CMV, a member of the genus Cucumovirus in the family Bromoviridae, is regarded as one of the most destructive viruses of chilli worldwide. It infects more than 1,000 plant species, including vegetables, ornamentals, pulses, and weeds, and is transmitted primarily by aphids in a non-persistent manner. The virus survives in alternate hosts and weed reservoirs, which facilitates rapid dissemination under field conditions, and seed transmission has been reported in several hosts, contributing to long-distance spread. Recent research has further shown that CMV infection can alter host volatile emissions and plant physiology in ways that enhance aphid-mediated transmission, adding to the virus’s epidemiological fitness.

The CMV genome consists of three positive-sense single-stranded RNA molecules, designated RNA1, RNA2, and RNA3, which encode proteins involved in replication, movement, pathogenicity, and suppression of host defense responses. Based on serological properties, nucleotide divergence, and phylogenetic relationships, CMV isolates are classified into subgroup I and subgroup II, with subgroup I further divided into IA and IB lineages. Previous Indian studies have shown that most CMV isolates infecting chilli and other crop hosts predominantly belong to subgroup IA, but the majority of these investigations were confined to southern, western, or central India. Molecular epidemiological information from eastern India, particularly Bihar, remained very limited before this study.

To address this gap, researchers led by Mahesh Kumar of Dr. Kalam Agricultural College, Bihar Agricultural University, Sabour, conducted extensive field surveys during 2019 and 2020 in the major chilli-growing districts of Kishanganj, Purnia, and Bhagalpur. The surveys revealed characteristic viral symptoms including mosaic, mottling, chlorosis, leaf deformation, vein clearing, shoestring leaves, and stunted growth. A total of 355 symptomatic samples were collected: 165 from Kishanganj district at locations including Kharkhari, Motihara, Ganiyabari, Lodhabari, and Arrabari; 70 from Purnia district at Kasba, Jalalgarh, and Dagarua; and 95 from the vegetable-growing belt of Bhagalpur district, particularly Sabour and Kahalgoan near the Ganga River.

Field diagnosis based solely on symptoms proved difficult because CMV symptoms frequently overlap with those induced by other chilli-infecting viruses such as Chilli veinal mottle virus and Capsicum chlorosis virus. Chilli leaf curl virus, which induces typical upward leaf curling, puckering, and reduced leaf lamina, could be visually differentiated and was excluded from the CMV-oriented investigation. All collected samples were screened using Direct Antigen Coating Enzyme-Linked Immunosorbent Assay (DAC-ELISA), a serological method in which clarified leaf sap is adsorbed onto polystyrene microplate wells and probed with CMV polyclonal antiserum followed by an enzyme-conjugated secondary antibody, with absorbance recorded at 405 nm. The assay confirmed CMV infection in 115 of the 355 samples: 64 out of 165 from Kishanganj, 32 out of 70 from Purnia, and 19 out of 95 from Bhagalpur. The highest optical density value, 0.421, was recorded for an isolate from Jalalgarh in Purnia district that exhibited pronounced shoestring leaf symptoms, a frequent association that validates leaf narrowing and filiform symptoms as characteristic indicators of CMV infection in chilli.

Six representative isolates, four from Zone II (Belwa, Kharkhari, Purnia, and Kasba) and two from Zone IIIB (Sabour and Kahalgoan), were selected for molecular characterization based on confirmed CMV positivity, geographic representation, confirmed sap transmission, variation in symptom expression, and availability of high-quality RNA. The isolates were biologically maintained through mechanical sap inoculation on indicator hosts. Inoculated Chenopodium plants developed mild mosaic symptoms from the sixth day after inoculation, progressing to severe mosaic by fifteen days post-inoculation, while Nicotiana glutinosa proved suitable for routine long-term maintenance of the virus cultures. Total RNA extracted from infected plants was subjected to reverse transcription polymerase chain reaction (RT-PCR) using coat protein gene-specific primers, which consistently amplified the expected 657 bp fragment from all six isolates. The amplified products were cloned into the pGEM-T Easy vector, transformed into Escherichia coli, confirmed by colony PCR and restriction digestion, and bidirectionally sequenced before submission to GenBank under accession numbers OQ756157, PV364315, PV364316, PV364319, PV364320, and PV370354.

Comparative nucleotide and deduced amino acid sequence analyses revealed a high degree of sequence conservation among the Bihar isolates, with sequence identity ranging from 95.8% to 100%. Several intra-study comparisons showed complete identity, particularly between Kahalgoan and Sabour, Purnia and Kasba, and Belwa and Purnia isolates, suggesting these isolates are either identical or differ by only single nucleotide substitutions. The limited variation was restricted to a few point mutations, indicating a highly conserved coat protein gene within the local CMV population. When compared with previously reported sequences, all isolates showed strong genetic affinity, exceeding 96% identity, with Indian CMV isolates such as MN341000.1, MN340995.1, and MN340989.1, and considerable similarity of around 97% with geographically distant isolates from Europe, Africa, and other parts of Asia, indicating strong global conservation of the coat protein gene. At the amino acid level, BLASTp analysis revealed only minor conservative substitutions with no major insertions, deletions, or frame-shift mutations.

Despite the overall conservation, the comparative analysis identified specific nucleotide substitutions at key positions. In the Sabour and Kahalgoan isolates, a cytosine was observed at nucleotide position 378, in contrast to the thymine found at the same position in the other isolates, a single nucleotide polymorphism that suggests a possible mutation unique to the isolates from these locations. At the protein level, a notable amino acid substitution was detected at position 173, where valine replaced methionine in the Kahalgoan isolate. Although limited in number, such substitutions may have biological significance, as the coat protein is a key determinant of virus encapsidation, vector transmission, host interaction, and viral evolution. Previous studies have demonstrated that even single amino acid substitutions in the coat protein can alter symptom expression, host response, and transmission efficiency, and the observed variation in the Bihar isolates may represent a region-specific adaptive response driven by host selection pressure and agro-climatic conditions in eastern India, although functional validation was not performed in this study.

Phylogenetic analysis based on the coat protein gene sequences grouped all six Bihar isolates within CMV subgroup IA, indicating close genetic relatedness with previously reported Indian CMV isolates. The evolutionary history was inferred using the Neighbor-Joining method with a dataset of 30 nucleotide sequences, including reference isolates from Thailand, Germany, Japan, Taiwan, and Italy, as well as CMV isolates from diverse hosts such as banana, jasmine, tobacco, capsicum, chilli, and tomato. Bootstrap analysis with 1,000 replications assessed the reliability of the tree topology. The clustering of Purnia, Kasba, Belwa, and Kharkhari isolates was supported by comparatively high bootstrap values, suggesting a recent common evolutionary origin with minimal genetic divergence, while Sabour and Kahalgoan formed a closely related cluster with high bootstrap support, reflecting stable phylogenetic association and probable localized spread within neighboring chilli-growing regions. The broader subgroup IA clade containing the Bihar isolates together with previously reported Indian and Asian isolates showed moderate bootstrap support, indicating reliable evolutionary grouping alongside gradual diversification among geographically separated CMV populations.

The observed sequence conservation suggests that CMV populations infecting chilli in Bihar are genetically stable and may have originated from a common ancestral source followed by regional dissemination. The low genetic diversity indicates a stable viral population, but the presence of minor sequence variation points to ongoing microevolution that may allow gradual adaptation to local hosts and environments. The widespread occurrence of CMV across multiple districts highlights the role of alternative hosts, weeds, and aphid vectors in maintaining virus reservoirs, and the virus’s broad host range facilitates its persistence in agro-ecosystems, making eradication impractical. The predominance of subgroup IA has significant implications for chilli improvement programs in Bihar: resistance breeding efforts should focus on screening germplasm against subgroup IA isolates, while integrated disease management involving virus-free seedlings, removal of weed reservoirs, and effective aphid vector management remains essential. Future studies should characterize additional genomic regions or pursue complete genome sequencing to obtain deeper insights into recombination events, strain evolution, and subgroup differentiation, and continuous molecular surveillance will be important for detecting the emergence of new variants under changing agro-climatic conditions.

Subject of Research: Molecular characterization and phylogenetic analysis of cucumber mosaic virus isolates infecting chilli in Zone II and Zone IIIB of Bihar, India, showing predominance of CMV subgroup IA with high coat protein gene conservation.

Article Title: Genetic diversity and characterization of cucumber mosaic virus infecting chilli in Zone II and Zone IIIB of Bihar, India

Article References: Kumar, M., Kumar, A., Shamim, M., Arzoo, K., & Srivastava, D. (2026). Genetic diversity and characterization of cucumber mosaic virus infecting chilli in Zone II and Zone IIIB of Bihar, India. Frontiers in Virology, 6, Article 1869218. https://doi.org/10.3389/fviro.2026.1869218

Image Credits: AI Generated

DOI: 10.3389/fviro.2026.1869218

Keywords: cucumber mosaic virus, chilli, Capsicum annuum, CMV subgroup IA, coat protein gene, DAC-ELISA, RT-PCR, phylogenetic analysis, genetic diversity, Bihar, plant virology, aphid transmission