Molecular docking and simulation studies of 3‐(1‐chloropiperidin‐4‐yl)‐6‐fluoro benzisoxazole 2 against VP26 and VP28 proteins of white spot syndrome virus

White spot syndrome virus (WSSV), an aquatic virus infecting shrimps and other crustaceans, is widely distributed in Asian subcontinents including India. The infection has led to a serious economic loss in shrimp farming. The WSSV genome is approximately 300 kb and codes for several proteins mediating the infection. The envelope proteins VP26 and VP28 play a major role in infection process and also in the interaction with the host cells. A comprehensive study on the viral proteins leading to the development of safe and potent antiviral therapeutic is of adverse need. The novel synthesized compound 3‐(1‐chloropiperidin‐4‐yl)‐6‐fluoro benzisoxazole 2 is proved to have potent antiviral activity against WSSV. The compound antiviral activity is validated in freshwater crabs (Paratelphusa hydrodomous). An in silico molecular docking and simulation analysis of the envelope proteins VP26 and VP28 with the ligand 3‐(1‐chloropiperidin‐4‐yl)‐6‐fluoro benzisoxazole 2 are carried out. The docking analysis reveals that the polar amino acids in the pore region of the envelope proteins were involved in the ligand binding. The influence of the ligand binding on the proteins is validated by the molecular dynamics and simulation study. These in silico approaches together demonstrate the ligand's efficiency in preventing the trimers from exhibiting their physiological function.     

Development and validation of a novel Taqman‐based real‐time RT‐PCR assay suitable for demonstrating freedom from viral haemorrhagic septicaemia virus

Viral haemorrhagic septicaemia (VHS) is a serious disease in several fish species. VHS is caused by the rhabdovirus viral haemorrhagic septicaemia virus (VHSV). To prevent spreading of the pathogen, it is important to use a fast, robust, sensitive and specific diagnostic tool to identify the infected fish. Traditional diagnosis based on isolation in cell culture followed by identification using, for example, ELISA is sensitive and specific but slow. By switching to RT‐PCR for surveillance and diagnosis of VHS the time needed before a correct diagnosis can be given will be considerably shortened and the need for maintaining expensive cell culture facilities reduced. Here we present the validation, according to OIE guidelines, of a sensitive and specific Taqman‐based real‐time RT‐PCR. The assay detects all isolates in a panel of 79 VHSV isolates covering all known genotypes and subtypes, with amplification efficiencies of approximately 100%. The analytical and diagnostic specificity of the real‐time RT‐PCR is close to 1, and the analytical and diagnostic sensitivity is comparable with traditional cell‐based methods. In conclusion, the presented real‐time RT‐PCR assay has the necessary qualities to be used as a VHSV surveillance tool on par with cell culture assays.     

Comparative genome analysis reveals a distinct influence of nucleotide composition on virus–host species‐specific interaction of prawn‐infecting nodavirus

Discovery of species‐specific interaction between the host and virus has drawn the interest of many researchers to study the evolution of the newly emerged virus. Comparative genome analysis provides insights of the virus functional genome evolution and the underlying mechanisms of virus–host interactions. The analysis of nucleotide composition signified the evolution of nodavirus towards host specialization in a host‐specific mutation manner. GC‐rich genome of betanodavirus was significantly deficient in UpA and UpU dinucleotides composition, whilst the AU‐rich genome of gammanodavirus was deficient in CpG dinucleotide. The capsid of MrNV and PvNV of gammanodavirus retains the highest abundance of adenine and uracil at the second codon position, respectively, which were found to be very distinctive from the other genera. ENC‐GC3 plot inferred the influence of natural selection and mutational pressure in shaping the evolution of MrNV RdRp and capsid, respectively. Furthermore, CAI/eCAI analysis predicts a comparable adaptability of MrNV in squid, Sepia officinalis than its natural host, Macrobrachium rosenbergii. Thus, further study is warranted to investigate the capacity of MrNV replication in S. officinalis owing to its high codon adaptation index.     

Immunity induced by recombinant attenuated IHNV (infectious hematopoietic necrosis virus)‐GN438A expresses VP2 gene‐encoded IPNV (infectious pancreatic necrosis virus) against both pathogens in rainbow trout

Infectious hematopoietic necrosis virus (IHNV) and infectious pancreatic necrosis virus (IPNV) are important pathogens in rainbow trout farming worldwide. Their co‐infection is also common, which causes great economic loss in juvenile salmon species. Development of a universal virus vaccine providing broadly cross‐protective immunity will be of great importance. In this study, we generated two recombinant (r) virus (rIHNV‐N438A‐ΔNV‐EGFP and rIHNV‐N438A‐ΔNV‐VP2) replacing the NV gene of the backbone of rIHNV at the single point mutation at residue 438 with an efficient green fluorescent protein (EGFP) reporter gene and antigenic VP2 gene of IPNV. Meanwhile, we tested their efficacy against the wild‐type (wt) IHNV HLJ‐09 virus and IPNV serotype Sp virus challenge. The relative per cent survival rates of two recombinant viruses against (wt) IHNV HLJ‐09 virus challenge were 84.6% and 81.5%, respectively. Simultaneously, the relative per cent survival rate of rIHNV‐N438A‐ΔNV‐VP2 against IPNV serotype Sp virus challenge was 88.9%. It showed the two recombinant viruses had high protection rates and induced a high level of antibodies against IHNV or IPNV. Taken together, these results suggest the VP2 gene of IPNV can act as candidate gene for vaccine and attenuated multivalent live vaccines and molecular marker vaccines have potential application for viral vaccine.     

High prevalence of betanodavirus barfin flounder nervous necrosis virus as well as red‐spotted grouper nervous necrosis virus genotype in shellfish

Using two serially executed PCRs, the discriminative multiplex two‐step RT‐PCR (DMT‐2 RT‐PCR) following the detection seminested two‐step RT‐PCR (DSN‐2 RT‐PCR), we found a high frequency presence of BFNNV genotype as well as RGNNV in various domestic and imported shellfish. This was definitely different from the previous reports of outbreaks and asymptomatic infection only by the RGNNV genotype in cultured finfish in Korea. Cultivation of NNV entrapped in shellfish was performed successfully by a blind passage. Thus, in an attempt to elucidate the epidemiology of betanodavirus, experiments conducted on 969 shellfish samples concluded that (i) distribution of NNV genotype, especially BFNNV, in shellfish is clearly different from that found in finfish of the world; (ii) unlike RGNNV, which showed a high rate in summer, BFNNV showed no seasonal variation and this result suggests BFNNVs in the marine environment remain fairly constant throughout the year; and (iii) the entrapped virus in shellfish was alive and culturable in vitro. These results are the first report of high level prevalence of in vitro culturable NNV in shellfish, for both BFNNV and RGNNV, which may present a potential risk in transmitting nodaviruses to host species in a marine environment.     

Molecular cloning of MDA5, phylogenetic analysis of RIG‐I‐like receptors (RLRs) and differential gene expression of RLRs,interferons and proinflammatory cytokines after in vitro challenge with IPNV,ISAV and SAV in the salmonid cell line TO

The RIG‐I receptors RIG‐I, MDA5 and LGP2 are involved in viral recognition, and they have different ligand specificity and recognize different viruses. Activation of RIG‐I‐like receptors (RLRs) leads to production of cytokines essential for antiviral immunity. In fish, most research has focused on interferons, and less is known about the production of proinflammatory cytokines during viral infections. In this study, we have cloned the full‐length MDA5 sequence in Atlantic salmon, and compared it with RIG‐I and LGP2. Further, the salmonid cell line TO was infected with three fish pathogenic viruses, infectious pancreatic necrosis virus (IPNV), infectious salmon anaemia virus (ISAV) and salmonid alphavirus (SAV), and differential gene expression (DEG) analyses of RLRs, interferons (IFNa‐d) and proinflammatory cytokines (TNF‐α1, TNF‐α2, IL‐1β, IL‐6, IL‐12 p40s) were performed. The DEG analyses showed that the responses of proinflammatory cytokines in TO cells infected with IPNV and ISAV were profoundly different from SAV‐infected cells. In the two aforementioned, TNF‐α1 and TNF‐α2 were highly upregulated, while in SAV‐infected cells these cytokines were downregulated. Knowledge of virus recognition by the host and the immune responses during infection may help elucidate why and how some viruses can escape the immune system. Such knowledge is useful for the development of immune prophylactic measures.     

In vitro assembly of Penaeus monodon densovirus (PmDNV)‐like particles produced in a prokaryote expression system

Viral diseases are a significant problem in the shrimp aquaculture industry as outbreaks can cause significant mortality and economic loss. While it has been shown that triggering the shrimp RNA interference pathway through dsRNA is a potentially viable treatment pathway, this approach is hampered by the lack of a suitable delivery mechanism. Virus‐like particles (VLPs), which are structurally similar to native viruses but lack the genetic material, could possibly be developed as a delivery vehicle. To generate a candidate VLP, the Penaeus monodon densovirus (PmDNV) capsid protein was cloned with an added histidine tag and expressed in an E. coli expression system. While the protein was expressed in inclusion bodies, the recombinant PmDNV capsid protein could be dissolved and subsequently purified by nickel affinity column chromatography. The formation of VLP from this purified rPmDNV capsid protein was investigated by transmission electron microscopy, and PmDNV‐VLPs were observed that looked similar to the native PmDNV virion. Our results suggest that the PmDNV‐like particle could be promisingly applied towards vaccination and that this PmDNV‐like particle can potentially serve as a system for delivery of nucleic acids to trigger innate immunity in shrimp.     

Histology,immunocytochemistry and qRT‐PCR analysis of Atlantic salmon,Salmo salar L., post‐smolts following infection with infectious pancreatic necrosis virus (IPNV)

Infectious pancreatic necrosis (IPN) is a very serious viral disease in terms of its impact on production of Atlantic salmon, Salmo salar L., fry and post‐smolts. Post‐smolts of Atlantic salmon were injected with infectious pancreatic necrosis virus (IPNV) and cohabited with naive fish to produce natural infection. Cohabitant fish were sampled every 2 days, up to day 36 post‐infection (p.i.). From 90 cohabitant fish, 11 (12.2%) were positive by immunohistochemistry (IHC). The first detection of IPNV by IHC occurred on day 16 p.i. which coincided with the onset of mortality in this group. Besides the pancreas, the liver was found to be a key target organ for IPNV. For the first time, the virus was observed in the islets of Langerhans and in the kidney corpuscles of Stannius which suggests that the virus could affect the fish’s metabolism. The liver of two fish, which showed the most widespread presence of IPNV by IHC, had a pathology including focal necrosis and widespread presence of apoptotic hepatocytes, many of which did not stain for virus by IHC. Up‐regulation of cytokine gene expression was found only in the IHC‐positive (IHC+ve) fish and reflected the level of infection as determined by IHC positivity of the liver. In most fish, interferon (IFN), Mx, γIFN and γIP were up‐regulated in liver and kidney, while only IFN and Mx were up‐regulated in gill. IL1β and TNFα were not induced in any tissue. The gill showed variable levels of constitutive expression of IL1β and γIFN. The two fish with liver pathology had the highest level of IFN expression, especially relative to the level of Mx expression, in the liver compared with the other IHC+ve fish which did not have a liver pathology. The results suggest that following widespread infection of hepatocytes, the cells may over‐produce IFN, resulting in apoptosis of neighbouring cells with subsequent death from liver failure.     

Construction of KHV‐CJ ORF25 DNA vaccine and immune challenge test

KHV ORF25 fragments were cloned from Koi Herpes Virus‐CJ (KHV‐CJ) strains isolated in our laboratory. The amplified products were inserted into the eukaryotic expression vector pIRES‐neo, forming recombinant plasmid pIRES‐ORF25. The recombinant plasmid pIRES‐ORF25 at 1 μg per koi, 10 μg per koi and 50 μg per koi was intramuscularly injected into healthy kois, respectively. The results showed that the recombinant pIRES‐ORF25 could induce the production of specific antibodies in koi determined by indirect ELISA. The differences of immune effect between three doses were not significant (P > 0.05), but all of them could induce the production of neutralizing antibodies. The immune challenge test showed that the mortality of koi injected with PBS, blank pIRES‐neo vector and nothing was 90%, 92.5% and 85% at 25 days. While the mortalities of koi injected with eukaryotic expression plasmid pIRES‐ORF25 were 20%, 17.5% and 12.5%. Differences in comparison with the control group were highly significant (P < 0.01). Histopathological staining revealed that the tissues of the immunized koi did not change apparently. In conclusion, the DNA vaccine pIRES‐ORF25 construct could well protect koi against KHV and had the potential to be applied in practice.     

Cross‐neutralization studies with salmonid alphavirus subtype 1–6 strains: results with sera from experimental studies and natural infections

The serological reactivity between strains of each of the six currently genetically defined subtypes of salmonid alphavirus (SAV) was examined by comparison of homologous and heterologous virus neutralization titres on sera from experimentally infected fish. With the exception of the level of SAV subtype 6 neutralization by heterologous sera, good cross‐neutralization was detected between all subtypes, albeit with variation in geometric mean titres when each subtype‐specific serum set was tested against the panel of virus subtypes. A similar pattern was evident with field sera, except that heterologous neutralization of the SAV6 strain was more evident. In only 23% of available pairwise comparisons was the homologous titre recorded with an experimentally derived serum fourfold or greater than the heterologous titre, and in only two instances was this difference demonstrated in both directions. No virus strains consistently met the old serology‐based criteria (Sub‐committee on Inter‐relationships Among Catalogued Alphaviruses) to be considered separate subtypes within an alphavirus species. Only when testing with an SAV subtype‐2‐specific monoclonal antibody was a major difference between homologous and heterologous neutralization capacity evident. These results provide new direct or indirect information in terms of SAV classification, vaccine efficacy and the selection and validation of reagents for serological and immunological diagnostic purposes.     

Non‐specific cellular defence mechanisms of rainbow trout (Oncorhynchus mykiss) in intensive and extensive rearing technologies

Water as a habitat is highly variable in terms of the physico‐chemical properties. The aim of the studies was to analyze the quality of the technology by evaluating the non‐specific cellular defence potential of rainbow trout (Oncorhynchus mykiss) in diversified intensity culture systems and in different periods of the breeding cycle. The evaluation was conducted in order to establish the scientific basis for the development of principles for recognition of the culture of this species as ‘organic’. Six rainbow trout rearing farms were selected for the studies and divided into two equal groups according to the production technology: farms with a flow through system (with single water usage – OOH) and facilities with multiple water usage via its backflow (re‐circulating system – RAS). The samples for tests were taken from 20 individuals from each fish farm. In all fish that originated both from OOH‐type farms and RAS operations, an increase in the activity of non‐specific cellular immunity in the autumn was observed. Statistically significant reduction in the activity of cellular defensive mechanisms in small (300–500 g) and big (501–850 g) fish occurred on two 3‐OOH and 3‐RAS farms in spring and autumn in 2011. During these periods, the research has shown the presence of Infectious Pancreatic Necrosis Virus (IPNV), which provoked an immune suppression. Based on the obtained results it is concluded that there were no significant differences in the parameters of non‐specific cellular immunity in rainbow trout originating from different farming systems OOH and RAS.     

Salmon cells SHK‐1 internalize infectious pancreatic necrosis virus by macropinocytosis

We have previously shown that infectious pancreatic necrosis virus (IPNV) enters the embryo cell line CHSE‐214 by macropinocytosis. In this study, we have extended our investigation into SHK‐1 cells, a macrophage‐like cell line derived from the head kidney of Atlantic salmon, the most economically important host of IPNV. We show that IPNV infection stimulated fluid uptake in SHK‐1 cells above the constitutive macropinocytosis level. In addition, upon infection of SHK‐1 cells, IPNV produced several changes in actin dynamics, such as protrusions and ruffles, which are important features of macropinocytosis. We also observed that the Na+/H+ pump inhibitor EIPA blocked IPNV infection. On the other hand, IPNV entry was independent of clathrin, a possibility that could not be ruled out in CHSE 214 cells. In order to determine the possible role of accessory factors on the macropinocytic process, we tested several inhibitors that affect components of transduction pathways. While pharmacological intervention of PKI3, PAK‐1 and Rac1 did not affect IPNV infection, inhibition of Ras and Rho GTPases as well as Cdc42 resulted in a partial decrease in IPNV infection. Further studies will be required to determine the signalling pathway involved in the macropinocytosis‐mediated entry of IPNV into its target cells.