Tiny Flies: A Mighty Pest That Threatens Agricultural Productivity-A Case for Next-Generation Control Strategies of Whiteflies.

Whiteflies are a group of universally occurring insects that are considered to be a serious pest in their own way for causing both direct and indirect damages to crops. A few of them serve as vectors of plant viruses that are detrimental to the crop in question and cause an actual loss in productivity. A lot of attention is focused on pest control measures under the umbrella of IPM. In this review, we attempt to summarize the existing literature on how and why whiteflies are a serious concern for agriculture and society. We reviewed why there could be a need for fresh insight into the ways and means with which the pest can be combated. Here, we have emphasized next-generation strategies based on macromolecules, i.e., RNA interference and genetic engineering (for the expression of anti-whitefly proteins), as these strategies possess the greatest scope for research and improvement in the future. Recent scientific efforts based on nanotechnology and genome editing, which seem to offer great potential for whitefly/crop pest control, have been discussed. Comprehensive apprehensions related to obstacles in the path of taking lab-ready technologies into the farmers' field have also been highlighted. Although the use of RNAi, GM crops, nanotechnologies, for the control of whiteflies needs to be evaluated in the field, there is an emerging range of possible applications with promising prospects for the control of these tiny flies that are mighty pests.

Waste candle soot derived carbon nanoparticles: A competent alternative for the management of Helicoverpaarmigera.

Helicoverpaarmigera (Lepidoptera: Noctuidae) is considered as one of the foremost pests of global agriculture. This pest is contemplated for substantial economic loss apart from the socio-economic and environmental costs associated with its control. Farmers adopt several strategies for the control of this pest but the cost associated with these strategies is always a big question. This is the first time when waste-candle soot (CS) derived carbon nanoparticles (CNPs) are explored for the putative toxicity to H. armigera. In the present study, the entomotoxic effects of CNPs on H. armigera were investigated and compared with that of commercially available multi-walled carbon nanotubes (MWCNTs). Larvae fed on both the nanomaterials exhibited significant weight reduction and enhanced levels of antioxidant enzymes. Moths developed from the treated larvae exhibit very poor egg-laying capacity and poor egg hatchability. However, these entomotoxic effects were found more noticeable in larvae and moths fed on CNPs that eventually led to the complete cessation of the population build-up of H. armigera. These findings advocate the candidature of CNPs as a cost-effective alternative for efficient control of H. armigera in pest management programs.

CRISPR-Cas System: An Approach With Potentials for COVID-19 Diagnosis and Therapeutics.

COVID-19, the human coronavirus disease caused by SARS-CoV-2, was reported for the first time in Wuhan, China in late 2019. COVID-19 has no preventive vaccine or proven standard pharmacological treatment, and consequently, the outbreak swiftly became a pandemic affecting more than 215 countries around the world. For the diagnosis of COVID-19, the only reliable diagnostics is a qPCR assay. Among other diagnostic tools, the CRISPR-Cas system is being investigated for rapid and specific diagnosis of COVID-19. The CRISPR-Cas-based methods diagnose the SARS-CoV-2 infections within an hour. Apart from its diagnostic ability, CRISPR-Cas system is also being assessed for antiviral therapy development; however, till date, no CRISPR-based therapy has been approved for human use. The Prophylactic Antiviral CRISPR in huMAN cells (PAC-MAN), which is Cas 13 based strategy, has been developed against coronavirus. Although this strategy has the potential to be developed as a therapeutic modality, it may face significant challenges for approval in human clinical trials. This review is focused on describing potential use and challenges of CRISPR-Cas based approaches for the development of rapid and accurate diagnostic technique and/or a possible therapeutic alternative for combating COVID-19. The assessment of potential risks associated with use of CRISPR will be important for future clinical advancements.

First report and genetic characterization of porcine astroviruses of lineage 4 and 2 in diarrhoeic pigs in India.

Porcine astroviruses (PAstVs) have extended their distribution globally and have a high prevalence; however, their clinical significance is still under investigation. Thus far, information about their prevalence and diversity in the Indian pig population is unknown. This study is the first report on the prevalence and genetic characterization of PAstVs in diarrhoeic piglets in India. From January 2013 to December 2017, 757 samples were screened using an RT-PCR assay and PAstV infection was detected in 17.6% (133/757) pigs. Of the 133 positive samples, 79 (59.4%) were positive for PAstV alone, whereas 54 (40.6%) were found to be co-infected with porcine rotavirus A (PoRVA). Phylogenetic analysis of RdRp/capsid gene region revealed high genetic heterogeneity among PAstV sequences, with a predominance of PAstV lineage 4 and detection of lineage 2. The lineage 4 PAstVs exhibited 61.2%-94.5% sequence similarity at the nucleotide level to other reported sequences, whereas lineage 2 strain shared 66.0%-71.6% sequence identity with cognate sequences of the same lineage. This is the first report on PAstV and circulation of lineages 4 and 2 in India. Further, phylogenetic analysis indicates a multiphyletic origin of PAstV strains and suggests cross-border circulation of PAstVs with a similar genetic configuration in Asian countries.

Epidemiologic Status of Picobirnavirus in India, A Less Explored Viral Disease.

Since the unexpected discovery of picobirnaviruses (PBV) in 1988, they have been reported in many animals including mammals and birds, which comprises both terrestrial and marine species. Due to their divergent characteristics to other viral taxa they are classified into a new family Picobirnaviridae. Although their pathogenicity and role in causing diarrhea still remains a question since they have been discovered in symptomatic and asymptomatic cases both. Recent studies employing state-of-art molecular tools have described their presence in various clinical samples, like stool samples from different mammals and birds, respiratory tracts of pigs and humans, sewage water, different foods, etc. Furthermore, their epidemiological status from different parts of the world in different hosts has also increased. Due to their diverse host and irregular host pattern their role in causing diarrhea remains alien. The heterogeneity nature can be ascribed to segmented genome of PBV, which renders them prone to continuous reassortment. Studies have been hampered on PBVs due to their non-adaptability to cell culture system. Here, we describe the molecular epidemiological data on PBVs in India and discusses the overall status of surveillance studies carried out till date in India.

Frequency distribution of porcine rotavirus-A and capsid protein gene based sequence and phylogenetic analysis indicating marked heterogeneity among prevailing strains, India.

Rotavirus (RV), is an etiological agent of acute infantile diarrhea in both humans and animals including poultry. Among the eight accepted species/types of RVs, RV-A is genetically and antigenically the most diverse. RV-A associated enteritis is a major problem in the weaning and post-weaning piglets. Due to high genetic variability in the antigenic regions, RV-A is thought to have high interspecies jumping probability. In this study, comparatively a large sized sample (n = 757) was screened, where the samples were collected from diarrheic porcine population of north (Uttar Pradesh), North eastern (Assam, Nagaland, Meghalaya, Tripura, Manipur, Mizoram and Arunachal Pradesh) and Southern states of India (Kerala, Karnataka and Tamil Nadu). The VP6 gene based reverse-transcription (RT)-PCR based screening of the samples for RV-A identified 42.4% (321/757) positivity, where highest identification was from Uttar Pradesh 119 (37.07%), followed by 74 (23.05%), 34 (10.6%), 31 (9.65%), 21 (6.54%), 15 (4.67%), 11 (3.43%), 8 (2.49%), 3 (0.93%), 3 (0.93%) and 2 (0.62%) from Assam, Nagaland, Meghalaya, Tripura, Kerala, Manipur, Mizoram, Arunachal Pradesh, Karnataka and Tamil Nadu, respectively. Percentage identity calculation of the VP6 gene sequences from different porcine RV-A revealed 77.1-97.3% identity within the Indian porcine RV-A strains of the current study. Phylodendrogram and percent identity based analysis of the amplified and sequenced full length VP6 gene confirmed the presence of new VP6 genotypes (I1 and I5). Although, there are reports of detection of porcine RV-A based on VP6 gene from India, no lineage/genotype based characterization is available for the target gene. Till date, only a single VP6 type (I2) has been confirmed from pig population of India. Here, the findings confirm the circulation of diverse RV-A strains in porcine population in India.

Analysis of structure-function relationship in porcine rotavirus A enterotoxin gene.

Rotavirus (RV)-infected piglets are presumed to be latent sources of heterologous RV infection in humans and other animals. In RVs, non-structural protein 4 (NSP4) is the major virulence factor with pleiotropic properties. In this study, we analyzed the nsp4 gene from porcine RVs isolated from diarrheic and non-diarrheic cases at different levels of protein folding to explore correlations to diarrhea-inducing capabilities and evolution of nsp4 in the porcine population. Full-length nsp4 genes were amplified, cloned, sequenced, and then analyzed for antigenic epitopes, RotaC classification, homology, genetic relationship, modeling of NSP4 protein, and prediction of post-translational modification. RV presence was observed in both diarrheic and non-diarrheic piglets. All nsp4 genes possessed the E1 genotype. Comparison of primary, secondary, and tertiary structure and the prediction of post-translational modifications of NSP4 from diarrheic and non-diarrheic piglets revealed no apparent differences. Sequence analysis indicated that nsp4 genes have a multi-phyletic evolutionary origin and exhibit species independent genetic diversity. The results emphasize the evolution of the E9 nsp4 genotype from the E1 genotype and suggest that the diarrhea-inducing capability of porcine RVs may not be exclusively linked to its enterotoxin gene.

Unexpected detection of porcine rotavirus C strains carrying human origin VP6 gene.

BACKGROUND: Rotavirus C (RVC), a known etiological agent of diarrheal outbreaks, mainly inflicts swine population globally with sporadic incidence in human, cattle, ferret, mink and dog. OBJECTIVE: To demonstrate the presence of RVC in Indian swine population and characterization of its selected structural (VP6) and non-structural (NSP4 and NSP5) genes. METHODS: A total of 108 diarrheic samples from different regions of India were used. Isolated RNA was loaded onto polyacrylamide gel to screen for the presence of RVs through the identification of specific electrophoretic genomic migration pattern. To characterize the RVC strains, VP6 gene and NSP4 and NSP5 genes were amplified, sequenced and analyzed. RESULTS: Based on VP6 gene specific diagnostic RT-PCR, the presence of RVC was confirmed in 12.0% (13/108) piglet fecal specimens. The nucleotide sequence analysis of VP6 gene, encoding inner capsid protein, from selected porcine RVC (PoRVC) strains revealed more than 93% homologies to human RVC strains (HuRVC) of Eurasian origin. These strains were distant from hitherto reported PoRVCs and clustered with HuRVCs, owning I2 genotype. However, the two non-structural genes, i.e. NSP4 and NSP5, of these strains were found to be of swine type, signifying a re-assortment event that has occurred in the Indian swine population. CONCLUSION: The findings indicate the presence of human-like RVC in Indian pigs and division of RVC clade with I2 genotype into further sub-clades. To the best of our knowledge, this appears to be the first report of RVC in Indian swine population. Incidence of human-like RVC VP6 gene in swine supports its subsequent zoonotic prospective.

Expression of an insecticidal fern protein in cotton protects against whitefly.

Whitefly (Bemisia tabaci) damages field crops by sucking sap and transmitting viral diseases. None of the insecticidal proteins used in genetically modified (GM) crop plants to date are effective against whitefly. We report the identification of a protein (Tma12) from an edible fern, Tectaria macrodonta (Fee) C. Chr., that is insecticidal to whitefly (median lethal concentration = 1.49 µg/ml in in vitro feeding assays) and interferes with its life cycle at sublethal doses. Transgenic cotton lines that express Tma12 at ∼0.01% of total soluble leaf protein were resistant to whitefly infestation in contained field trials, with no detectable yield penalty. The transgenic cotton lines were also protected from whitefly-borne cotton leaf curl viral disease. Rats fed Tma12 showed no detectable histological or biochemical changes, and this, together with the predicted absence of allergenic domains in Tma12, indicates that Tma12 might be well suited for deployment in GM crops to control whitefly and the viruses it carries.

Peptide-Recombinant VP6 Protein Based Enzyme Immunoassay for the Detection of Group A Rotaviruses in Multiple Host Species.

We developed a novel enzyme immunoassay for the detection of group A rotavirus (RVA) antigen in fecal samples of multiple host species. The assay is based on the detection of conserved VP6 protein using anti-recombinant VP6 antibodies as capture antibodies and anti-multiple antigenic peptide (identified and constructed from highly immunodominant epitopes within VP6 protein) antibodies as detector antibodies. The clinical utility of the assay was evaluated using a panel of 914 diarrhoeic fecal samples from four different host species (bovine, porcine, poultry and human) collected from diverse geographical locations in India. Using VP6- based reverse transcription-polymerase chain reaction (RT-PCR) as the gold standard, we found that the diagnostic sensitivity (DSn) and specificity (DSp) of the new assay was high [bovine (DSn = 94.2% & DSp = 100%); porcine (DSn = 94.6% & DSp = 93.3%); poultry (DSn = 74.2% & DSp = 97.7%) and human (DSn = 82.1% & DSp = 98.7%)]. The concordance with RT-PCR was also high [weighted kappa (k) = 0.831-0.956 at 95% CI = 0.711-1.0] as compared to RNA-polyacrylamide gel electrophoresis (RNA-PAGE). The performance characteristics of the new immunoassay were comparable to those of the two commercially available ELISA kits. Our results suggest that this peptide-recombinant protein based assay may serve as a preliminary assay for epidemiological surveillance of RVA antigen and for evaluation of vaccine effectiveness especially in low and middle income settings.

Zika virus - emergence, evolution, pathology, diagnosis, and control: current global scenario and future perspectives - a comprehensive review.

This review converses the Zika virus which has attained global concern due to its rapid pandemic potential and impact on humans. Though Zika virus was first isolated in 1947, till the recent large-scale outbreak which occurred in Micronesia, in 2007, the virus was placed into the innocuous pathogen category. The World Health Organization on 1 February 2016 declared it as a 'Public Health Emergency of International Concern.' Of the note, American as well as Pacific Island strains/isolates is relatively closer to Asian lineage strains. The African and American strains share more than 87.5% and 95% homologies with Asian strains/isolates, respectively. Asian strains form independent clusters, except those isolated from China, suggesting relatively more diversity than African strains. Prevention and control are mainly aimed at the vector population (mosquitoes) with Aedes aegypti being the main species. Surveys in Africa and Asia indicated seropositivity in various animal species. However, so far its natural reservoir is unknown. There is an urgent need to understand why Zika virus has shifted from being a virus that caused mild illness to unforeseen birth defects as well as autoimmune-neurological problems. Unfortunately, an effective vaccine is not available yet. Availability of cryo-electron microscopy based on 3.8 Å resolution revealing mature Zika virus structure and the probable virus attachment site to host cell would provide critical insights into the development of antiviral treatments and vaccines.

Multispecies reassortant bovine rotavirus strain carries a novel simian G3-like VP7 genotype.

Rotavirus-A (RVAs), are the major cause of severe gastroenteritis in the young of mammals and birds. RVA strains possessing G6, G8, and G10 genotypes in combination with P[1] or P[11] have been commonly detected in cattle. During a routine surveillance for enteric viruses in a bovine population on North-Western temperate Himalayan region of India, an uncommon bovine RVA strain, designated as RVA/Cow-wt/IND/M1/09/2009 was detected in a diarrhoeic crossbred calf. The examination of nearly complete genome sequence of this RVA strain revealed an unusual G-P combination (G3P[11]) on a typical bovine RVA genotype backbone (I2-R2-C2-M2-A11-N2-T6-E2-H3). The VP7 gene of M1/09 isolate displayed a maximum nucleotide sequence identity of 73.8% with simian strain (RVA/Simian-tc/USA/RRV/1975/G3P[3]). The VP4 and NSP5 genes clustered with an Indian pig strain, RVA/Pig-wt/IND/AM-P66/2012/G10P[11] (99.6%), and a caprine strain, RVA/Goat-tc/BGD/GO34/1999/G6P[1] (98.9%) from Bangladesh, respectively, whilst the, VP6, NSP1, NSP3 and NSP4 genes were identical or nearly identical to Indian bovine strains (RVA/Cow-wt/IND/B-72/2008/G10P[X], RVA/Cow-wt/IND/B85/2010/GXP[X], and RVA/Cow-wt/IND/C91/2011/G6P[X]). The remaining four genes (VP1, VP2, VP3 and NSP2) were more closely related to RVA/Human-wt/ITA/PAI11/1996/G2P[4] (93.5%), RVA/Sheep-wt/CHN/LLR/1985/G10P[15] (88.8%), RVA/Human-tc/SWE/1076/1983/G2P2A[6] (93.2%) and RVA/Human-wt/AUS/CK20003/2000/G2P[4] (91.2%), respectively. Altogether, these findings are suggestive of multiple independent interspecies transmission and reassortment events between co-circulating bovine, porcine, ovine and human rotaviruses. The complete genome sequence information is necessary to establish the evolutionary relationship, interspecies transmission and ecological features of animal RVAs from different geographical regions.

Proteome analysis of Bemisia tabaci suggests specific targets for RNAi mediated control.

RNA interference offers effective control of several economically important insect pests. Bemisia tabaci is an important field crop pest, which causes significant yield loss worldwide. In our earlier study, we have demonstrated successful control of B. tabaci through transgenic plant mediated RNAi. However, selection of target genes without off-target effect(s) has been major concern so far and therefore, a critical exploration for B. tabaci specific targets is frantically required. In this study, we have followed proteomics approach to discover B. tabaci specific targets for RNAi and identified unique nucleotide sequences in functional genes (n=11) of the pest. For this, we have developed proteome profile of B. tabaci extract using two-dimensional electrophoresis. A total of 504 protein spots were analyzed on mass-spectrometer and 453 proteins including 246 non-redundant proteins have been identified successfully. Complementation of the proteome data with available nucleotide database has helped us to interpret the unique nucleotide sequences. These nucleotide stretches may serve as environmentally safe targets for RNAi mediated control of the pest through crop genetic engineering. To the best of our knowledge, it is the most complete proteome of any whitefly species. We have also demonstrated application of proteomics in the identification of functional transcripts for RNAi. BIOLOGICAL SIGNIFICANCE: Insects cause major loss to crop productivity through direct and indirect damages. Among them, hemipteran group of insects are major contributor of global crop yield loss. In current study, gel based proteome profile of B. tabaci (one of the major hemipteran crop insect pest) is developed and characterized, which is a gap area in field of whitefly biology. It is an important data set of future whitefly studies like insect-plant interaction, virulence of whiteflies, their control program and discovery of new pesticides. Out of various control strategies, RNA interference offers a great potential to combat the whitefly successfully. However, the uniqueness of target genes and off target impact of the technology remains a challenge to scientific community. We used our proteome data set for the identification of B. tabaci specific gene targets for RNAi mediated control. The identified genes are critical for the life cycle of B. tabaci hence, could be proven as good molecules for making transgenic crop plant for efficient control of whiteflies in the field.

Purification and characterization of a lectin with high hemagglutination property isolated from Allium altaicum.

A lectin was purified from the leaves of Allium altaicum and corresponding gene was cloned. The lectin namely Allium altaicum agglutinin (AAA) was ~24 kDa homodimeric protein and similar to a typical garlic leaf lectin. It was synthesized as 177 amino acid residues pre-proprotein, which consisted of 28 and 43 amino acid long N and C-terminal signal peptides, respectively. The plant expressed this protein more in scapes and flowers in comparison to the bulbs and leaves. Hemagglutination activity (with rabbit erythrocytes) was 1,428 fold higher as compared to Allium sativum leaf agglutinin (ASAL) although, the insecticidal activity against cotton aphid (Aphis gossypii) was relatively low. Glycan array revealed that AAA had higher affinity towards GlcAb1-3Galb as compared to ASAL. Homology analysis showed 57-94% similarity with other Allium lectins. The mature protein was expressed in E. coli as a fusion with SUMO peptide in soluble and biologically active form. Recombinant protein retained high hemagglutination activity.

SUMO fusion facilitates expression and purification of garlic leaf lectin but modifies some of its properties.

Over expression of lectin genes in E. coli often gives inclusion bodies that are solubilised to characterize lectins. We made N-terminal fusion of the Allium sativum leaf agglutinin (ASAL) with SUMO (small ubiquitin related modifier) peptide. The SUMO peptide allowed expression of the recombinant lectin in E. coli, predominantly in soluble form. The soluble fusion protein could be purified by immobilized metal affinity column (IMAC), followed by size exclusion chromatography. The SUMO protease failed to cleave the SUMO peptide from ASAL. This may be due to steric hindrance caused by the homodimer structure of the chimeric ASAL. Some properties like dimerization, haemagglutination and insecticidal properties of the recombinant SUMO-ASAL fusion protein were comparable to the plant derived native lectin. However, glycan array analysis revealed that the carbohydrate binding specificity of the recombinant SUMO-ASAL was altered. Further, the fusion protein was not toxic to E. coli (native ASAL exhibited toxicity). The recombinant lectin was more thermo-labile as compared to the native lectin. Three important findings of this study are: (1) sugar specificity of ASAL can be altered by amino-terminal fusion; (2) anti-E. coli activity of ASAL can be eliminated by N-terminal SUMO fusion and (3) SUMO-ASAL may be a preferred candidate insecticidal protein for the development of transgenic plants.