Citrus tristeza virus (CTV), a member of the aphid-transmitted closterovirus group, is the causal agent of the notorious tristeza disease in several citrus species worldwide. The codon usage patterns of viruses reflect the evolutionary changes for optimization of their survival and adaptation in their fitness to the external environment and the hosts. The codon usage adaptation of CTV to specific citrus hosts remains to be studied; thus, its role in CTV evolution is not clearly comprehended. Therefore, to better explain the host–virus interaction and evolutionary history of CTV, the codon usage patterns of the coat protein (CP) genes of 122 CTV isolates originating from three economically important citrus hosts (55 isolate from Citrus sinensis, 38 from C. reticulata, and 29 from C. aurantifolia) were studied using several codon usage indices and multivariate statistical methods. The present study shows that CTV displays low codon usage bias (CUB) and higher genomic stability. Neutrality plot and relative synonymous codon usage analyses revealed that the overall influence of natural selection was more profound than that of mutation pressure in shaping the CUB of CTV. The contribution of high-frequency codon analysis and codon adaptation index value show that CTV has host-specific codon usage patterns, resulting in higheradaptability of CTV isolates originating from C. reticulata (Cr-CTV), and low adaptability in the isolates originating from C. aurantifolia (Ca-CTV) and C. sinensis (Cs-CTV). The combination of codon analysis of CTV with citrus genealogy suggests that CTV evolved in C. reticulata or other Citrus progenitors. The outcome of the study enhances the understanding of the factors involved in viral adaptation, evolution, and fitness toward their hosts. This information will definitely help devise better management strategies of CTV.

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Begomoviruses are transmitted by whitefly (Bemisia tabaci Gennadius, Hemiptera: Aleyrodidae) in a persistent-circulative way. Once B. tabaci becomes viruliferous, it remains so throughout its life span. Not much is known about the copies of begomoviruses ingested and/or released by B. tabaci during the process of feeding. The present study reports the absolute quantification of two different begomoviruses viz. tomato leaf curl New Delhi virus (ToLCNDV, bipartite) and chilli leaf curl virus (ChiLCV, monopartite) at different exposure of active acquisition and inoculation feeding using a detached leaf assay. A million copies of both the begomoviruses were acquired by a single B. tabaci with only 5 min of active feeding and virus copy number increased in a logarithmic model with feeding exposure. Whereas, a single B. tabaci could inoculate 8.21E+09 and 4.19E+11 copies of ToLCNDV and ChiLCV, respectively in detached leaves by 5 min of active feeding. Virus copies in inoculated leaves increased with an increase in feeding duration. Comparative dynamics of these two begomoviruses indicated that B. tabaci adult acquired around 14-fold higher copies of ChiLCV than ToLCNDV 24 hrs post feeding. Whereas, the rate of inoculation of ToLCNDV by individual B. tabaci was significantly higher than ChiLCV. The study provides a better understanding of begomovirus acquisition and inoculation dynamics by individual B. tabaci and would facilitate research on virus-vector epidemiology and screening host resistance.

Chilli leaf curl virus (ChiLCV; genus: Begomovirus), transmitted by Bemisia tabaci (Gennadius) (Hemiptera: Aleyrodidae) in a persistent-circulative manner, is a major constraint in chilli production. The present study demonstrates for the first time that a topical spray of naked double-stranded RNA (dsRNA) on chilli plants causes mortality and inability to acquire and transmit ChiLCV in B. tabaci. dsRNA targeting heat shock protein 70 (hsp70) and fasciclin 2 (fas2) of B. tabaci Asia II 1 was first assessed under controlled conditions through oral delivery. Hsp70 and fas2 dsRNA resulted in up to 82.22% and 72% mortality of B. tabaci and around 12.4- and 8.5-fold decreases in mRNA levels, respectively, 24 h post-ingestion. ChiLCV copies in hsp70 dsRNA-fed B. tabaci steadily decreased with an increase in dsRNA concentration and were undetectable at a higher concentration of dsRNA. However, ChiLCV copies significantly increased in fas2 dsRNA-fed B. tabaci. Transmission of ChiLCV by B. tabaci was completely inhibited post-24 h feeding on hsp70 dsRNA at 3 μg/mL. Naked hsp70 dsRNA was topically sprayed on ChiLCV-infected chilli plants like an insecticide. 67.77% mortality of B. tabaci, 4.6-fold downregulation of hsp70 mRNA, and 1.34 × 1015-fold decreased ChiLCV copies in B. tabaci were recorded when adults were exposed to the dsRNA-treated plants under semi-field conditions. Foliar application of naked dsRNA reduced the ChiLCV transmission by 75% without any visible symptoms in the inoculated plants. A total of 2 consecutive sprays of dsRNA provided significant protection to B. tabaci for up to 20 days under semi-field conditions.

Sub-Himalayan West Bengal is favorable for the production of several fruits and vegetables. Papaya is one of the common plants cultivated in the area. Most of the papaya plants of the area are susceptible to Papaya ringspot virus (PRSV). Coat protein genes of 6 PRSV isolates of the area were sequenced following RT-PCR. Phylogenetic study of the PRSV isolates showed about 80%-90% similarity with Cuban isolates. The codon usage pattern of our isolates was also analyzed, along with several other isolates. PRSV isolates of our study showed a preference for 8 putative optimal codons. Correspondence analysis of the genes of different isolates along the first 2 major axes were done, as the first 2 axes contributed more in shaping codon usage pattern. In the phylogenetic tree constructed by the neighbour-joining method, our isolates clustered together with the east Indian, north Indian, and Bangladeshi isolates. The diversity and codon usage pattern of the PRSV isolates of different regions were studied, and it was observed that the codon usage pattern of PRSV isolates is probably influenced by translational selection along with mutational bias.

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Bemisia tabaci (Hemiptera: Aleyrodidae) is a highly efficient vector in the spread of chilli leaf curl virus (ChiLCV, Begomovirus) which is a major constraint in the production of chilli in South Asia. Transcriptome analysis of B. tabaci post-6 h acquisition of ChiLCV showed differential expression of 80 (29 upregulated and 51 downregulated) genes. The maximum number of DEGs are categorized under the biological processes category followed by cellular components and molecular functions. KEGG analysis of DEGs showed that the genes are involved in the functions like metabolism, signaling pathways, cellular processes, and organismal systems. The expression of highly expressed 20 genes post-ChiLCV acquisition was validated in RT-qPCR. DEGs such as cytosolic carboxypeptidase 3, dual-specificity protein phosphatase 10, 15, dynein axonemal heavy chain 17, fasciclin 2, inhibin beta chain, replication factor A protein 1, and Tob1 were found enriched and favored the virus infection and circulation in B. tabaci. The present study provides an improved understanding of the networks of molecular interactions between B. tabaci and ChiLCV. The candidate genes of B. tabaci involved in ChiLCV transmission would be novel targets for the management of the B. tabaci-begomovirus complex.

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