Investigation on Pathological Aspects, Mode of Transmission, and Tissue Tropism of Antheraea proylei Nucleopolyhedrovirus Infecting Oak Tasar Silkworm.

The temperate oak tasar silkworm, Antheraea proylei, is frequently infested with Antheraea proylei nucleopolyhedrovirus (AnprNPV) causing tiger band disease. This disease is one of the key factors that obstructs production and productivity of oak tasar sericulture. The current study aimed to investigate the pathogenicity of AnprNPV, its mode of transmission, and detection of AnprNPV in different tissues. Transmission electron micrographs of AnprNPV showed single rod-shaped bodies and occlusion derived virus (ODV) enclosed within multiple envelopes. The infecting AnprNPV displayed tissue tropism with higher copy numbers detected in the insect fat body and ovary. The virus was observed to multiply in all developmental stages of the silkworm such as egg, larva, pupa, and moth, confirming its ability to spread throughout the silkworm lifecycle. Baculovirus isolated from infected A. proylei showed cross-infectivity in other Saturniidae wild silkworm species such as Antheraea pernyi, A. frithi, and Samia ricini, widening their probable host range for infection. Baculoviruses generally display a horizontal mode of transmission, mainly through ingestion of occlusion bodies (OBs); however, the present study revealed a trans-ovum vertical mode of transmission in addition to a horizontal mode. The observations made in this study aid a detailed understanding of the tiger band disease and its causative pathogen AnprNPV, which will support future studies and disease management in oak tasar sericulture.

Cloning, Expression and Characterization of Spore Wall Protein 5 (SWP5) of Indian Isolate NIK-1S of Nosema bombycis.

SWPs are the major virulence component of microsporidian spores. In microsporidia, SWPs can be found either in exospore or endospore to serve as a putative virulence factor for host cell invasion. SWP5 is a vital protein that involves in exospore localization and supports the structural integrity of the spore wall and this action potentially modulates the course of infection in N. bombycis. Here we report recombinant SWP5 purification using Ni-NTA IMAC and SEC. GFC analysis reveals SWP5 to be a monomer which correlates with the predicted theoretical weight and overlaps with ovalbumin peak in the chromatogram. The raised polyclonal anti-SWP5 antibodies was confirmed using blotting and enterokinase cleavage experiments. The resultant fusion SWP5 and SWP5 in infected silkworm samples positively reacts to anti-SWP5 antibodies is shown in ELISA. Immunoassays and Bioinformatic analysis reveal SWP5 is found to be localized on exospore and this action could indicate the probable role of SWP5 in host pathogen interactions during spore germination and its contribution to microsporidian pathogenesis. This study will support development of a field-based diagnostic kit for the detection N. bombycis NIK-1S infecting silkworms. The analysis will also be useful for the formulation of drugs against microsporidia and pebrine disease.

An investigation into the effects of infection and ORF expression patterns of the Indian bidensovirus isolate (BmBDV) infecting the silkworm Bombyx mori.

The Indian isolate of Bombyx mori bidensovirus (BmBDV) is a bipartite virus that comprises of a segmented, non-homologous, two linear single-strands of DNA molecules (VD1 and VD2). It is one of the causative agents of the fatal silkworm disease 'Flacherie' that causes severe crop loss for the sericulture farmers. Genome analyses of the Indian isolate of BmBDV revealed that it consists of 6 putative ORFs similar to the Japanese and Chinese isolates. VD1 consists of 4 ORFs while VD2 has 2 ORFs that code for 4 non- structural (NS) and 2 structural (VP) proteins, in total. In this study, we investigated, in detail, the impact of BmBDV pathogenesis on growth and development of the silkworm Bombyx mori, at different developmental stages. Mortality rate and weight uptake analyses were also performed on newly ecdysed 4th instar larvae. BmBDV infection was not found to be developmental stage specific and it occurred at all stages. Onset of mortality took place 8 days post infection (dpi) and 100% mortality occurred at 11 dpi. The infected larvae showed a significant difference in weight uptake wherein from 7 dpi the larvae stopped gaining weight and from 8th dpi started demonstrating the typical symptoms of flacherie. Further, the expression pattern of the 6 viral ORFs were also investigated in the newly ecdysed 4th instar BmBDV infected silkworms. Among all the six ORFs, VD2 ORF 1 and 2 revealed the highest transcript numbers, which was followed by VD1 ORF 4 that encodes for the viral DNA polymerase enzyme. This was the first ever attempt to understand the pathogenesis and the expression pattern of all the six ORF transcripts of the Indian isolate of BmBDV.

Genetic characterisation of an Iflavirus associated with a vomiting disease in the Indian tropical tasar silkworm, Antheraea mylitta.

Antheraea mylitta, the Tropical tasar silkworm, is frequently affected by a vomiting disease called Virosis by sericulturists although not confirmed being of viral origin. Based on the symptoms and the disease pattern, the causal agent is however suspected to be a virus. The condition involves a series of characteristic and progressive symptoms that generally culminates in the death of the larva. The disease is common in autumn season (Sep- Oct), with widespread distribution causing severe damage to the tasar silk industry. The leads for this study were obtained from a transcript identified in the EST database in a different study, which matched the positive strand of Iflavirus, an RNA virus known to infect insects. In the present study the genome of this novel Iflavirus was characterised and the full length of the genome was found to be 9728 nucleotides long encoding for a single large open reading frame (ORF) with flanking NTR regions at 5' and 3' ends and a natural poly A tail at the 3' end. The ORF encoded structural proteins at the N-terminal end and non-structural proteins at the C-terminal end with a predicted 2967 amino acid long polyprotein. The structural proteins consisted of 4 proteins (VP1-VP4) and the non-structural proteins consisted of helicase, RNA-dependent RNA polymerase and 3C-protease. The virus is found in various tissues (midgut, fatbody, trachea, Malpighian tubules and silk gland) and also has a vertical route of transmission, i.e., from gravid females to the offspring. Based on the available data, the virus is a new member of Iflaviridae for which we propose the name Antheraea mylitta Iflavirus (AmIV).

Development and optimization of a TaqMan assay for Nosema bombycis, causative agent of pébrine disease in Bombyx mori silkworm, based on the ß-tubulin gene.

"Pébrine" is a devastating disease of Bombyx mori silkworms that is highly contagious and can completely destroy an entire crop of silkworms and is thus a serious threat for the viability and profitability of sericulture. The disease is most commonly attributed to microsporidians of the genus Nosema, which are obligate intracellular parasites that are transmitted through spores. Nosema infections in silkworms are diagnosed primarily through light microscopy, which is labour intensive and less reliable, sensitive, and specific than PCR-based techniques. Here, we present the development and optimization of a new TaqMan based assay targeting the ß-tubulin gene in the pébrine disease causing agent Nosema bombycis in silkworms. The assay displayed excellent quantification linearity over multiple orders of magnitude of target amounts and a limit of detection (LOD) of 6.9 × 102 copies of target per reaction. The method is highly specific to N. bombycis with no cross-reactivity to other Nosema species commonly infecting wild silkworms. This specificity was due to three nucleotides in the probe-binding region unique to N. bombycis. The assay demonstrated a high reliability with a Coefficient of variation (CV) <5% for both intra-assay and inter-assay variability. The assay was used to trace experimental N. bombycis infection of silkworm larvae, in the fat body, midgut and ovary tissues, through pupation and metamorphosis to the emerging female moth, and her larval off-spring, confirming the vertical transmission of N. bombycis in silkworms. The TaqMan assay revealed a gradual increase in infection levels in the post-infection samples. The assay is reliable and simple to implement and can be a suitable complement to microscopy for routine diagnostics and surveillance in silkworm egg production centres with appropriate infrastructure.

Host Response against Virus Infection in an Insect: Bidensovirus Infection Effect on Silkworm (Bombyx mori).

Silk cocoons obtained from silkworms are the primary source of commercial silk, making the silkworm an economically important insect. However, the silk industry suffers significant losses due to various virus infections. Bombyx mori bidensovirus (BmBDV) is one of the pathogens that cause flacherie disease in silkworms. Most silkworm strains die after BmBDV infection. However, certain silkworm strains show resistance to the virus, which is determined by a single recessive gene, nsd-2. The +nsd-2 gene (allele of nsd-2; the susceptibility gene) encodes a putative amino acid transporter expressed only in the insect's midgut, where BmBDV can infect, suggesting that this membrane protein may function as a receptor for BmBDV. Interestingly, the expression analysis revealed no changes in the +nsd-2 gene expression levels in virus-uninfected silkworms, whereas the gene expression drastically decreased in the virus-infected silkworm. This condition indicates that the host factor's expression, the putative virus receptor, is affected by BmBDV infection. It has recently been reported that the expression levels of some host genes encoding cuticle, antioxidant, and immune response-related proteins were significantly regulated by BmBDV infection. In this review, we discuss the host response against virus infection based on our knowledge and long-term research experience in this field.

Transcriptome analysis of Nosema assamensis infecting muga silkworms (Antheraea assamensis) reveals insights into candidate pathogenicity related genes and molecular pathways required for pathogenesis.

Muga silkworms are often prone to many diseases since, these are non-domesticated and are reared outdoors. Microsporidia, an obligate intracellular pathogen with spore as its active form, causes pebrine disease in these silkworms. The study has attempted to categorise the transcript data of the Nosema obtained from the infected muga silkworm using gene ontology and KEGG pathway studies. A total of 2850 unigene sets were identified out of which 2739 unigenes were placed under biological, cellular as well as molecular function categories based on the gene ontology (GO) terms. 1620 out of these unigenes sets found their orthologous partner in the corresponding Nosema bombycis transcriptome. The unigenes were found to be enriched under organic substance metabolic process, organic cyclic compound binding and intracellular anatomical structure for biological process, molecular function and cellular components respectively. The KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway analysis indicated majority of the enzymes were found to be mapped under purine and thiamine metabolic pathways, indicating an increase in the energy metabolism required to establish the infection in the silkworms. The putative virulence genes identified in this study are PTP2, PTP3, SWP12 and SWP26 which were found to be expressed in other Nosema species indigenous to India, indicating a probable conservation of these genes, which are primarily involved in establishing host pathogen interactions. The expression of these genes was in detectable levels in the infected silkworm samples. These genes may be validated further through bioassay in order understand their roles in establishing the infection and propagation of the spores. The identified virulence genes may be further targeted to develop diagnostic tools for identification of the pathogen at early stages of infection.

Targeting essential genes of Nosema for the diagnosis of pebrine disease in silkworms.

Pebrine is one of the devastating diseases mostly caused by notorious Nosema - a microsporidian infecting silkworms. Identification of novel genes associated with the pathogen plays a key role in developing a reliable diagnostic tool for the detection of disease. Targeting potential biomarkers will help in developing strategies for fast and efficient control measures, which can prevent the spread of infection. This study was to identify genes present commonly in Nosema such as Nosema bombycis, Nosema mylitta, Nosema assamensis and Nosema ricini infecting Indian silkworms in order to find potential gene markers for early pebrine disease diagnosis. Real time PCR was used to validate the genes active early during the infection cycle, confirming the expression of genes and their order of expression. 16S rRNA and ß-tubulin were found to be expressed early in infection followed by PTP1 and PTP2, PTP3, SWP5 and MetAP2 genes. These identified molecular markers can be used in addition to conventional gene primers which are traditionally used for the detection of pebrine.

Genome wide characterization revealed MnMLO2 and MnMLO6A as candidate genes involved in powdery mildew susceptibility in mulberry.

Mulberry is a fast growing economically important tree for sericulture industry and contains compounds for preventing and treating several diseases and ailments. The quality and quantity of mulberry leaf available to produce silk fibre and for medicinal purpose is greatly affected by number of foliar diseases, out of which powdery mildew is the major one. Imparting genetic resistance becomes an important approach in disease management in mulberry as spraying of fungicides has harmful effects on silkworm growth and development. Deployment of non-functional susceptible genes such as Mildew resistance Locus O (MLO) against powdery mildew in few crops stimulated to identify and characterize MLO genes in mulberry. In this study, genome wide analysis identified 16 MLO genes in Morus notabilis. Phylogenetic analysis found that MnMLO2, MnMLO6A, MnMLO6B, MnMLO12A and MnMLO12B clustered with functionally characterized MLOs associated with powdery mildew susceptibility in dicot species. Gene expression analysis indicated increased transcript abundance of MnMLO2, MnMLO6A, and MnMLO12A in response to powdery mildew infection. Further, conserved motifs exclusive to functionally characterized MLOs were identified in MnMLO1C, MnMLO2 and MnMLO6A proteins. Combined analysis of the phylogenetic relationship, conserved motif analysis and gene expression in response to infection identified MnMLO2 and MnMLO6A as potential candidate genes involved in powdery mildew susceptibility in mulberry. Identification and deployment of natural and induced mutations in the candidate genes can be useful for mulberry breeding programs to develop powdery mildew resistant varieties.

Evolutionary Diversity in the Intracellular Microsporidian Parasite Nosema sp. Infecting Wild Silkworm Revealed by IGS Nucleotide Sequence Diversity.

Intracellular microsporidian Nosema mylitta infects Indian wild silkworm Antheraea mylitta causing pebrine disease. Genetic structure and phylogeny of N. mylitta are analysed using nucleotide variability in 5S ribosomal DNA and intergenic spacer (IGS) sequence from 20 isolates collected from Southern, Northern and Central regions of Jharkhand State. Nucleotide diversity (π) and genetic differentiation Gst were highest in the Central isolates whereas lowest in the North. Among the isolates, absence of nucleotides, transitions and transversions were observed. Haplotyping showed nucleotide variability at 83 positions in IGS and 13 positions in 5S rDNA. Haplotype-based genetic differentiation was 0.96 to 0.97 whereas nucleotide sequence-based genetic differentiation was higher (Ks = 22.29) between Southern and Central isolates. Bottleneck analysis showed negative value for Tajima's D and other summary statistics revealing induction of loss of rare alleles and population explosion. From IGS, 17 ancestral sequences were inferred by Network algorithm. Core of nine closely related nodes having ancient nucleotides and peripheral nodes with highly divergent nucleotides were derived. Most diverged peripheral haplotype was Bero (H11) from the Central region whereas Deoghar (H3) of the Northern region diverged early. Phylogeny of N. mylitta grouped Southern and Northern isolates together revealed weak phylogenetic signal for these locations. Phylogeny of N. mylitta with Nosema sp. infecting other lepidopterans clustered N. mylitta isolates with N. antheraea and N. philosamiae of China indicating genetic similarity whereas other species were dissimilar showing diversity irrespective of country of origin.

Development and comparison of real-time and conventional PCR tools targeting ß-tubulin gene for detection of Nosema infection in silkworms.

Microsporidiosis (Pebrine) caused by the microsporidian parasite is one of the important devastating disease which affect the silk production leading to an unprofitable harvest. Till date ribosomal RNA (rRNA) gene was used as a target for detection of microsporidian species. In this study, we describe conventional and SYBR green based real-time PCR techniques alternatively targeting ß-tubulin gene for quantitative detection of microsporidia infecting both the mulberry and non-mulberry silkworms. The modified DNA extraction method followed in our study was found to be easy, economical and could be used for both conventional and real time PCR as template. The real time qPCR revealed the expression of ß-tubulin gene in different infected tissues of the silkworm Bombyx mori. The sensitivity of the SYBR green based real time PCR was found to be 100 times more than the conventional PCR and PCR was found more sensitive than the microscopic examination. The developed method did not produce any false positive results with the other silkworm pathogens and healthy silkworm. The data suggest that both the developed PCR methods targeting ß-tubulin gene could be used effectively in quarantine process at seed centres for early detection of microsporidian infection in silkworms.

Selection of suitable reference genes for quantitative real-time PCR gene expression analysis in Mulberry (Morus alba L.) under different abiotic stresses.

Mulberry (Morus alba L.) is the sole food source for the mulberry silkworm, Bombyx mori and therefore important for sericulture industry. Different abiotic stress conditions like drought, salt, heat and cold stress adversely affect the productivity and quality of mulberry leaves. Quantitative real time PCR (qPCR) is a reliable and widely used method to identify abiotic stress responsive genes and molecular mechanism in different plant species. Selection of suitable reference genes is important requirement for normalizing the expression of genes through qRT-PCR study. In the present study, we have selected eight candidate reference genes in mulberry for analyzing their expression stability in different abiotic stress treatments including drought, salt, heat and cold stresses. The expression stability of these reference genes was determined using geNorm, NormFinder and RefFinder statistical algorithms. The results showed that Ubiquitin and protein phosphatase 2A regulatory subunit A (PP2A) were the most stable genes across all the treatment samples. However, analysis of individual stresses revealed different expression profiles and stability of reference genes. Actin3 and PP2A were most stable in drought and salt conditions respectively. RPL3 most preferred in heat stress and Ubiquitin was most stable in cold stress. We propose the ubiquitin and PP2A are the preferred reference genes for normalization of gene expression data from abiotic stresses. In addition, Actin3 are preferred for drought stress, PP2A for salt stress, RPL3 for heat stress and Ubiquitin for cold stress studies.

Molecular characterization of Nosema bombycis methionine aminopeptidase 2 (MetAP2) gene and evaluation of anti-microsporidian activity of Fumagilin-B in silkworm Bombyx mori.

Nosema bombycis is a spore-forming parasite causing microsporidiosis in silkworm Bombyx mori. Methionine aminopeptidase 2 (MetAP2), an essential gene of N. bombycis, is a target for the anti-microsporidian drug Fumagillin, an antibiotic derived from Aspergillus fumigatus. In this study, a 1077 bp full-length cDNA of the MetAP2 gene of N. bombycis was cloned and characterized. Furthermore, the expression study of the MetAP2 gene revealed a ubiquitous expression during all the developmental stages of the silkworm B. mori. The phylogenetic analysis of the MetAP2 gene of N. bombycis revealed the MetAP2 gene sequences to be highly conserved in nature. The present study also includes the validation of the anti-microsporidian drug Fumagillin against the MetAP2 gene of N. bombycis. The findings revealed that Fumagilin-B could also suppress the N. bombycis multiplication in the silkworm B. mori, thereby proving the therapeutic role of Fumagillin against microsporidian infection. This is the first-ever report regarding the characterization of the MetAP2 gene in the Indian isolate of N. bombycis and also towards the usage of Fumagillin in the control of microsporidiosis in B. mori.

Characterization and genome comparison of an Indian isolate of bidensovirus infecting the silkworm Bombyx mori.

The bipartite genome of an Indian isolate of Bombyx mori bidensovirus (BmBDV), one of the causative agents of the fatal silkworm disease 'Flacherie', was cloned and completely sequenced. Nucleotide sequence analysis of this Indian isolate of BmBDV revealed two viral DNA segments, VD1 and VD2 as well as a DNA polymerase motif which supports its taxonomical status as the type species of a new family of Bidnaviridae. The Indian isolate of BmBDV was found to have a total of six putative ORFs four of which were located on the VD1 with the other two being on the VD2 DNA segment. The VD1 DNA segment was found to code for three non-structural proteins including a viral DNA polymerase as well as one structural protein, while the VD2 DNA segment was found to code for one structural and one non-structural protein, similar to that of the Japanese and Zhenjiang isolates of BmBDV. A BmBDV ORF expression study was done through real time qPCR wherein the VD2 ORF 1 and 2 showed the maximum transcript levels. This is the first report of the genome characterization of an Indian isolate of BmBDV, infecting silkworm B. mori.

Oxidative stress and cytotoxicity elicited lipid peroxidation in hemocytes of Bombyx mori larva infested with dipteran parasitoid, Exorista bombycis.

Parasitization of silkworm, Bombyx mori by invasive larva of dipteran parasitoid Exorista bombycis caused upto 20% revenue loss in sericulture. The parasitism was successful by suppressing host immune system however mechanism of immune suppression induced by E. bombycis is unknown which is unravelled here. The infestation induced cytotoxic symptoms in host hemocytes, such as vacuolated cytoplasm, porous plasma membrane, indented nuclei with condensed chromatin and dilated RER. One of the markers of necrosis is cell permeabilization, which can be measured as released lactate dehydrogenase (LDH). LDH level showed significantly (P<0.01) high release into extracellular medium in vitro after exposure of hemocytes to parasitoid larval tissue protein compared with control revealing membrane permeability and loss of cell integrity. At five minutes after exposure, cytotoxicity was 43% and was increased to 99% at 3h. The cytotoxicity is signalled by increased content of hydrogen peroxide (H2O2) causing lipid peroxidation followed by porosity in plasma membrane. A test for lipid peroxidation by measurement of lipid peroxidation breakdown product, malondialdehyde (MDA) revealed significant increase in peroxidation from one to 24 h post-invasion, with maximum at 12 h (P<0.008). Level of reactive oxygen species measured as H2O2 production increased from 6 to 12 h post-invasion and continued to increase significantly (P<0.03) reaching maximum at 48 h. These observations reveal that dipteran endoparasitoid invasion induced H2O2 production in the hemocytes causing cytotoxicity, lipid peroxidation and membrane porosity that suppressed both humoral- and cell-mediated immune responses of hemocytes in B. mori.

The dipteran parasitoid Exorista bombycis induces pro- and anti-oxidative reactions in the silkworm Bombyx mori: Enzymatic and genetic analysis.

Hymenopteran parasitoids inject various factors including polydnaviruses along with their eggs into their host insects that suppress host immunity reactions to the eggs and larvae. Less is known about the mechanisms evolved in dipteran parasitoids that suppress host immunity. Here we report that the dipteran, Exorista bombycis, parasitization leads to pro-oxidative reactions and activation of anti-oxidative enzymes in the silkworm Bombyx mori larva. We recorded increased activity of oxidase, superoxide dismutase, thioredoxin peroxidase, catalase, glutathione-S-transferase (GST), and peroxidases in the hemolymph plasma, hemocytes, and fat body collected from B. mori after E. bombycis parasitization. Microarray and qPCR showed differential expression of genes encoding pro- and anti-oxidant enzymes in the hemocytes. The significance of this work lies in increased understanding of dipteran parasitoid biology.

Genome-wide identification, characterization of sugar transporter genes in the silkworm Bombyx mori and role in Bombyx mori nucleopolyhedrovirus (BmNPV) infection.

Sugar transporters play an essential role in controlling carbohydrate transport and are responsible for mediating the movement of sugars into cells. These genes exist as large multigene families within the insect genome. In insects, sugar transporters not only have a role in sugar transport, but may also act as receptors for virus entry. Genome-wide annotation of silkworm Bombyx mori (B. mori) revealed 100 putative sugar transporter (BmST) genes exists as a large multigene family and were classified into 11 sub families, through phylogenetic analysis. Chromosomes 27, 26 and 20 were found to possess the highest number of BmST paralogous genes, harboring 22, 7 and 6 genes, respectively. These genes occurred in clusters exhibiting the phenomenon of tandem gene duplication. The ovary, silk gland, hemocytes, midgut and malphigian tubules were the different tissues/cells enriched with BmST gene expression. The BmST gene BGIBMGA001498 had maximum EST transcripts of 134 and expressed exclusively in the malphigian tubule. The expression of EST transcripts of the BmST clustered genes on chromosome 27 was distributed in various tissues like testis, ovary, silk gland, malphigian tubule, maxillary galea, prothoracic gland, epidermis, fat body and midgut. Three sugar transporter genes (BmST) were constitutively expressed in the susceptible race and were down regulated upon BmNPV infection at 12h post infection (hpi). The expression pattern of these three genes was validated through real-time PCR in the midgut tissues at different time intervals from 0 to 30hpi. In the susceptible B. mori race, expression of sugar transporter genes was constitutively expressed making the host succumb to viral infection.

Attacin gene sequence variations in different ecoraces of tasar silkworm Antheraea mylitta.

Attacin gene exists as paralogous conversion and is being used for identification of strain variations in insects based on the sequence variation. Hence, a study was undertaken to analyze the sequence variation of the attacin gene isoforms in the tasar silkworm Anthereae mylitta that exists in the form of different ecoraces depending upon the environment, food plant and location. Comparison of the previously reported attacin sequences with the DNA sequences of attacin A and B genes revealed six amino acid substitutions among the sequences of the ecoraces which however did not affect the functional domain of Attacin. The generated dendrogram clearly indicated unique branches for each ecorace with two separate gene clusters for attacin A and B. The Sarihan ecorace formed a separate sub-group under both the gene clusters. The present study also revealed the presence of Attacin_N Superfamily domain exclusively in Exon I separated from the Attacin_C Superfamily domain that was present in Exon II and part of Exon III, a prominent character of attacin gene. The phylogenetic reconstruction analysis of attacin gene in A.mylitta supported the common evolutionary origin of attacin genes belonging to the Lepidoteran and Dipteran families that formed two separate clusters.

Genome wide microarray based expression profiles associated with BmNPV resistance and susceptibility in Indian silkworm races of Bombyx mori.

The molecular mechanism involved in BmNPV resistance was investigated using a genome wide microarray in midgut tissue of Indian silkworm Bombyx mori. In resistant race (Sarupat), 735 genes up-regulated and 589 genes down-regulated at 12 h post BmNPV infection. Similarly, in case of susceptible race (CSR-2), 2183 genes up-regulated and 2115 genes down-regulated. Among these, nine up-regulated and eight down-regulated genes were validated using real-time qPCR analysis. In Sarupat, vacuolar protein sorting associated, Xfin-like protein and carboxypeptidase E-like protein genes significantly up-regulated in infected midgut; prominently down-regulated genes were glutamate receptor ionotropic kainite 2-like, BTB/POZ domain and transferrin. Considerably up-regulated genes in the CSR-2 were peptidoglycan recognition protein S6 precursor and rapamycin while the conspicuous down-regulated genes were facilitated trehalose transporter and zinc transporter ZIP1-like gene. The up-regulation of genes in resistant race after BmNPV infection indicates their possible role in antiviral immune response.

The Indian isolate of Densovirus-2 - Impact of infection and mechanism of resistance in Bombyx mori L.

The Indian Bombyxmori Densovirus type 2 isolate (DNV-2), revealed closer homology with Japanese Yamanashi isolate. PCR and qPCR analyses indicated severe and widespread prevalence of the virus in flacherie diseased B. mori under Indian field conditions. Viral inoculation revealed typical flacherie disease symptoms and transmission electron microscopy revealed damage of infected midgut tissue cells. The nsd-2 gene for resistance to DNV-2 restricted viral proliferation in B. mori. This study indicates possible major role of the Indian DNV-2 isolate in causing flacherie disease in B. mori leading to crop loss. A detailed molecular characterization of the whole viral genome including nsd-2 gene expression profiling is essential to develop appropriate diagnostic tools and control strategies.