Demonstration of seed inoculation of dimer infectious clones of mungbean yellow mosaic India virus-soybean isolate.

In this study, the full-length components of mungbean yellow mosaic India virus (MYMIV) DNA-A (MW590720 & MW600934) and DNA-B (MW659819 & MW659820) from a soybean isolate were cloned and sequenced. Nucleotide sequence analysis of both MYMIV components revealed > 96% identity and close ancestry with MYMIV isolates from legumes in Southeast Asia. Furthermore, dimeric infectious clones of MYMIV were generated in the pCAMBIA1302 vector, and a seed infiltration protocol was established for mungbean, soybean, and Nicotiana tabacum. Agroinfiltration induced yellow mosaic symptoms in mungbean and N. tabacum plants 3 weeks post-infiltration, which were further confirmed by PCR using MYMIV-specific DNA primers. Supplementary Information: The online version contains supplementary material available at 10.1007/s13205-023-03778-7.

First report of apple stem grooving virus infection in loquat from India.

Loquat, commonly known as Eriobotrya japonica, is a major subtropical fruit from the Rosaceae family that is native to China but also found in most of Europe and Asia, including India. Apple stem grooving virus (ASGV) infecting loquat was detected using leaf samples collected from Himachal Pradesh (India) through DAC-ELISA followed by RT-PCR assays targeting coat protein (CP), movement protein (MP) and replicase (Rep) regions of ASGV genome. Sequencing of RT-PCR amplicons and sequence analyses revealed that CP, MP and Rep sequences of ASGV loquat Indian isolate of the current study shared a maximum of 98-100% nucleotide sequence identities with the corresponding sequences of available ASGV Indian isolates [LN559078, HE978837, MZ127820, MN912568]. Phylogenetic tree based on each sequenced gene confirmed the genetic diversity of ASGV. To the best of our knowledge, and based on review of the literature, this is the first report of ASGV infection in loquat from India.

Development of a new Collateral Cleavage-independent CRISPR/Cas12a based easy detection system for plant viruses.

Plant virus spread through various means, from mechanically to the insect vectors and act as obligate parasite, therefore, are extremely challenging to eradicate. Geminiviruses are an important class of viruses which have reported extensively in last two decades on several new hosts. They infect wide range of annual crops and perineal shrubs, therefore, essentially required to detect them on field and dispose to check their vector transmission to healthy crops. In this study, we have chosen two important begomovirus viz. Mungbean yellow mosaic India virus which infect wide range of leguminous crops while Ageratum enation virus is reported to infect a wide range of crops from weed to opium poppy. Here, we have utilized the binding and cleaving ability of LbaCas12a protein with target to detect the virus infection on field. We proposed here a new Collateral Cleavage Independent CRISPR/Cas12a based detection system (CCI-CRISPR) for plant viruses.

An Overview of Recent Advances in Biomedical Applications of Click Chemistry.

Cu(I)-catalyzed azide-alkyne cycloaddition (CuAAC) is a modular and bio-orthogonal approach that is being adopted for the efficient synthesis of organic and bioorganic compounds. It leads to the selective formation of 1,4-disubstituted 1,2,3-triazole units connecting readily accessible building blocks via a stable and biocompatible linkage. The vast array of the bioconjugation applications of click chemistry has been attributed to its fast reaction kinetics, quantitative yields, minimal byproducts, and high chemospecificity and regioselectivity. These combined advantages make click reactions quite suitable for the lead identification and the development of pharmaceutical agents in the fields of medicinal chemistry and drug discovery. In this review, we have outlined the key aspects, the mechanistic details and merits and demerits of the click reaction. In addition, we have also discussed the recent pharmaceutical applications of click chemistry, ranging from the development of anticancer, antibacterial, and antiviral agents to that of biomedical imaging agents and clinical therapeutics.

Genomic Analysis of an Indian G8P[1] Caprine Rotavirus-A Strain Revealing Artiodactyl and DS-1-Like Human Multispecies Reassortment.

The surveillance studies for the presence of caprine rotavirus A (RVA) are limited in India, and the data for the whole-genome analysis of the caprine RVA is not available. This study describes the whole-genome-based analysis of a caprine rotavirus A strain, RVA/Goat-wt/IND/K-98/2015, from a goat kid in India. The genomic analysis revealed that the caprine RVA strain K-98, possess artiodactyl-like and DS-1 human-like genome constellation G8P[1]-I2-R2-C2-M2-A3-N2-T6-E2-H3. The three structural genes (VP2, VP4, and VP7) were close to caprine host having nucleotide-based identity range between 97.5 and 98.9%. Apart from them, other gene segments showed similarity with either bovine or human like genes, ultimately pointing toward a common evolutionary origin having an artiodactyl-type backbone of strain K-98. Phylogenetically, the various genes of the current study isolate also clustered inside clades comprising Human-Bovine-Caprine isolates from worldwide. The current findings add to the knowledge on caprine rotaviruses and might play a substantial role in designing future vaccines or different alternative strategies combating such infections having public health significance. To the best of our knowledge, this is the first report on the whole-genome characterization of a caprine RVA G8P[1] strain from India. Concerning the complex nature of the K-98 genome, whole-genome analyses of more numbers of RVA strains from different parts of the country are needed to comprehend the genomic nature and genetic diversity among caprine RVA.

Prophylactic interferon-γ and interleukin-17 facilitate parasite clearance in experimental visceral leishmaniasis.

BACKGROUND AND OBJECTIVE: The synergy of interleukin (IL)-17 along with other pro-inflammatory cytokines is well known in various autoimmune and infectious diseases. A longitudinal study in the Sudanese population showed an association of IL-17 with the protection of kala-azar outbreak. The protective role of IL-17 is also known in terms of expansion of IL-17-producing cells in vaccine-induced immunity. However, the prophylactic role of IL-17 in visceral leishmaniasis has still not been validated. In the present study, we evaluated the prophylactic efficacy of IL-17A and interferon (IFN)-γ in Leishmania donovani-challenged Balb/c mice. MATERIALS AND METHODS: Two doses of recombinant IL (rIL)-17A and/or IFN-γ were administered intraperitoneally after/at 1 week interval and then the mice were challenged with amastigote form of L. donovani. At 45 days of postchallenge, mice were sacrificed and evaluated for change in the body and organ weight, parasitic load in visceral organs, and fold change in gene expression of cytokines. RESULTS: We observed that the prophylactic use of rIL-17A and IFN-γ alone or in combination significantly inhibited the parasitic load in visceral organs. Furthermore, pro-inflammatory cytokine gene expression increased up to 2-4-folds in mice treated with recombinant cytokines. CONCLUSION: Our results suggest that prophylactic use of recombinant IFN-γ and IL-17A inhibits parasitic growth in visceral organs of L. donovani-challenged experimental mice model, especially through upregulation of pro-inflammatory cytokines' gene expression.

A new variant of Croton yellow vein mosaic virus naturally infecting wild sunflower in India.

During 2015-2016, wild sunflower (Verbesina encelioides) was observed with bright yellow vein mosaic symptims in Noida, Uttar Pradesh, India. The initial analysis by PCR with a pair of coat protein based primers revealed the association of a begomovirus. Further, the virus was identified by rolling circle amplification and cloning of the complete genome. The DNA-A (2671 nucleotides, MH359168) with the genome organisation typical of the Old World begomovirus shared 98% sequence identity with that of Croton yellow vein mosaic virus (CYVMV) reported from India. The betasatellite (MH359169) associated with the disease shared 92% sequence identity with papaya leaf curl betasatellite (KY825245). The results showed that the wild sunflower is a new alternate host of CYVMV. The study also revealed a natural association of heterologous betasatellite with CYVMV in wild sunflower exhibiting yellow vein mosaic symptoms.

Inhibiting HLA-G restores IFN-γ and TNF-α producing T cell in pleural Tuberculosis.

Human Leukocyte Antigen-G (HLA-G), a non-classical, class Ib molecule, has been shown to mediate immunoregulatory functions by inducing apoptosis, inhibits cytotoxicity and differentiation by modulating cytokine secretion. Due to its immune-suppressive function, it facilitates tolerance in feto-maternal interface and transplantation. In contrary, it favours immune evasion of microbes and tumors by inhibiting immune and inflammatory responses. In Tuberculosis (TB), we previously reported differential expression of HLA-G and its receptor Ig-like transcript -2 (ILT-2) in disseminated vs. localized Tuberculosis. The present study explores the impact of HLA-G inhibition on the function of T cells and monocytes, in TB Pleural Effusion (PE), a localized form of TB. Blocking of HLA-G resulted in significant increase in IFN-γ and TNF-α production by CD3+ T cells. Additionally, we observed that HLA-G influences the apoptosis and cytotoxic effect of T cells from TB- PE patients. Next, we checked the impact of interaction between HLA-G and ILT-4 receptor in monocytes derived from TB-PE patients upon blocking and observed significant increase in IFN-γ production. The present study reveals for the first time HLA-G mediated suppression of Th1 cytokines, especially, IFN-γ and TNF-α in TB-PE patients.

Efficient immunodiagnosis of Citrus yellow mosaic virus using polyclonal antibodies with an expressed recombinant virion-associated protein.

Citrus yellow mosaic virus (CYMV) is a member of genus Badnavirus of the family Caulimoviridae. It is the causal agent of citrus yellow mosaic disease in citrus and causes reduction in yield. As the virus is vegetative propagated by grafting, development of high-throughput diagnosis methods based on serological techniques is a prerequisite for production of healthy virus-free planting material. The current study describes the development of polyclonal antibodies raised in rabbits against purified recombinant virion-associated protein (rVAP) encoded by ORF-II of CYMV. The specificity of developed antiserum was evaluated in immunosorbent electron microscopy (ISEM), antigen-coated plate-enzyme linked immunosorbent assay (ACP-ELISA) and immunocapture PCR (IC-PCR). The antiserum specifically reacted up to a dilution of 1:2000 in ACP-ELISA for detection of CYMV-infected plants. The antiserum was validated by screening CYMV-infected plants maintained in the glass house through ACP-ELISA. To the best for our knowledge, this is the first report on production of polyclonal antiserum using recombinant virion-associated protein as fusion protein, which could be used for screening CYMV-infected plants by ELISA and IC-PCR. These immunodiagnostic methods can be effectively employed in routine indexing of citrus and in quarantine process.

Functional characterization of CD4 and CD8 T cell responses among human papillomavirus infected patients with ano-genital warts.

Ano-genital warts are considered one of the commonest and highly infectious sexually transmitted infections. These warts are primarily caused by the human papillomavirus (HPV) of the family Papillomaviridae, genus alpha-papillomavirus, species 10 and types 6 and 11. However the high recurrence rate of warts is a matter of serious concern to the patients and a challenge for the treating physician. The conventional treatment options are targeted only to the local site of warts. There is no systemic treatment modality as there is limited understanding of the disease immune-pathogenesis. The role of cell-mediated immunity in combating HPV infection is not clearly defined. Hence the present study is aimed at investigating the CD4+ T helper (Th1 and Th2) and CD8+ T cell responses among wart patients. In this study, we compared HPV6 and HPV11 antigen-specific T cell responses among venereal wart patients relative to healthy controls. Significant decrease in percent frequencies of IFN-γ producing CD4+ and CD8+ T cells were observed in HPV infected wart patients. On the other hand, the frequency of CD4+ T cells expressing IL-4 was significantly increased in these patients as compared to healthy controls. The observed functional skewing of HPV specific T cells from Th1 to Th2 response in patients indicated suppressed immunity against the HPV. Moreover, decrease in CD8 T cell function correlated with poor wart clearance. Our findings open future avenues for exploring potential immunomodulation strategies as an adjunct to standard treatment for better management of these patients and prevention of recurrence.

Comparison of ELISA and dual stage real time RT-PCR to differentiate Sabin like and non-Sabin like poliovirus isolates.

Environmental surveillance of polioviruses has been used as an important tool in monitoring circulation of wild polioviruses and/or Vaccine derived polioviruses in sewage samples. It is important to distinguish Sabin like isolates from non-Sabin like; ELISA & dual stage real time RT-PCR have been used for the same. Current study was carried out on sewage isolates to compare ELISA & RT-PCR with sequencing to distinguish Sabin like from non-Sabin like. Out of 468 sewage specimens, 91 (19.44%) were non-polio enteroviruses positive and 377 (80.56%) were polio positive by virus isolation method. A total of 488 polio virus isolates were detected by L20B and RD route which were further subjected to ELISA and RT-PCR. The results were compared with sequencing. On comparison, the specificity of ELISA was only 66.67% in spite of very low sensitivity (3.43%). The sensitivity of RT-PCR was 97.71% which makes it a good primary screening test for detection of non-Sabin like viruses. However, the specificity was only 33.33%. RT-PCR appears to be a sensitive tool for detecting non-Sabin like viruses however; the isolates which are non-Sabin like by RT-PCR may not necessarily be mutated viruses. ELISA cannot be used for differentiation of Sabin likes from non-Sabin likes due to low sensitivity.

Distinct clinico-immunological profile of patients infected with human papilloma virus genotypes 6 and 11.

Anogenital warts are primarily caused by Human Papillomavirus (HPV) type 6 and 11, which belong to the taxonomic family Papillomaviridae, genus alpha-papillomavirus and species 10. The presentation of the warts is varied and most of the patients have high recurrence rate of wart lesions. Studies had shown that an effective cellular immune response is required for the control of HPV infection. Here, we report distinct clinico-immunological profile of two patients presenting with venereal warts caused by HPV genotypes 6 and 11. The Case 1 manifested greater number of verrucous warts and case 2 had fewer subtle lesions. Further, evaluation of HPV antigen-specific cellular immune response revealed a robust T cell response against HPV6 peptide and a weak response against HPV11 in case 1. Interestingly, HPV genotyping revealed type 6 in case 1 with greater severity of infection and robust immune response against HPV6 peptide. In contrast, case 2 presented with milder infection and weak immune response and was positive for genotype 11. More extensive study with larger cohorts will strengthen our observation and could be relevant for designing immunotherapeutic adjunct strategies along with the standard treatment for rapid clearance of HPV infections in these patients. This communication reports immune status of two patients with venereal warts and their correlation with clinical presentation and the genotyping.

Environmental surveillance of polioviruses with special reference to L20B cell line.

With the eradication of poliovirus, the focus has now shifted to environmental surveillance of poliovirus to determine the circulating polioviruses in an area. L20B and RD cell lines are used for isolation of polioviruses. It is imperative to study the efficacy of these cell line in isolating polioviruses from environmental samples. The present study was carried out to determine the sensitivity and specificity of L20B cell line for isolation of polioviruses from environmental samples. L20B and RD cell lines are used for isolation of polioviruses. Molecular characterization was done by using real time RT-PCR. A total of 432 sewage samples from Delhi and Punjab were processed for the isolation of polioviruses during Jan-Dec 2015. 96.76% of the samples were positive in either of the cell lines. Non-polio enteroviruses were obtained in 50 samples on primary isolation. On RT-PCR, 347 (94.29%) samples yielded polioviruses and the rest (21) non-polio enteroviruses or non-enteroviruses. A total of 703 isolates were obtained. 635 isolates were found polioviruses by PCR (90.33%), 20 isolates were found to be NPEV (2.84%) and 48 (6.83%) were found to be NEV. Out of the 20 NPEV isolates, 14 were from RLR (RD-L20B-RD) route and six isolates were from LR (L20B-RD) route. All 48 NEV isolates were from LR route. Thus L20B cell line is more sensitive as compared to RD cell line for isolation of polioviruses however it is not absolutely specific for polioviruses.

Physicochemical characterization and immunological properties of Pichia pastoris based HPV16L1 and 18L1 virus like particles.

There continues to be an urgent need for cost-effective prophylaxis for HPV-associated cancers in socio-economically underdeveloped nations. Presently HPV vaccines, which are commercially available, are adjuvanted virus-like particles (VLPs) expressed from various recombinant expression systems. They have been characterized by different methods as safe, pure, and potent HPV vaccine antigens. We cloned and expressed L1 proteins of HPV16 & 18 in Pichia pastoris and tested their immunogenicity. We observed that HPVL1 proteins (16L1 and 18L1) are expressed in Pichia pastoris at high levels. Critical physicochemical parameters of these HPV recombinant L1 proteins were characterized by SDS PAGE, western blotting, peptide mapping, glycosylation pattern, mass spectrometry, host cell DNA and protein analysis, electron microscopy, and immunogenicity analysis. These data establish a blueprint of HPV recombinant protein antigens for standardizing & developing an alternative high-quality, cost-effective vaccine for HPV as well as similar recombinant protein-based vaccines.

Immunogenicity of next-generation HPV vaccines in non-human primates: Measles-vectored HPV vaccine versus Pichia pastoris recombinant protein vaccine.

Human papillomavirus (HPV) infection is the most common sexually transmitted disease worldwide. HPVs are oncogenic small double-stranded DNA viruses that are the primary causal agent of cervical cancer and other types of cancers, including in the anus, oropharynx, vagina, vulva, and penis. Prophylactic vaccination against HPV is an attractive strategy for preventing cervical cancer and some other types of cancers. However, there are few safe and effective vaccines against HPV infections. Current first-generation commercial HPV vaccines are expensive to produce and deliver. The goal of this study was to develop an alternate potent HPV recombinant L1-based vaccines by producing HPV virus-like particles into a vaccine that is currently used worldwide. Live attenuated measles virus (MV) vaccines have a well-established safety and efficacy record, and recombinant MV (rMV) produced by reverse genetics may be useful for generating candidate HPV vaccines to meet the needs of the developing world. We studied in non-human primate rMV-vectored HPV vaccine in parallel with a classical alum adjuvant recombinant HPV16L1 and 18L1 protein vaccine produced in Pichia pastoris. A combined prime-boost approach using both vaccines was evaluated, as well as immune interference due to pre-existing immunity against the MV. The humoral immune response induced by the MV, Pichia-expressed vaccine, and their combination as priming and boosting approaches was found to elicit HPV16L1 and 18L1 specific total IgG and neutralizing antibody titres. Pre-existing antibodies against measles did not prevent the immune response against HPV16L1 and 18L1.

Molecular cloning of coat protein gene of an Indian cotton leaf curl virus (CLCuV-HS2) isolate and its phylogenetic relationship with others members of Geminiviridae.

Cotton leaf curl geminivirus is a whitefly (Bemisia tabaci Genn.) transmitted Begomovirus (Family Geminiviridae) causing a serious disease of cotton in northern India. The very typical symptoms of present isolate (CLCuV-HS2) showed thickened veins, dark green discoloration of the leaves with upward curling and leaf like structure known as enation (one in number), which develops into cup-shaped on the reverse side of leaves. The polymerase chain reaction (PCR) based technique can detect the viral DNA in samples stored upto 3 days after the collection and have wide application for the field diagnosis. The complete nucleotide sequence of the Indian isolate of cotton leaf curl geminivirus (CLCuV-HS2) coat protein (CP) gene component was determined using CP specific primers through PCR amplification from field infected cotton plants growing in Haryana, India. Comparison of the amino acid sequence of the putative CP with some other mono and bipartite geminiviruses revealed a maximum of 97.3% identity with Pakistan cotton leaf curl virus (CLCuV-62). A nuclear localization signal located close to the N-terminal of CP gene was determined.

Diversity of DNA 1: a satellite-like molecule associated with monopartite begomovirus-DNA beta complexes.

DNA 1 components are satellite-like, single-stranded DNA molecules associated with begomoviruses (family Geminiviridae) that require the satellite molecule DNA beta to induce authentic disease symptoms in some hosts. They have been shown to be present in the begomovirus-DNA beta complexes causing cotton leaf curl disease (CLCuD) and okra leaf curl disease (OLCD) in Pakistan as well as Ageratum yellow vein disease (AYVD) in Singapore. We have cloned and sequenced a further 17 DNA 1 molecules from a diverse range of plant species and geographical origins. The analysis shows that DNA 1 components are associated with the majority of begomovirus-DNA beta complexes, being absent from only two of the complexes examined, both of which have their origins in Far East Asia. The sequences showed a high level of conservation as well as a common organization consisting of a single open reading frame (ORF) in the virion sense, a region of sequence rich in adenine and a predicted hairpin structure. In phylogenetic analyses, there was some evidence of grouping of DNA 1 molecules according to geographic origin, but less evidence for grouping according to host plant origin. The possible origin and function of DNA 1 components are discussed in light of these findings.

Diversity of DNA beta, a satellite molecule associated with some monopartite begomoviruses.

DNA beta molecules are symptom-modulating, single-stranded DNA satellites associated with monopartite begomoviruses (family Geminiviridae). Such molecules have thus far been shown to be associated with Ageratum yellow vein virus from Singapore and Cotton leaf curl Multan virus from Pakistan. Here, 26 additional DNA beta molecules, associated with diverse plant species obtained from different geographical locations, were cloned and sequenced. These molecules were shown to be widespread in the Old World, where monopartite begomoviruses are known to occur. Analysis of the sequences revealed a highly conserved organization for DNA beta molecules consisting of a single conserved open reading frame, an adenine-rich region, and a region of high sequence conservation [the satellite conserved region (SCR)]. The SCR contains a potential hairpin structure with the loop sequence TAA/GTATTAC; similar to the origins of replication of geminiviruses and nanoviruses. Two major groups of DNA beta satellites were resolved by phylogenetic analyses. One group originated from hosts within the Malvaceae and the second from a more diverse group of plants within the Solanaceae and Compositae. Within the two clusters, DNA beta molecules showed relatedness based both on host and geographic origin. These findings strongly support coadaptation of DNA beta molecules with their respective helper begomoviruses.