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Qadiri, Syed Shariq Nazir,Kim, Soo-Jin,Krishnan, Rahul,Kim, Jae-Ok,Kole, Sajal,Kim, Wi-Sik,Oh, Myung-Joo Elsevier 2019 Aquaculture Vol.505 No.-
<P><B>Abstract</B></P> <P>An insight on localization of viral pathogen is imperative to better understand the host-pathogen interaction. Previously, we developed an <I>in-situ</I> hybridization (RNA-ISH) assay to detect viral hemorrhagic septicemia virus (VHSV), an OIE reportable fish pathogen, in an <I>in vitro</I> model. Here, we utilized its potential and applicability <I>in vivo</I> to further our understanding about the localization and tissue tropism of VHSV in experimentally infected juvenile olive flounder (<I>Paralichthys olivaceus</I>), an economically important flatfish in Asian aquaculture. Two separate digoxigenin (DIG) labeled antisense RNA probes targeting a fragment of viral nucleoprotein (N) and glycoprotein (G) gene segments were employed to localize VHSV in five infected flounder tissues <I>viz.</I>, kidney, spleen, heart, liver and brain. Further, immunohistochemistry (IHC) assay was also used to observe and substantiate the spatial localization of the viral particles using a monoclonal antibody (MAb) against the viral nucleoprotein (N). Following the RNA-ISH and IHC assays, VHSV localization was observed in different cells and areas of the tested tissues. The positive reaction in both the assays correlated with histopathological alterations confirming the presence of VHSV within the tested tissues. Information gained from the present study using both genome and protein based localization sheds light on viral tropism which can help to further elucidate VHSV pathogenesis in olive flounder.</P> <P><B>Highlights</B></P> <P> <UL> <LI> RNA-ISH assay was used for the first time to localize VHSV in olive flounder tissues. </LI> <LI> Viral mRNAs were visualized in different areas of tested tissues using two DIG-labeled riboprobes (VHSV-N and G). </LI> <LI> Protein based detection (IHC) and histopathological alterations also confirmed the viral localization. </LI> <LI> The present study sheds light on VHSV tissue tropism and pathogenesis in olive flounder. </LI> </UL> </P>
Qadiri, Syed Shariq Nazir,Kim, Soo-Jin,Krishnan, Rahul,Kim, Jae-Ok,Kim, Wi-Sik,Oh, Myung-Joo Elsevier 2019 Journal of virological methods Vol.264 No.-
<P><B>Abstract</B></P> <P>An <I>in situ</I> hybridization (RNA-ISH) assay has been developed and optimized to detect viral haemorrhagic septicemia virus (VHSV), an OIE listed piscine rhabdovirus, in infected fish cells using fathead minnow (FHM) as a model cell line. Two antisense riboprobes (RNA probes) targeting viral transcripts from a fragment of nucleoprotein (N) and glycoprotein (G) genes were generated by reverse transcription polymerase chain reaction (RT-PCR) using VHSV specific primers followed by a transcription reaction in the presence of digoxigenin dUTP. The synthesized RNA probes were able to detect viral mRNAs in formalin fixed VHSV infected FHM cells at different time points post inoculation (pi). To correlate the signal intensity, a time dependent quantitation of the viral mRNA transcript and infectivity titer was done by quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) and 50% tissue culture infectivity dose (TCID<SUB>50</SUB>), respectively, from the infected cells and culture supernatants. Further, we compared the diagnostic sensitivity of ISH assay with immunocytochemistry (ICC). Both the riboprobes used in the ISH assay detected VHSV as early as 6 hpi in the FHM cells inoculated with a multiplicity of infection (moi) of 2. Also, the signal detection in ISH was at an early stage in comparison to ICC, wherein, signal was first detected at 12 hpi. Our results clearly highlight that current ISH assay can be of value as a diagnostic tool to localize and detect VHSV in conjunction with conventional virus isolation in cell culture.</P> <P><B>Highlights</B></P> <P> <UL> <LI> RNA-ISH assay was developed and optimized to complement VHSV detection in FHM cells. </LI> <LI> Two antisense riboprobes (N and G) efficiently localized VHSV mRNAs in infected cells. </LI> <LI> RNA-ISH signal intensity correlated with viral quantification assays (qRT-PCR and TCID<SUB>50</SUB>). </LI> <LI> Diagnostic sensitivity of RNA-ISH assay was more than ICC. </LI> <LI> The assay will aid in physical visualization of VHSV in routine cell culture isolation. </LI> </UL> </P>
Kole, Sajal,Qadiri, Syed Shariq Nazir,Shin, Su-Mi,Kim, Wi-Sik,Lee, Jehee,Jung, Sung-Ju ACADEMIC PRESS LTD 2019 FISH AND SHELLFISH IMMUNOLOGY Vol.91 No.-
<P><B>Abstract</B></P> <P>Viral haemorrhagic septicaemia virus (VHSV), a (−) ssRNA virus belonging to the genus <I>Novirhabdovirus</I> of rhabdoviridae family, is the aetiological agent of viral haemorrhagic septicaemia (VHS) disease which causes huge economic losses in farmed olive flounder (<I>Paralichthys olivaceus</I>) and significant mortalities among several other marine fish species in Korea, Japan, and China. Previously, we developed an inactivated vaccine viz., formalin-inactivated VHSV mixed with squalene as adjuvant which was effective in conferring protective immunity (58–76% relative percentage survival) against VHSV but the mode of administration was intraperitoneal injection which is not feasible for small sized fingerling fish. To overcome this limitation, we presently focused on replacing the injection route of vaccine delivery by oral and immersion routes. In this context, we encapsulated the inactivated VHSV vaccine with chitosan nanoparticles (CNPs-IV) by water-in-oil (W/O) emulsification method. After encapsulation, two sets of <I>in vivo</I> vaccination trials were conducted viz., preliminary trial-I and final trial-II. In preliminary trial-I, olive flounder fingerlings (10.5 ± 1.7 g) were vaccinated with CNPs-IV by different delivery strategies involving oral and immersion routes (single/booster dose) followed by challenge with VHSV (1 × 10<SUP>6</SUP> TCID<SUB>50</SUB> virus/fish) to evaluate an effective method amongst different applied delivery strategies. Subsequently, a final trial-II was conducted to better understand the immune mechanism behind the efficacy of the employed delivery strategy and also to further improvise the delivery mechanism with prime-boost (primary immersion and oral boosting) combination in order to improve the transient anti-VHSV response in the host. Evaluation of RPS analysis in trial-I revealed higher RPS of 46.7% and 53.3% in the CNPs-IV (immersion) and CNPs-IV (immersion/immersion) groups, respectively compared to 0% RPS in the CNPs-IV (oral) group and 20% RPS in the CNPs-IV (oral/oral) group when calculated against 100% cumulative mortality percentage in the NVC (non-vaccinated challenged) control group, whereas, in the trial-II, RPS of 60% and 66.6% were obtained for CNPs-IV (immersion/immersion) and CNPs-IV (immersion/oral) groups, respectively. In addition, specific (anti-VHSV) antibody titre in the fish sera, skin mucus and intestinal mucus of the immunized groups were significantly (p < 0.05) enhanced following vaccination. Furthermore, CNPs-IV immunized fish showed significant (p < 0.05) upregulation of different immune gene transcripts (IgM, IgT, pIgR, MHC-I, MHC-II, IFN-γ, and Caspase3) compared to control, in both the systemic (kidney) and mucosal (skin and intestine) immune compartments of the host post immunization as well as post challenge. To conclude, mucosal immunization with CNPs-IV vaccine can orchestrate an effective immunization strategy in organizing a coordinative immune response against VHSV in olive flounder thereby exhibiting higher protective efficacy to the host with minimum stress.</P> <P><B>Highlights</B></P> <P> <UL> <LI> Encapsulation of inactivated VHSV in chitosan NPs for preparation of CNPs-IV vaccine. </LI> <LI> Olive flounder fingerlings immunized with CNPs-IV through mucosal routes. </LI> <LI> CNPs-IV vaccinated fish exhibited high RPS (60–66.6%) post VHSV challenged. </LI> <LI> CNPs-IV immunized group showed higher specific antibody response in sera and mucus. </LI> <LI> Immune genes were significantly upregulated in immunized fish. </LI> </UL> </P>
Krishnan, Rahul,Qadiri, Syed Shariq Nazir,Kim, Jong-Oh,Kim, Jae-Ok,Oh, Myung-Joo The Korean Society of Fisheries and Aquatic Scienc 2019 Fisheries and Aquatic Sciences Vol.22 No.12
Background: In the present study, we evaluated four commonly used housekeeping genes, viz., actin-β, elongation factor-1α (EF1α), acidic ribosomal protein (ARP), and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) as internal references for quantitative analysis of immune genes in nervous necrosis virus (NNV)-infected seven-band grouper, Hyporthodus septemfasciatus. Methods: Expression profiles of the four genes were estimated in 12 tissues of healthy and infected seven-band grouper. Expression stability of the genes was calculated using the delta Ct method, BestKeeper, NormFinder, and geNorm algorithms. Consensus ranking was performed using RefFinder, and statistical analysis was done using GraphpadPrism 5.0. Results: Tissue-specific variations were observed in the four tested housekeeping genes of healthy and NNV-infected seven-band grouper. Fold change calculation for interferon-1 and Mx expression using the four housekeeping genes as internal references presented varied profiles for each tissue. EF1α and actin-β was the most stable expressed gene in tissues of healthy and NNV-infected seven-band grouper, respectively. Consensus ranking using RefFinder suggested EF1α as the least variable and highly stable gene in the healthy and infected animals. Conclusions: These results suggest that EF1α can be a fairly better internal reference in comparison to other tested genes in this study during the NNV infection process. This forms the pilot study on the validation of reference genes in Hyporthodus septemfasciatus, in the context of NNV infection.
Rahul Krishnan,Syed Shariq Nazir Qadiri,김종오,Jae-Ok Kim,오명주 한국수산과학회 2019 Fisheries and Aquatic Sciences Vol.22 No.4
Background: In the present study, we evaluated four commonly used housekeeping genes, viz., actin-β, elongation factor-1α (EF1α), acidic ribosomal protein (ARP), and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) as internal references for quantitative analysis of immune genes in nervous necrosis virus (NNV)-infected seven-band grouper, Hyporthodus septemfasciatus. Methods: Expression profiles of the four genes were estimated in 12 tissues of healthy and infected seven-band grouper. Expression stability of the genes was calculated using the delta Ct method, BestKeeper, NormFinder, and geNorm algorithms. Consensus ranking was performed using RefFinder, and statistical analysis was done using GraphpadPrism 5.0. Results: Tissue-specific variations were observed in the four tested housekeeping genes of healthy and NNVinfected seven-band grouper. Fold change calculation for interferon-1 and Mx expression using the four housekeeping genes as internal references presented varied profiles for each tissue. EF1α and actin-β was the most stable expressed gene in tissues of healthy and NNV-infected seven-band grouper, respectively. Consensus ranking using RefFinder suggested EF1α as the least variable and highly stable gene in the healthy and infected animals. Conclusions: These results suggest that EF1α can be a fairly better internal reference in comparison to other tested genes in this study during the NNV infection process. This forms the pilot study on the validation of reference genes in Hyporthodus septemfasciatus, in the context of NNV infection.