Molecular characterization of genome segments 1 and 3 encoding two capsid proteins of Antheraea mylitta cytoplasmic polyhedrosis virus

<p>Abstract</p> <p>Background</p> <p><it>Antheraea mylitta </it>cytoplasmic polyhedrosis virus (AmCPV), a cypovirus of <it>Reoviridae </it>family, infects Indian non-mulberry silkworm, <it>Antheraea mylitta</it>, and contains 11 segmented double stranded RNA (S1-S11) in its genome. Some of its genome segments (S2 and S6-S11) have been previously characterized but genome segments encoding viral capsid have not been characterized.</p> <p>Results</p> <p>In this study genome segments 1 (S1) and 3 (S3) of AmCPV were converted to cDNA, cloned and sequenced. S1 consisted of 3852 nucleotides, with one long ORF of 3735 nucleotides and could encode a protein of 1245 amino acids with molecular mass of ~141 kDa. Similarly, S3 consisted of 3784 nucleotides having a long ORF of 3630 nucleotides and could encode a protein of 1210 amino acids with molecular mass of ~137 kDa. BLAST analysis showed 20-22% homology of S1 and S3 sequence with spike and capsid proteins, respectively, of other closely related <it>cypoviruses </it>like <it>Bombyx mori </it>CPV (BmCPV), <it>Lymantria dispar </it>CPV (LdCPV), and <it>Dendrolimus punctatus </it>CPV (DpCPV). The ORFs of S1 and S3 were expressed as 141 kDa and 137 kDa insoluble His-tagged fusion proteins, respectively, in <it>Escherichia coli </it>M15 cells via pQE-30 vector, purified through Ni-NTA chromatography and polyclonal antibodies were raised. Immunoblot analysis of purified polyhedra, virion particles and virus infected mid-gut cells with the raised anti-p137 and anti-p141 antibodies showed specific immunoreactive bands and suggest that S1 and S3 may code for viral structural proteins. Expression of S1 and S3 ORFs in insect cells via baculovirus recombinants showed to produce viral like particles (VLPs) by transmission electron microscopy. Immunogold staining showed that S3 encoded proteins self assembled to form viral outer capsid and VLPs maintained their stability at different pH in presence of S1 encoded protein.</p> <p>Conclusion</p> <p>Our results of cloning, sequencing and functional analysis of AmCPV S1 and S3 indicate that S3 encoded viral structural proteins can self assemble to form viral outer capsid and S1 encoded protein remains associated with it as inner capsid to maintain the stability. Further studies will help to understand the molecular mechanism of capsid formation during cypovirus replication.</p

Analysis of Transcripts Expressed in One-Day-Old Larvae and Fifth Instar Silk Glands of Tasar Silkworm, Antheraea mylitta

Antheraea mylitta is one of the wild nonmulberry silkworms, which produces tasar silk. An EST project has been undertaken to understand the gene expression profile of A. mylitta silk gland. Two cDNA libraries, one from the whole bodies of one-day-old larvae and the other from the silkglands of fifth instar larvae, were constructed and sequenced. A total of 2476 good-quality ESTs (1239 clones) were obtained and grouped into 648 clusters containing 390 contigs and 258 singletons to represent 467 potential unigenes. Forty-five sequences contained putative coding region, and represented potentially novel genes. Among the 648 clusters, 241 were categorized according to Gene Ontology hierarchy and showed presence of several silk and immune-related genes. The A. mylitta ESTs have been organized into a freely available online database “AmyBASE”. These data provide an initial insight into the A. mylitta transcriptome and help to understand the molecular mechanism of silk protein production in a Lepidopteran species

Patterns and rates of viral evolution in HIV-1 subtype B infected females and males.

Biological sex differences affect the course of HIV infection, with untreated women having lower viral loads compared to their male counterparts but, for a given viral load, women have a higher rate of progression to AIDS. However, the vast majority of data on viral evolution, a process that is clearly impacted by host immunity and could be impacted by sex differences, has been derived from men. We conducted an intensive analysis of HIV-1 gag and env-gp120 evolution taken over the first 6-11 years of infection from 8 Women's Interagency HIV Study (WIHS) participants who had not received combination antiretroviral therapy (ART). This was compared to similar data previously collected from men, with both groups infected with HIV-1 subtype B. Early virus populations in men and women were generally homogenous with no differences in diversity between sexes. No differences in ensuing nucleotide substitution rates were found between the female and male cohorts studied herein. As previously reported for men, time to peak diversity in env-gp120 in women was positively associated with time to CD4+ cell count below 200 (P = 0.017), and the number of predicted N-linked glycosylation sites generally increased over time, followed by a plateau or decline, with the majority of changes localized to the V1-V2 region. These findings strongly suggest that the sex differences in HIV-1 disease progression attributed to immune system composition and sensitivities are not revealed by, nor do they impact, global patterns of viral evolution, the latter of which proceeds similarly in women and men

Molecular characterization of genome segment 2 encoding RNA dependent RNA polymerase of Antheraea mylitta cytoplasmic polyhedrosis virus

AbstractGenome segment 2 (S2) from Antheraea mylitta cypovirus (AmCPV) was converted into cDNA, cloned and sequenced. S2 consisted of 3798 nucleotides with a long ORF encoding a 1116 amino acid long protein (123kDa). BLAST and phylogenetic analysis showed 29% sequence identity and close relatedness of AmCPV S2 with RNA dependent RNA polymerase (RdRp) of other insect cypoviruses, suggesting a common origin of all insect cypoviruses. The ORF of S2 was expressed as 123kDa soluble His-tagged fusion protein in insect cells via baculovirus recombinants which exhibited RdRp activity in an in vitro RNA polymerase assay without any intrinsic terminal transferase activity. Maximum activity was observed at 37°C at pH 6.0 in the presence of 3mM MgCl2. Site directed mutagenesis confirmed the importance of the conserved GDD motif. This is the first report of functional characterization of a cypoviral RdRp which may lead to the development of anti-viral agents

p21(WAF1/CIP1) RNA expression in highly HIV-1 exposed, uninfected individuals.

Some individuals remain HIV-1 antibody and PCR negative after repeated exposures to the virus, and are referred to as HIV-exposed seronegatives (HESN). However, the causes of resistance to HIV-1 infection in cases other than those with a homozygous CCR5Δ32 deletion are unclear. We hypothesized that human p21WAF1/CIP1 (a cyclin-dependent kinase inhibitor) could play a role in resistance to HIV-1 infection in HESN, as p21 expression has been associated with suppression of HIV-1 in elite controllers and reported to block HIV-1 integration in cell culture. We measured p21 RNA expression in PBMC from 40 HESN and 40 low exposure HIV-1 seroconverters (LESC) prior to their infection using a real-time PCR assay. Comparing the 20 HESN with the highest exposure risk (median = 111 partners/2.5 years prior to the 20 LESC with the lowest exposure risk (median = 1 partner/2.5 years prior), p21 expression trended higher in HESN in only one of two experiments (P = 0.11 vs. P = 0.80). Additionally, comparison of p21 expression in the top 40 HESN (median = 73 partners/year) and lowest 40 LESC (median = 2 partners/year) showed no difference between the groups (P = 0.84). There was a weak linear trend between risk of infection after exposure and increasing p21 gene expression (R2 = 0.02, P = 0.12), but again only in one experiment. Hence, if p21 expression contributes to the resistance to viral infection in HESN, it likely plays a minor role evident only in those with extremely high levels of exposure to HIV-1

Cloning, overexpression, purification, crystallization and preliminary X-ray diffraction analysis of glyceraldehyde-3-phosphate dehydrogenase from Antheraea mylitta

The cloning, overexpression, purification, crystallization and preliminary X-ray crystallographic analysis of glyceraldehyde-3-phosphate dehydrogenase from A. mylitta are reported

Potential of nanobiosensor in sustainable agriculture: the state-of-art

A rapid surge in world population leads to an increase in worldwide demand for agricultural products. Nanotechnology and its applications in agriculture have appeared as a boon to civilization with enormous potential in transforming conventional farming practices into redefined farming activities. Low-cost portable nanobiosensors are the most effective diagnostic tool for the rapid on-site assessment of plant and soil health including plant biotic and abiotic stress level, nutritional status, presence of hazardous chemicals in soil, etc. to maintain proper farming and crop productivity. Nanobiosensors detect physiological signals and convert them into standardized detectable signals. In order to achieve a reliable sensing analysis, nanoparticles can aid in signal amplification and sensor sensitivity by lowering the detection limit. The high selectivity and sensitivity of nanobiosensors enable early detection and management of targeted abnormalities. This study identifies the types of nanobiosensors according to the target application in agriculture sector

p21^WAF1/CIP1 RNA Expression in Highly HIV-1 Exposed, Uninfected Individuals

<div><p>Some individuals remain HIV-1 antibody and PCR negative after repeated exposures to the virus, and are referred to as HIV-exposed seronegatives (HESN). However, the causes of resistance to HIV-1 infection in cases other than those with a homozygous CCR5Δ32 deletion are unclear. We hypothesized that human p21^WAF1/CIP1 (a cyclin-dependent kinase inhibitor) could play a role in resistance to HIV-1 infection in HESN, as p21 expression has been associated with suppression of HIV-1 in elite controllers and reported to block HIV-1 integration in cell culture. We measured p21 RNA expression in PBMC from 40 HESN and 40 low exposure HIV-1 seroconverters (LESC) prior to their infection using a real-time PCR assay. Comparing the 20 HESN with the highest exposure risk (median = 111 partners/2.5 years prior to the 20 LESC with the lowest exposure risk (median = 1 partner/2.5 years prior), p21 expression trended higher in HESN in only one of two experiments (P = 0.11 vs. P = 0.80). Additionally, comparison of p21 expression in the top 40 HESN (median = 73 partners/year) and lowest 40 LESC (median = 2 partners/year) showed no difference between the groups (P = 0.84). There was a weak linear trend between risk of infection after exposure and increasing p21 gene expression (R² = 0.02, P = 0.12), but again only in one experiment. Hence, if p21 expression contributes to the resistance to viral infection in HESN, it likely plays a minor role evident only in those with extremely high levels of exposure to HIV-1.</p></div

p21 expression in HESN and LESC.

<p>Shown are comparisons of p21 expression between: A) the top 20 ranking HIV-uninfected individuals with the greatest risk exposure (HESN) and the top 20 ranking HIV-infected individuals with the lowest risk exposure (LESC) (sampled prior to HIV infection); and B) the top 40 ranking HESN and LESC. All individuals were of European ancestry and none possessed a CCR5Δ32 deletion. p21 count refers to the number of p21 transcripts per ng of cDNA derived from total PBMC RNA.</p