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 (SepOct), 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 pebrine disease in Bombyx mori silkworm, based on the beta-tubulin gene

"Pe ' brine" 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 beta-tubulin gene in the pe ' brine 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 x 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

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

Development and optimization of a TaqMan assay for Nosema bombycis, causative agent of pebrine disease in Bombyx mori silkworm, based on the β-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 β-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) &lt;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

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 (SepOct), 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)