Geraniol Suppresses Oxidative Stress, Inflammation, and Interstitial Collagenase to Protect against Inflammatory Arthritis.

Geraniol (GER) is a plant-derived acyclic isoprenoid monoterpene that has displayed anti-inflammatory effects in numerous in vivo and in vitro models. This study was therefore designed to evaluate the antiarthritic potential of GER in complete Freund's adjuvant (CFA)-induced inflammatory arthritis (IA) model in rats. IA was induced by intraplantar injection of CFA (0.1 mL), and a week after CFA administration, rats were treated with various doses of methotrexate (MTX; 1 mg/kg) or GER (25, 50, and 100 mg/kg). Treatments were given on every alternate day, and animals were sacrificed on the 35th day. Paw volume, histopathological, hematological, radiographic, and qPCR analyses were performed to analyze the severity of the disease. GER significantly reduced paw edema after 35 days of treatment, and these results were comparable to the MTX-treated group. GER-treated animals displayed a perfect joint structure with minimal inflammation and no signs of cartilage or bone damage. Moreover, GER restored red blood cell and hemoglobin levels, normalized erythrocyte sedimentation rate, platelet, and c-reactive protein values, and also attenuated the levels of rheumatoid factor. RT-qPCR analysis demonstrated that GER decreased mRNA expression of pro-inflammatory cytokines like tumor necrosis factor-alpha (TNF-α) and interleukin-1 beta. GER also down-regulated the transcript levels of cyclooxygenase-2 (COX-2), microsomal prostaglandin E synthase-1, prostaglandin D2 synthase, and interstitial collagenase (MMP-1). Molecular docking of GER with COX-2, TNF-α, and MMP-1 also revealed that the antiarthritic effects of GER could be due to its direct interactions with these mediators. Based on our findings, it is conceivable that the antiarthritic effects of GER could be attributed to downregulation of pro-inflammatory mediators and protease like MMP-1.

Identification and primary distribution of Citrus viroid V in citrus in Punjab, Pakistan.

BACKGROUND: Citrus plants are prone to infection by different viroids which deteriorate their vigor and production. Citrus viroid V (CVd-V) is among the six citrus viroids, belongs to genus Apscaviroid (family Pospiviroidae) which induces symptoms of mild necrotic lesions on branches and cracks on trunk portion. METHODS AND RESULTS: A survey was conducted to evaluate the prevalence of CVd-V in core and non-core citrus cultivated areas of Punjab, Pakistan. A total of 154 samples from different citrus cultivars were tested for CVd-V infection by RT-PCR. The results revealed 66.66% disease incidence of CVd-V. Citrus cultivars Palestinia Sweet lime, Roy Ruby, Olinda Valencia, Kaghzi lime, and Dancy were identified as new citrus hosts of CVd-V for the first time from Pakistan. The viroid infection was confirmed by biological indexing on indicator host Etrog citron. The reported primers used for the detection of CVd-V did not amplify, rather showed non-specific amplification, which led to the designing of new primers. Whereas, new back-to-back designed primers (CVd-V AF1/CVd-V AR1) detected CVd-V successfully and obtained an expected amplified product of CVd-V with 294 bp. Sequencing analysis confirmed the new host of CVd-V showing 98-100% nucleotide sequence homology with those reported previously from other countries while 100% sequence homology to the isolates reported from Pakistan. Based on phylogenetic analysis using all CVd-V sequences in GenBank, two main CVd-V groups (I and II) were identified, and newly identified isolates during this study fall in the group I. CONCLUSION: The study revealed that there are some changes in the nucleotide sequences of CVd-V which made difficult for their detection using reported primers. All isolates of Pakistan showed high sequence homology with other isolates of CVd-V from Iran and USA whereas; the isolates from China, Japan, Tunisia, and Africa are distantly related. It is evident that CVd-V is spreading in all citrus cultivars in Pakistan.

Prevalence, transmission and molecular characterization of Cotton leaf curl Multan virus infecting hollyhock plants in Pakistan.

BACKGROUND: Hollyhock (Alcea rosea) is an ornamental plant belonging to the Malvaceae family and has a remarkable aesthetic and medicinal value. A number of distinct infectious entity including fungi, nematode, bacteria and most importantly both single and double stranded DNA and RNA viruses are reported from infected hollyhock plant. Begomoviruses, the well reputed member of the family Geminiviridae infected the hollyhock recently with a new hollyhock vein yellowing virus and in the present study it infected the hollyhock plant with Cotton leaf curl Multan virus (CLCuMV) which cause the disease of leaf curling. METHODS AND RESULTS: The symptomatic leaves of the hollyhock plants were collected based on the characteristic symptoms of leaf curling, puckering as well as vein thickening. DNA was extracted by using the recommended 2× CTAB protocol and PCR technique was optimized for the detection of begomovirus followed by sequencing. The data of disease incidence of infection location wise was collected based on the positive results of PCR amplification. Virus free whitefly collected from cotton field and feed on infected hollyhock plant in cage for few days then used for the transmission study of begomovirus on healthy hollyhock plants. Results of PCR amplification indicated that the primers Av/Ac core, Begomo 01/02, and CLCV 01/02 showed the bands of 579 bp, 2.8 kb and 1.1 kb respectively. The betasatellite was amplified by using beta01/02 and CLCuMuBF11/R33, which showed the band of 1400 bp and 481 bp. Disease incidence and Transmission study confirmed the begomovirus in hollyhock plants at molecular level. The sequence obtained with Av/Ac core primers showed the 99% identity with Cotton leaf curl Multan virus-Rajasthan strain and betasatellite primers showed 98% identity with Cotton leaf curl Multan betasatellite. CONCLUSION: Hollyhock plants infected by CLCuMV and associated betasatellite has been reported as a possible source of virus inoculum from Pakistan. These findings extend the range of Begomoviruses and betasatellites known to infect A. rosea and highlight this hollyhock species as an important reservoir of agriculturally important Begomoviruses and betasatellites.

Novel acetamide derivatives of 2-aminobenzimidazole prevent inflammatory arthritis in rats via suppression of pro-inflammatory mediators.

Benzimidazole ring system is an important pharmacophore with diverse pharmacological activities. In this study, we explored the anti-arthritic effects of newly synthesized acetamide derivatives of 2-aminobenzimidazole (N1 and N2) in rats. FTIR and NMR spectroscopies were used to characterize these compounds. Carrageenan (CRG) induced paw edema model was used to test the acute anti-inflammatory activity of various doses (10, 20 and 30 mg/kg) of N1 and N2 compounds. Based on acute anti-inflammatory effects, the most potent dose of each compound was selected and investigated in complete freund's adjuvant (CFA) induced inflammatory arthritis (RA) model (n = 4 in each group). Histopathological, hematological, radiographic, and RT-qPCR analyses were performed to assess the progression or resolution of inflammatory arthritis. The tested compounds produced a dose-dependent anti-inflammatory activity against CRG induced paw inflammation and similarly reduced edema in CFA induced inflammatory arthritis model. Histopathological and X-ray analyses of ankle joints revealed minimal inflammation and normal joint structures in N1 and N2 treated groups. The tested compounds also reduced the levels of autoantibodies and restored hematological parameters. Interestingly, the tested compounds did not elevate aspartate aminotransferase and alanine transaminase levels and displayed a better safety profile than methotrexate. N1 and N2 compounds also attenuated the transcript levels of IRAK1, NF-kB1, TNF-α, IL-1ß, IL17 and MMP1. In addition, N1 displayed a greater inhibition of mRNA levels of COX1, COX2, mPGES1 and PTGDS as compared to N2. Our findings demonstrate that N1 and N2 compounds possess strong anti-arthritic activity which can be attributed to the suppression of pro-inflammatory mediators.

Molecular characterization of divergent isolates of Citrus bent leaf viroid (CBLVd) from citrus cultivars of Punjab, Pakistan.

Citrus viroid infection is emerging as a serious threat because of its efficient systemic movement within the host plant and its quick spread due to contaminated pruning tools. A survey was conducted to investigate the primary distribution and molecular characterization of Citrus bent leaf viroid (CBLVd) and its variants in different citrus cultivars. A total of 154 symptomatic citrus samples were collected and detected by RT‒PCR with newly designed specific primers with the incidence of 36.33%. During biological indexing study on Etrog citron, expressions of reduced leaf size, yellowing with a light green pattern, and bending were observed. Amplified products were sequenced and analyzed using a nucleotide BLAST search, which showed 98% homology with other CBLVd isolates. The results of the phylogenetic tree analysis showed the presence of two main groups (A and B), with the predominant variants of CBLVd, i.e., CVd-I-LSS (Citrus viroid Low Sequence Similarity) sequences, clustering in subgroup A1 along with newly detected CVd-I-LSS from Palestinian sweet lime (Citrus limettioides), which has been identified as a new host of CVd-I-LSS in Pakistan. Further analysis of the sequences in subgroup A1 showed that the variant of CVd-I-LSS infecting citrus cultivars had a close relationship with isolates reported from China, Japan, and Iran, which may have resulted from the exchange of planting material. This study also unveiled the variability in nucleotide sequences of CBLVd, which made it unable to be detected by old primers. The results of this study indicate that the widespread presence of divergent variants of CBLVd is a major concern for the citrus industry in Pakistan and other countries where virulent isolates of CBLVd are prevalent. These findings suggest the need for future research on effective management and quarantine measures to stop the spread of CBLVd.

Amplicon-based RNAi construct targeting beta-C1 gene gives enhanced resistance against cotton leaf curl disease.

Cotton leaf curl disease (CLCuD) is one of the major limiting factors affecting cotton production in Pakistan for the last three decades. The disease is caused by begomoviruses of the family Geminiviridae. RNA interference (RNAi) is a promising tool that has been proved effective against several pathogens. Using RNAi, different genomic regions of geminiviruses have been targeted to attain sustainable resistance. However, the silencing of the transgene upon virus infection is a limiting factor. Here, we have developed for the first time an amplicon-based RNAi construct to target ßC1 gene of betasatellite associated with cotton leaf curl begomoviruses. In addition to producing short interfering (si) RNAs, Rep-based activation or looping out of the construct induced upon virus infection produces multiple copies of transgene that results in accumulation of defective molecules of betasatellite. Subsequent transcription gives rise to increased number of siRNAs that gives enhanced resistance. Transgenic Nicotiana benthamiana plants having RCß (RNAi construct for betasatellite) were challenged against Cotton leaf curl Khokran virus (CLCuKV) and Cotton leaf curl Multan betasatellite (CLCuMB). Reduced titer of the virus and betasatellite were detected through Southern blot hybridization. Significance of the study has been discussed. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s13205-021-02816-6.

CRISPR technology to combat plant RNA viruses: A theoretical model for Potato virus Y (PVY) resistance.

RNA viruses are the most diverse phytopathogens which cause severe epidemics in important agricultural crops and threaten the global food security. Being obligatory intracellular pathogens, these viruses have developed fine-tuned evading mechanisms and are non-responsive to most of the prophylactic treatments. Additionally, their sprint ability to overcome host defense demands a broad-spectrum and durable mechanism of resistance. In context of CRISPR-Cas discoveries, some variants of Cas effectors have been characterized as programmable RNA-guided RNases in the microbial genomes and could be reprogramed in mammalian and plant cells with guided RNase activity. Recently, the RNA variants of CRISPR-Cas systems have been successfully employed in plants to engineer resistance against RNA viruses. Some variants of CRISPR-Cas9 have been tamed either for directly targeting plant RNA viruses' genome or through targeting the host genes/factors assisting in viral proliferation. The new frontiers in CRISPR-Cas discoveries, and more importantly shifting towards RNA targeting will pyramid the opportunities in plant virus research. The current review highlights the probable implications of CRISPR-Cas system to confer the pathogen-derived or host-mediated resistance against phytopathogenic RNA viruses. Furthermore, a multiplexed CRISPR-Cas13a methodology is proposed here to combat Potato virus Y (PVY); a globally diverse phytopathogen infecting multiple crops.

Identification of a dicot infecting mastrevirus along with alpha- and betasatellite associated with leaf curl disease of spinach (Spinacia oleracea) in Pakistan.

Spinach is a common vegetable crop and very little data is available about its virus infection. Symptomatic leaves of spinach were collected during field survey. Circular DNA molecules were amplified from symptomatic samples using rolling circle amplification (RCA). After restriction analysis, presumed bands of virus and satellites were cloned, sequenced and analyzed. Analysis of sequenced RCA product revealed the presence of chickpea chlorotic dwarf virus (CpCDV; Mastrevirus). Further analyses of the cloned virus showed that strain "C" of CpCDV was present in symptomatic samples of spinach collected from field associated with vein darkening, curling and enations on leaves. Amplification of alpha- and betasatellites with universal primers was performed. CpCDV showed association with cotton leaf curl Multan betasatellite (CLCuMB) and cotton leaf curl Multan alphasatellites (CLCuMA). Infectivity analysis of CpCDV and CpCDV/CLCuMB were done in N. benthamiana using particle bombardment method and the results showed that CpCDV was able to transreplicates CLCuMB in this host. To our knowledge, this is the first report of a dicot infecting mastrevirus (CpCDV) along with CLCuMB and CLCuMA associated with leaf curl disease of spinach in Pakistan. The significance of the results is discussed.

Characterization of a Begomovirus-Betasatellite Complex, Producing Defective Molecules in Spinach (Spinacia oleracea L.), a New Host for Begomovirus and Betasatellite Complex in Pakistan.

Spinach is a vegetable crop which is widely grown over a large area especially in Punjab province of Pakistan. Leaf curling and enations on spinach plant collected shown to be associated with the begomovirus Pedilanthus leaf curl virus (PeLCV) and Shahdadpur strain of Cotton leaf curl Multan betasatellite (CLCuMBSha). Defective molecules of half and quarter size derived from monopartite begomoviruses are usually generated by the deletion of virion-sense sequences. Characterization of defective molecules of PeLCV from spinach revealed that the molecules of half the size are derived from the deletion of complementary-sense genes while quarter size molecule appears to have evolved by further deletion. This is the first report of the begomovirus-betasatellite complex on spinach and unusual defective molecules derived from deletion of complementary-sense genes in Pakistan.

RNAi-Mediated Simultaneous Resistance Against Three RNA Viruses in Potato.

RNA interference (RNAi) technology has been successfully applied in stacking resistance against viruses in numerous crop plants. During RNAi, the production of small interfering RNAs (siRNAs) from template double-standard RNA (dsRNA) derived from expression constructs provides an on-switch for triggering homology-based targeting of cognate viral transcripts, hence generating a pre-programmed immunity in transgenic plants prior to virus infection. In the current study, transgenic potato lines (Solanum tuberosum cv. Desiree) were generated, expressing fused viral coat protein coding sequences from Potato virus X (PVX), Potato virus Y (PVY), and Potato virus S (PVS) as a 600-bp inverted repeat expressed from a constitutive 35S promoter. The expression cassette (designated Ec1/p5941) was designed to generate dsRNAs having a hairpin loop configuration. The transgene insertions were confirmed by glufosinate resistance, gene-specific PCR, and Southern blotting. Regenerated lines were further assayed for resistance to virus inoculation for up to two consecutive crop seasons. Nearly 100% resistance against PVX, PVY, and PVS infection was observed in transgenic lines when compared with untransformed controls, which developed severe viral disease symptoms. These results establish the efficacy of RNAi using the coat protein gene as a potential target for the successful induction of stable antiviral immunity in potatoes.

Amplicon-Based RNA Interference Targeting V2 Gene of Cotton Leaf Curl Kokhran Virus-Burewala Strain Can Provide Resistance in Transgenic Cotton Plants.

The conserved coat or V2 gene of begomoviruses is responsible for viral movement in the plant cells. RNAi technology was used to silence V2 gene for resistance against these viruses in transgenic plants. The transformation of the RNAi-based gene construct targeting V2 gene of CLCuKoV-Bur, cloned under 35S promoter, was done in two elite cotton varieties MNH-786 and VH-289 using shoot apex cut method of gene transformation. The transformation efficiency was found to be 3.75 and 2.88 % in MNH-786 and VH-289, respectively. Confirmation of successful transformation was done through PCR in T 0, T 1, and T 2 generations using gene-specific primers. Transgenic cotton plants were categorized on the basis of the virus disease index in T 1 generation. Copy number and transgene location were observed using FISH and karyotyping in T 2 generation which confirmed random integration of V2 RNAi amplicon at chromosome 6 and 16. Real-time quantitative PCR analyses of promising transgenic lines showed low virus titer compared to wild-type control plants upon challenging them with viruliferous whiteflies in a contained environment. From the results, it was concluded that amplicon V2 RNAi construct was able to limit virus replication and can be used to control CLCuV in the field.

Regional changes in the sequence of cotton leaf curl multan betasatellite.

Cotton leaf curl disease (CLCuD) in Pakistan and northwestern India is caused by monopartite begomoviruses in association with an essential, disease-specific satellite, Cotton leaf curl Multan betasatellite (CLCuMB). Following a recent upsurge in CLCuD problems in Sindh province (southern Pakistan), sequences of clones of CLCuMB were obtained from Sindh and Punjab province (central Pakistan), where CLCuD has been a problem since the mid-1980s. The sequences were compared to all sequences of CLCuMB available in the databases. Analysis of the sequences shows extensive sequence variation in CLCuMB, most likely resulting from recombination. The range of sequence variants differ between Sindh, the Punjab and northwestern India. The possible significance of the findings with respect to movement of the CLCuD between the three regions is discussed. Additionally, the lack of sequence variation within the only coding sequence of CLCuMB suggests that the betasatellite is not involved in resistance breaking which became a problem after 2001 in the Punjab and subsequently also in northwestern India.

The merging of two dynasties--identification of an African cotton leaf curl disease-associated begomovirus with cotton in Pakistan.

Cotton leaf curl disease (CLCuD) is a severe disease of cotton that occurs in Africa and Pakistan/northwestern India. The disease is caused by begomoviruses in association with specific betasatellites that differ between Africa and Asia. During survey of symptomatic cotton in Sindh (southern Pakistan) Cotton leaf curl Gezira virus (CLCuGV), the begomovirus associated with CLCuD in Africa, was identified. However, the cognate African betasatellite (Cotton leaf curl Gezira betasatellite) was not found. Instead, two Asian betasatellites, the CLCuD-associated Cotton leaf curl Multan betasatellite (CLCuMB) and Chilli leaf curl betasatellite (ChLCB) were identified. Inoculation of the experimental plant species Nicotiana benthamiana showed that CLCuGV was competent to maintain both CLCuMB and ChLCB. Interestingly, the enations typical of CLCuD were only induced by CLCuGV in the presence of CLCuMB. Also in infections involving both CLCuMB and ChLCB the enations typical of CLCuMB were less evident. This is the first time an African begomovirus has been identified on the Indian sub-continent, highlight the growing threat of begomoviruses and particularly the threat of CLCuD causing viruses to cotton cultivation in the rest of the world.

Cotton leaf curl disease in Sindh province of Pakistan is associated with recombinant begomovirus components.

Cotton leaf curl disease (CLCuD) is a devastating disease of cotton causing severe losses to cotton across the Punjab province of Pakistan and northeastern India. Although the disease has been reported as occurring sporadically in Sindh province, Pakistan, this has not caused significant losses. However, in the last few years the disease has become more significant in Sindh province. CLCuD is caused by begomoviruses in association with a disease-specific symptom determining satellite (Cotton leaf curl Multan betasaellite [CLCuMB]) and, in some cases, a non-essential alphasatellite (the function of which remains unclear). These components were cloned from six samples collected in Sindh. Analysis of the full-length sequences of six begomovirus clones showed one to be an isolate of Cotton leaf curl Kokhran virus (CLCuKV), a virus previously shown to be associated with CLCuD in the Punjab, whereas the other five clones showed less than approximately 90% nucleotide sequence identity to several known begomoviruses associated with CLCuD. We take this to indicate that these are isolates of a newly identified begomovirus, for which we propose the name Cotton leaf curl Shahdadpur virus (CLCuShV). Closer inspection of the sequence of CLCuShV showed it to have a recombinant origin. For only two of the cotton samples was the presence of an alphasatellite detected. The sequences of clones of these alphasatellites indicate them to be newly identified species. A betasatellite was shown to be present in all six plants examined and sequence analysis of seven full-length clones indicated that two types of CLCuMB are present in Sindh and both are recombinant. These results indicate that the virus complex causing CLCuD in Sindh is distinct from that in the adjacent Punjab province. Possible reasons for these differences are discussed.