple viral proteins, including Rep, C2, C4, C5, and V2, act as suppressors of post-transcriptional gene silencing. The presence of multiple suppressor proteins in OELCuV, together with those encoded by BYVMV, likely enhances the ability of the virus complex to counteract host antiviral defences mediated by RNA silencing. By combining symptom analysis, quantitative assessment of viral DNA accumulation, and functional characterization of viral silencing suppressors, this study provides novel insights into the mechanistic basis of OELCuV-associated disease development.
Cancer immunotherapy has transformed oncology; however, its efficacy remains limited in immunologically “cold” tumors with poor immune infiltration. Oncolytic virotherapy (OVT) offers a promising solution through dual mechanisms of selective tumor lysis and immune activation, with advances in genetic engineering further enhancing specificity and therapeutic potency. Despite encouraging clinical progress, the translation of OVT remains constrained by the limited predictive capacity of preclinical models. The concept of translational fidelity refers to the extent to which preclinical models accurately replicate human tumor biology and reliably predict clinical therapeutic responses. This review provides a systematic evaluation of OVT through a four-dimensional framework encompassing structural complexity, immune representation, microenvironmental gradients, and translational predictivity. Using this framework as an integrative lens, we compare conventional and advanced platforms, including three-dimensional organoids, hydrogels, and patient-derived xenografts, highlighting their ability to recapitulate tumor architecture and heterogeneity. However, persistent limitations such as incomplete immune integration, inadequate characterization of physicochemical gradients, and species-specific discrepancies continue to hinder reliable clinical translation and may lead to overestimation of therapeutic efficacy. We further examine emerging engineering strategies in oncolytic viruses and emphasize the need to incorporate immune dynamics, delivery constraints, and patient-specific variability into model design. Future directions focus on developing humanized, multi-dimensional platforms integrating bioengineering, artificial intelligence, and real-time microenvironmental monitoring. Collectively, this review underscores that improving translational fidelity through integrative modeling frameworks is critical for bridging the gap between preclinical findings and clinical outcomes, thereby advancing OVT as a reliable modality in cancer immunotherapy.
Pathogens are the agents responsible for these infections, and they use various mechanisms to invade and evade host defenses, emphasizing the importance of early detection. Molecular techniques such as polymerase chain reaction (PCR) have become the gold standard for detecting viral and bacterial pathogens due to their high sensitivity, specificity, and well-established validation. However, PCR requires thermal cycling equipment, a stable power supply, and trained personnel, which may limit its accessibility in resource-constrained settings. Isothermal amplification techniques are promising alternatives to PCR by providing simplicity, speed, and portability. Among these, recombinase polymerase amplification is particularly notable for its versatility, rapidity, and efficiency in amplifying nucleic acids at constant temperatures. This review discusses pathogen invasion mechanisms and recent advances in molecular diagnostics, with particular emphasis on RPA. The principles of RPA are compared with PCR, highlighting key advantages such as rapid amplification (typically within 20 min), low-temperature operation, minimal instrumentation, and compatibility with point-of-care (POC) platforms. At the same time, limitations of RPA, including primer design complexity, potential non-specific amplification, and current cost considerations, are addressed. RPA represents a versatile and promising tool that complements existing molecular methods and holds significant potential for expanding early infectious disease detection, particularly in decentralized and low-resource environments. By enabling timely and accessible diagnostics, RPA contributes substantially to improved disease surveillance, control, and patient outcomes. Traditional detection methodologies, such as culturing, PCR, and isothermal amplification techniques, with a focus on RPA. Due to its simple procedure, short detection time, high sensitivity and specificity, and suitability for point-of-care testing in remote areas, RPA can be the best alternative for the detection of infectious diseases.
Feline coronavirus (FCoV), the etiological agent of feline infectious peritonitis (FIP), represents a major clinical and economic challenge in feline populations worldwide. However, molecular prevalence and genetic diversity data from India remain limited, and the relatedness of circulating strains to vaccine isolates is poorly defined. In this study, 24 suspected feline cases from Chennai were screened by RT-PCR targeting a 698 bp fragment of the spike (S) gene, and FCoV was detected in 10 cats (41.7
HIV/AIDS is a significant health issue on the global stage and has impacted more than 38 million individuals. Despite the development of antiretroviral therapy (ART), socioeconomic barriers and stigma and discrimination can result in lower quality of life (QoL) in many people living with HIV (PLHIV). There is limited evidence on the determinants of QoL in PLHIV in Nepal. The purpose of the study was to evaluate QoL and its correlates among PLHIV who receive tertiary care services at hospitals in eastern Nepal. A cross-sectional study was conducted among 972 PLHIV aged 18 years and older who had been receiving ART for at least 6 months. A structured questionnaire consisting of sociodemographic, clinical, and psychosocial variables was used to collect data. The CES-D scale was used to measure depression, and the WHOQOL-HIV BREF tool was used to measure QoL. SPSS version 25 was used to analyze data, descriptive statistics, Pearson correlation, and ANOVA (p < 0.05). The mean age of the participants amounted to 41.6 ± 10.6; 59.6
