Screening and characterization of antimicrobial secondary metabolites from Halomonas salifodinae MPM-TC and its in vivo antiviral influence on Indian white shrimp Fenneropenaeus indicus against WSSV challenge

AbstractAntimicrobial secondary metabolites from extremophiles play a significant role in the pharmacological industry due to their stable and strong activity and it is used in the treatment of microbial infections. In the present work, Halomonas salifodinae MPM-TC (M. Peter Marian-T. Citarasu) was isolated from the solar salt works in India and identified by 16S rRNA sequencing. The secondary metabolites were extracted from H. salifodinae MPM-TC and tested for antibacterial activity against aquatic bacterial pathogens such as Vibrio harveyi, Vibrio parahaemolyticus, Pseudomonas aeruginosa and Aeromonas hydrophila isolated from infected fish/shrimp, and it effectively controlled them with more than 10mm of zone of inhibition. The metabolites were purified through silica column chromatography and in vitro antiviral activity was performed against White Spot Syndrome Virus (WSSV) using different fractions. Among the different tested fractions, fraction-III (F-III) was able to suppress WSSV replication. Shrimps challenged with a WSSV inoculum incubated with F-III and treated Fenneropenaeus indicus survived around twice as many as the controls. Gas chromatography–mass spectroscopic (GC–MS) analysis revealed that the antiviral active fraction contains around eight compounds including Perfluorotributylamine, Cyclopentane, 1-butyl-2-ethyl and 1,1′-Biphenyl]-3-amine. Further the active fraction F-III was incorporated in the artificial diets at the concentration of 200 (HS1), 400 (HS2) and 800 (HS3) μgkg−1 and fed to F. indicus for 30days. After 30days of culture, shrimps were challenged with virulent WSSV and studied for WSSV VP 28 gene expression, biochemical, haematological and immunological changes. Surprisingly, groups treated with lower concentrations of fraction F-III (HS1 or HS2) significantly (P<0.05) suppressed the viral replication. Different levels of protein and glucose, improved total haemocyte count (THC), coagulase activity and oxyhaemocyanin level all were comparable to controls. Also, immunological parameters such as prophenol oxidase and intracellular superoxide anion production were significantly increased (F=97.18; P⩽0.001 and F=5.70; P⩽0.05) in the groups treated with the three test concentrations. The presence of antiviral and immunostimulant active principles in the F-III fraction effectively suppressed the WSSV load and boosted F. indicus’s immune system. This research will help to develop novel antiviral drugs from plants against aquatic important pathogens

<span style="mso-bidi-font-style:italic" lang="EN-US">Screening and characterization of antiviral compounds from <i style="mso-bidi-font-style:normal"><span style="font-size:11.0pt; font-family:"Times New Roman";mso-fareast-font-family:"Times New Roman"; mso-bidi-font-family:Mangal;mso-ansi-language:EN-GB;mso-fareast-language:EN-US; mso-bidi-language:HI" lang="EN-GB">Psidium guajava </span></i><span style="font-size:11.0pt;font-family:"Times New Roman";mso-fareast-font-family: "Times New Roman";mso-bidi-font-family:Mangal;mso-ansi-language:EN-GB; mso-fareast-language:EN-US;mso-bidi-language:HI" lang="EN-GB">Linn.<i style="mso-bidi-font-style: normal"> </i>root bark<i style="mso-bidi-font-style:normal"> </i>against white<i style="mso-bidi-font-style:normal"> </i>spot syndrome virus</span></span>

208-214<span style="font-size:11.0pt;font-family: " times="" new="" roman";mso-fareast-font-family:"times="" roman";mso-bidi-font-family:="" mangal;mso-ansi-language:en-gb;mso-fareast-language:en-us;mso-bidi-language:="" hi"="" lang="EN-GB">White Spot Syndrome Virus (WSSV) has been reported to cause severe mortality and economic loss in shrimp culture industry especially black tiger shrimp in worldwide. In present study, Psidium guajava Linn. root bark was serially extracted with hexane, ethyl acetate and methanol then screened antiviral activity against WSSV by incubating the extracts with WSSV infected haemolymph of Penaeus monodon which was propagated earlier. The incubated haemolymph was intramuscularly injected to the second abdominal segment of Indian white shrimp Fennerropenaeus indicus and monitored the survival up to 10 days. Two step PCR detection was performed from the genomic DNA of treated shrimps using VP 28-WSSV diagnostic PCR primer. The survival and two step PCR detections revealed that, the ethyl acetate extract effectively suppressed the WSSV followed by methanol extracts and no antiviral activity was observed in the hexane extracts. Phytochemical analysis was performed with the active extracts and further this was purified through silica column chromatography and Thin Layer Chromatography. The elution was screened again for anti-WSSV activity and the antiviral active elution of P. <i style="mso-bidi-font-style: normal">guajava were analysed by GC-MS which revealed that the active elution contains active compounds such as phenol, 2,5-bis(1,1-dimethylethyl), diethyl phthalate, asarone, phthalic acid, butyldodecyl ester, phytol and 1,2-benzenedicarboxylic acid, mono(2-ethylhexyl) ester. The present study revealed that there is a possibility for developing new antiviral drugs from P. guajava against WSSV infection.</span