Identification and Characterization of Citrus Concave Gum-Associated Virus Infecting Citrus and Apple Trees by Serological, Molecular and High-Throughput Sequencing Approaches

Citrus concave gum-associated virus (CCGaV) is a negative-stranded RNA virus, first reported a few years ago in citrus trees from Italy. It has been reported in apple trees in the USA and in Brazil, suggesting a wider host range and geographic distribution. Here, an anti-CCGaV polyclonal antiserum to specifically detect the virus has been developed and used in a standard double antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA) that has been validated as a sensitive and reliable method to detect this virus both in citrus and apple trees. In contrast, when the same antiserum was used in direct tissue-blot immunoassay, CCGaV was efficiently detected in citrus but not in apple. Using this antiserum, the first apple trees infected by CCGaV were identified in Italy and the presence of CCGaV in several apple cultivars in southern Italy was confirmed by field surveys. High-throughput sequencing (HTS) allowed for the assembling of the complete genome of one CCGaV Italian apple isolate (CE-c3). Phylogenetic analysis of Italian CCGaV isolates from apple and citrus and those available in the database showed close relationships between the isolates from the same genus (Citrus or Malus), regardless their geographical origin. This finding was further confirmed by the identification of amino acid signatures specific of isolates infecting citrus or apple hosts. Analysis of HTS reads also revealed that the CE-c3 Italian apple tree, besides CCGaV, was simultaneously infected by several viruses and one viroid, including apple rubbery wood virus 2 which is reported for the first time in Italy. The complete or almost complete genomic sequences of the coinfecting agents were determined.

Development of Polyclonal Antibody-Based on Immunological Formats for Pathogen Detection in Groundnut Leaf Tissues

Immunological methods are highly useful to detect plant pathogens before symptom excrescence. Among the various immuno assay Enzyme Linked Immuno Sorbant Assay (ELISA) are well suited for detecting viral and fungal pathogens. The present investigation for the specific and early detection of groundnut leaf blight pathogen was standardized using polyclonal antiserum, the optimum dilution of antigen and antisera (antobody) was 1:1000 and 1:100 respectively with the titre value of 1:10. leaf blight infected groundnut plants could be detected 6 days before symptom expression. Prediction of infection well in advance help us to take remedial measures in time.

Erythrocyte Adhesion of Merozoite Surface Antigen 2c1 Expressed During Extracellular Stages of Babesia orientalis

Babesia orientalis, a major infectious agent of water buffalo hemolytic babesiosis, is transmitted by Rhipicephalus haemaphysaloides. However, no effective vaccine is available. Essential antigens that are involved in parasite invasion of host red blood cells (RBCs) are potential vaccine candidates. Therefore, the identification and the conduction of functional studies of essential antigens are highly desirable. Here, we evaluated the function of B. orientalis merozoite surface antigen 2c1 (BoMSA-2c1), which belongs to the variable merozoite surface antigen (VMSA) family in B. orientalis. We developed a polyclonal antiserum against the purified recombinant (r)BoMSA-2c1 protein. Immunofluorescence staining results showed that BoMSA-2c1 was expressed only on extracellular merozoites, whereas the antigen was undetectable in intracellular parasites. RBC binding assays suggested that BoMSA-2c1 specifically bound to buffalo erythrocytes. Cytoadherence assays using a eukaryotic expression system in vitro further verified the binding and inhibitory ability of BoMSA-2c1. We found that BoMSA-2c1 with a GPI domain was expressed on the surface of HEK293T cells that bound to water buffalo RBCs, and that the anti-rBoMSA2c1 antibody inhibited this binding. These results indicated that BoMSA-2c1 was involved in mediating initial binding to host erythrocytes of B. orientalis. Identification of the occurrence of binding early in the invasion process may facilitate understanding of the growth characteristics, and may help in formulating strategies for the prevention and control of this parasite.

Identification of antigenic epitopes for Guertu virus nucleocapsid protein

Guertu virus (GTV), a novel tick-borne virus with potential pathogenicity, was first isolated from Dermacentor nuttalli in Xinjiang, China, in 2014. GTV has been shown to infect animal and human cell lines and to be pathogenic in mice. The viral nucleoprotein (NP) is the most conserved immunogenic protein. Elucidating the B-cell epitopes (BCEs) in the immunodominant region of the NP is important for the development of virus detection methods and vaccines. In order to identify the minimal motifs of linear BCEs in the NP of the GTV DXM strain, we used an improved biosynthetic peptide (BSP) method to truncate GTV NP into 30 16mer-peptides with 8 overlapping amino acid residues spanning the full length of the protein. The peptides were analyzed by western blot using rabbit anti-GTV NP polyclonal antiserum, and four positive 16mer-peptides were obtained. The 16mer-peptides were then truncated into 31 8mer-peptides with 7 overlapping amino acid residues and 10mer-peptides with 9 overlapping amino acid residues to screen for BCEs that can react with the rabbit anti-GTV NP polyclonal antiserum. The results showed that there were 6 minimal BCE motifs, namely, Enp1, 88EKYGLVER95; Enp2, 88EKYGLVER95; Enp3, 162TTKILMEA169; Enp4, 187GASKAEVY194; Enp5, 191AEVYNSFR198; and Enp6, 236ETAAAAYRNL245. Positive sheep sera could recognize all six BCEs with anti-GTV antibodies. The BCEs were aligned with the sequences of eight representative severe fever with thrombocytopenia syndrome phlebovirus strains from different countries and regions that were evolutionarily closely related to GTV. The sequence identity of the BCEs ranged from 80–100%, thus showing high conservation. The fine epitope mapping of GTV NP can be used to explore the biological and immunological properties of GTV NP antigens and serve as basic data for the development of multi-epitope detection reagents and vaccine design for GTV.

Expression of Recombinant Protein of Coat Protein Gene of Sweet Potato Feathery Mottle Virus in Bacterial Expression System and Production of Polyclonal Antiserum for Its Diagnosis

Sweet potato (Ipomoea batatus L. (Lam), Family Convolvulaceae) is one of the most important tuber crops providing nutritional security because of its high consumption value and medicinal properties, and numerous agro-industrial uses. Sweet potato feathery mottle disease caused by Sweet potato feathery mottle virus (SPFMV) is one of the serious constrains in sweet potato cultivation in India. Effective diagnostic methods need to be developed to solve the problem due to these viruses. As part of the study, infected leaf samples from fields were collected, positive samples were screened for SPFMV using DAC-ELISA and confirmed through PCR. Coat protein gene of SPFMV was PCR amplified, cloned into TA cloning vector and then transformed into Escherichia coli DH5α cells. Positive clones were sub cloned into expression vector pET28A(+) and transformed into DH5α cells. Plasmid DNA from positive clones were isolated and transformed into BL21DE3 cells (NiCo21-DE3 cells). Positive clones were identified and confirmed in-frame position through sequence analysis. Selected colony was grown in Luria both medium at 37oC. Cells were collected and solubility of SPFMV coat protein (CP) was checked through SDS PAGE. Various standardisations were carried out for optimising expression of SPFMV CP and it was observed that 4 hr induction of 1.5 mM IPTG at 25oC gives maximum yield. Using these conditions, cells were grown on large scale and purified the protein (SPFMV CP) using Ni-NTA resin affinity chromatography. Purified protein was checked using SDS PAGE, confirmed the expression using Western Blotting and given for immunization into two New Zealand white rabbits for polyclonal antibody production. Serological tests like ELISA and DIBA were done for confirming the sensitivity and specificity of the raised antibody using field samples of SPFMV infected sweet potato along with healthy plants. Tested samples gave strong positive reactions at dilutions of 1:500 up to 1:6000. Also antibody reacted specifically at a dilution of 1:6000 in ELISA and DIBA. This is the first report of development of polyclonal antiserum against CP of SPFMV through recombinant technology in India and can be useful for the detection of virus from the field-grown samples.

Immunohistochemical Detection of Enteroviruses in Pancreatic Tissues of Patients with Type 1 Diabetes Using a Polyclonal Antibody Against 2A Protease of Coxsackievirus

The need for antiserum for immunohistochemical (IHC) detection of enterovirus (EV) in formaldehyde fixed and paraffin-embedded (FFPE) specimens is increasing. The standard monoclonal antibody against EV-envelope protein (VP1), clone 5D8/1, was proven to cross-react with other proteins. Another candidate marker of EV proteins is 2A protease (2Apro), which is coded by the EV gene and translated by host cells during EV replication. We raised polyclonal antiserum by immunizing rabbits with an 18-mer peptide of Coxsackievirus B1 (CVB1)-2A protease (2Apro) and examined the specificity and sensitivity for EV on FFPE tissue samples. ELISA study showed a high titer of antibody for CVB1-2Apro. IHC demonstrated that antiserum against 2Apro reacted with CVB1-infected Vero-cells. Confocal microscopy demonstrated that 2Apro labelled by the antibody located in the same cell with VP1 stained with 5D8/1. IHC demonstrated dense positive reactions pancreatic islets of EV-associated fulminant type 1 diabetes (FT1DM), and located in the same cell stained positive with 5D8/1. Specificity of IHC staining FT1DM pancreas was confirmed by absorption with an excessive concentration of immunized peptide. In conclusion, our study provides a new polyclonal antiserum against CVB1 2Apro which may be useful for detection of EV-infected human tissues stored as archive of FFPE tissue samples.

Cross-Reactive anti-Nucleocapsid Protein Immunity against Crimean-Congo Hemorrhagic Fever Virus and Hazara Virus in Multiple Species

The World Health Organization estimates that there may be three billion people at risk of infection by Crimean-Congo Hemorrhagic Fever Virus (CCHFV), a highly lethal, emerging orthonairovirus carried by ticks. On the other hand, the closely related Hazara virus (HAZV), a member of the same serogroup, has not been reported as a pathogen for humans. Given the structural and phylogenetic similarities between these two viruses, we evaluated the immunological similarities of the nucleocapsid protein (NP) of these two viruses in multiple species. Strong antigenic similarities were demonstrated in anti-NP humoral immune responses against HAZV and CCHFV in multiple species using convalescent human CCHF sera, rabbit and mouse polyclonal antiserum raised against CCHFV, and mouse polyclonal antiserum against CCHFV-NP in enzyme immunoassays. We also report a convincing cross-reactivity between NPs in Western blots using HAZV-infected cell lysate as antigen and inactivated CCHFV and CCHFV-NP-immunized mice sera. These results suggest that NPs of HAZV and CCHFV share significant similarities in humoral responses across species and underline the potential utility of HAZV as a surrogate model for CCHFV. IMPORTANCE CCHFV and HAZV, members of the Nairoviridae family, are transmitted to mammals by tick bites. CCHFV is considered to be a severe threat to public health and causes hemorrhagic diseases with a high mortality rate, and there are neither preventative nor therapeutic medications against CCHFV disease. HAZV, on the other hand, is not a pathogen to humans and can be studied under BSL-2 conditions. The antigenic relationship between these viruses is of interest for vaccines and for preventative investigations. Here, we demonstrate cross-reactivity in anti-NP humoral immune response between NPs of HAZV and CCHFV in multiple species. These results underline the utility of HAZV as a surrogate model to study CCHFV infection.

Recombinant Coat Protein of Banana Bract Mosaic Virus as a Potential Antigen for Serological Detection of the Virus

Banana bract mosaic disease caused by Banana bract mosaic potyvirus (BBrMV) is reported to instigate heavy loss in banana and plantain across Asia. Almost all the cultivars of banana succumb to the disease resulting in malformed bunches weighing less than half of normal ones. In the current study the coat protein (CP) gene segment present at the 3’ terminal region of the viral genome amplified by RT-PCR was cloned into expression vectors, pRSET-C and pGEX-4T-2 to use it for raising polyclonal antiserum which in turn will aid in developing assays to detect the virus. Recombinant BBrMV CP (rCP) in pRSET-C when expressed was insoluble whereas, it was in the soluble fraction when expressed from pGEX-4T-2. The GST-fusion protein was purified by GSH sepharose affinity column chromatography and western blot analysis was performed using anti GST antibodies. 360 µg/ml of protein was purified from 1 l of culture. The GST tag was cleaved from the purified protein by incubation with thrombin at 25°C overnight. The rCP was characterized using ultracentrifugation, fluorescence spectroscopy and electron microscopy. The tagless monomer failed to assemble to virus like particles (VLPs) in vitro which was substantiated by fluorescence spectroscopy. This study will be first step towards deciphering structure and functions of Banana bract mosaic virus coat protein.