Effect of immune gene silencing in WSSV infected tiger shrimp Penaeus monodon.

White spot syndrome virus, continues to cause huge economic loss to aquaculture industry. In the absence of effective therapeutics to control WSSV, it is important to understand the host pathogen interaction at the molecular level. Suppression subtractive hybridization (SSH) cDNA library was constructed which led to identification of several differentially expressed genes in response to WSSV infection in Penaeus monodon. The genes expressed in SSH cDNA library of shrimp gill and gut tissues belonged to a wide range of biological functions. The three differentially expressed genes, Single von Willebrand factor type C domain protein (pmSVC), P53 protein gene (pmP53) and ADP ribosylation factor (pmArf) were up-regulated against WSSV infection and were further characterized by gene silencing to study the role of these shrimp immune genes on WSSV multiplication. The sequence-specific knock down of pmSVC, pmP53 and pmArf using the dsRNA revealed that in pmSVC-dsRNA inoculated shrimps WSSV replication was more with increased viral copy numbers when compared with pmP53-dsRNA and pmArf -dsRNA inoculated shrimps. The varied response of immune genes to WSSV infection, indicated that host genes may either inhibit virus replication to some extent or might act as a target to facilitate viral pathogenesis.

Is periodontitis an independent risk factor for subclinical atherosclerosis?

OBJECTIVES: The aim of this study was to assess the interrelationship between periodontitis and atherosclerosis by comparing the ultrasound and clinical markers of atherosclerosis in systemically healthy patients with and without periodontitis and whether periodontitis can be an independent risk factor for atherosclerosis. MATERIALS AND METHODS: Total 40 subjects, of same socioeconomic status, belonging to age group of 35-65 years, were recruited and divided into two groups - Group I (Chronic Generalised Periodontitis without any systemic disease: CP-SH), Group II (Normal healthy patients without periodontitis and any systemic disease - SH). Clinical measurements and ultrasound examinations were carried out. Qualitative variables were analyzed using Chi square test and qualitative variables using Unpaired Student t test. Statistical significance was accepted for p≤0.05. RESULTS: Carotid ultrasound revealed right and left intima media thickness (IMT) of 0.626±0.016mm and 0.715±0.037mm respectively in cases versus 0.495±0.009mm and 0.518±0.009mm respectively in controls, with the difference being statistically significant. In cases, mean diastolic blood pressure (DBP) was 83.45±4.07mmHg versus 79.25±3.63mmHg in controls, with the difference being statistically significant. CONCLUSION: In this study, we found statistically significant differences in carotid IMT and DBP values between cases and controls. These findings suggest independent role of periodontal disease in subclinical atherosclerosis.

Phylogenetic Relationship Among Brackishwater Vibrio Species.

Vibriosis is regarded as an important disease of penaeid shrimps affecting larvae in hatcheries. Among the Vibrio species, Vibrio parahaemolyticus, Vibrio vulnificus, Vibrio furnissii, Vibrio campbellii, Vibrio harveyi, Vibrio alginolyticus, and Vibrio anguillarum are often associated with diseases in finfish and shellfish of brackishwater ecosystem. Accurate species differentiating methods for the organisms present in an ecosystem are required for precise classification of the species and to take steps for their management. Conventional methods like 16s rRNA phylogeny and multilocus sequence typing (MLST) have often failed to correctly identify Vibrio species. This has necessitated a comprehensive investigation on methodologies available to distinguish Vibrio species associated with brackishwater aquaculture system. To achieve this, 35 whole genomes belonging to 7 Vibrio species were subjected to phylogenetic analysis based on 16s rRNA gene, MLST genes, single-copy orthologous genes, and single-nucleotide polymorphisms. In addition, genome-based similarity indices like average nucleotide identity (ANI) and in silico DNA-DNA hybridization (DDH) were computed as confirmatory tests to verify the phylogenetic relations. There were some misclassifications occurred regarding phylogenetic relations based on 16s rRNA genes and MLST genes, while phylogeny with single-copy orthologous genes produced accurate species-level clustering. Study reveals that the species identification based on whole genome-based estimates or genome-wide variants are more precise than the ones done with single or subset of genes.

A comparative synthesis of transcriptomic analyses reveals major differences between WSSV-susceptible Litopenaeus vannamei and WSSV-refractory Macrobrachium rosenbergii.

Since the 1990s White Spot Syndrome Virus (WSSV) has severely affected shrimp aquaculture worldwide causing a global pandemic of White Spot Disease (WSD) in penaeid culture. However, not all decapod species that can be infected by WSSV show the same susceptibility to the virus, thus raising interesting questions regarding the potential genetic traits that might confer resistance to WSSV. In order to shed light into the genetic markers of WSSV resistance, we employed a dual approach: i) we initially analysed the transcriptomes derived from the hepatopancreas of two species, the susceptible white shrimp Litopenaeus vannamei and the refractory fresh water prawn Macrobrachium rosenbergii, both infected with WSSV. We found a large number of differentially expressed genes (DEGs) belonging to the immune system (mostly anti-microbial peptides and haemolymph clotting components) that were generally up-regulated in M. rosenbergii and down-regulated in L. vannamei. Further, in both species we identified many up-regulated DEGs that were related to metabolism (suggesting a metabolic shift during the infection) and, interestingly, in L. vannamei only, we found several DEGs that were related to moult and suggested an inhibition of the moult cycle in this species following WSSV infection. ii) we then identified a limited number of genetic markers putatively linked with WSD tolerance by employing an ecological genomics approach in which we compared published reports with our own RNA-seq datasets for different decapod species infected with WSSV. Using this second comparative approach, we found nine candidate genes which are consistently down-regulated in susceptible species and up-regulated in refractory species and which have a role in immune response. Together our data offer novel insights into gene expression differences that can be found in susceptible and refractory decapod species infected with WSSV and provide a valuable resource towards our understanding of the potential genetic basis of tolerance to WSSV.

First Report of a Complete Genome Sequence of White spot syndrome virus from India.

White spot syndrome virus is a major pathogen of shrimp, causing economic loss to the aquaculture industry. For the first time, a complete de novo genome of an Indian isolate of this virus has been deciphered using Illumina and Nanopore sequencing technologies. The genome has 280,591 bp with 442 predicted coding genes.

Transcript Analysis of White spot syndrome virus Latency and Phagocytosis Activating Protein Genes in Infected Shrimp (Penaeus monodon).

Viral latency has been recently observed to be associated with White spot syndrome virus (WSSV) infection in shrimp. In the present study, shrimp samples (Penaeus monodon) surviving WSSV infection were examined for presence of WSSV in latent phase. Virus latency was observed in shrimp which were either experimentally challenged with WSSV and survived the infection or those which survived the natural infection. Three viral transcripts (ORFs 427, 151, 366) associated with latency were analyzed by real-time PCR. The shrimp surviving the natural WSSV infection on estimation with RT-PCR were found to have low grade of WSSV infection (less than 56 copies of WSSV). All the shrimp samples were RT-PCR negative for structural protein genes of WSSV, VP24 and VP28, indicating that these samples were harboring latent phase virus. RT-PCR of all the shrimp samples which survived WSSV infection revealed amplification of phagocytosis activating protein (PAP) gene (435 bp) with higher gene expression levels in experimentally challenged shrimp when compared to naturally infected shrimp. The expression of PAP in WSSV infected shrimp samples indicates its possible role in host response for resistance against WSSV infection. PAP was cloned and expressed as recombinant protein for protection studies. Shrimp were injected with three doses (5, 15 and 20 µg g(-1) body weight) of recombinant PAP. Relative percent survival of 10 % was observed in shrimp immunized with the dose of 15 µg g(-1) body weight of recombinant PAP. The expression of both WSSV latency associated and PAP genes obtained from shrimp surviving the WSSV infection, indicates the possible role of these genes in host-pathogen interaction.