Anutosh Paria

Toll-pathway in tiger shrimp (Penaeus monodon) responds to white spot syndrome virus infection: evidence through molecular characterisation and expression profiles of MyD88, TRAF6 and TLR genes.

The Toll-pathway plays key roles in regulating the innate immune response in invertebrates. Myeloid differentiation factor 88 (MyD88) and Tumour necrosis factor receptor (TNFR)-associated factor 6 (TRAF6) are key molecules in this signalling pathway. To investigate the role of Toll-pathway in innate immune response of shrimp, Penaeus monodon, MyD88 (PmMyD88) and TRAF6 (PmTRAF6) were identified and characterised. PmMyD88 cDNA is 1716 bp long with an open reading frame (ORF) of 1449 bp encoding a putative protein of 482 amino acids, with a death domain, a TIR domain and C-terminal extension domain. PmTRAF6 cDNA is 2563 bp long with an ORF of 1785 bp (594 amino acids) with an N-terminal RING-type zinc finger domain, two TRAF-type zinc finger domains, a coiled region and a MATH domain. In healthy shrimp, PmMyD88, PmTRAF6 and PmToll were detected in 15 tissues with the highest expression in midgut, eyestalk and lymphoid organ, respectively. Responses of these genes to WSSV in experimentally-infected P. monodon as well as in cultured haemocytes and also effect of poly I:C on the gene expression in vitro was investigated at six time-points in seven tissues. PmToll showed significant up-regulation at all time-points of infection in six tissues and until 24 h post-infection in vitro. However, poly I:C-induced haemocytes showed up-regulation of the gene until 48 h post-exposure. WSSV caused significant up-regulation of PmMyD88 in most of the tissues tested. The virus challenge as well as poly I:C induction in vitro also resulted in significant up-regulation of the gene. Up-regulated expression of PmTRAF6 was detected in haemocytes and lymphoid organ at late stage of infection. In vitro virus challenge showed significant up-regulation of PmTRAF6 at almost all time-points whereas no significant change in the expression was observed on poly I:C induction. The responses of these key genes, observed in the present study, suggest that Toll-pathway as a whole may play a crucial role in the immune response against viruses in shrimp.

Molecular cloning, characterisation and expression analysis of melanoma differentiation associated gene 5 (MDA5) of green chromide, Etroplus suratensis.

Innate immune system recognises pathogen-associated molecular patterns (PAMPs) by limited number of germline encoded and non-clonally developed pathogen recognition receptors (PRRs). Retinoic acid-inducible gene I (RIG-I)-like receptors (RLRs) are important cytosolic PRRs for sensing viral RNAs. The receptor encoded by melanoma differentiation associated gene 5 (MDA5), an RLR, recognises viral RNA and enhances antiviral response in host cells. The full-length MDA5 cDNA in Etroplus suratensis was cloned and found to have 3673 nucleotides encoding a polypeptide of 978 amino acids. The deduced amino acid sequence contains four main structural domains: two CARD domains in the N-terminal region, a DExDc (DEAH/DEAD box helicase domain), HELICc (C-terminal helicase) domain and a C-terminal regulatory domain (RD). Phylogenetic analysis revealed a close relationship of E. suratensis MDA5 (EsMDA5) with MDA5 of Neolamprologus brichardi and Oreochromis niloticus, both belonging to Cichlidae family. EsMDA5 transcripts were ubiquitously expressed in all the 12 tissues tested in healthy fish. Although, transcript level was found to be the highest in muscle, high expression was also detected in the spleen, head kidney and hindgut. In poly I:C-injected fish, EsMDA5 transcripts showed peak expression in the spleen, intestine and heart at 12h post-injection (hpi). However, in gill and kidney tissues, maximum up-regulation of EsMDA5 was observed at 6 and 48 hpi, respectively. Further, liver tissue showed an increasing trend in expression profile from 6 to 48 hpi. Interferon promoter stimulator-1 (IPS-1) gene, an adaptor triggering RIG-I- and MDA5-mediated type I interferon induction, also showed up-regulated expression at initial time-points in poly I:C-injected E. suratensis. The constitutive expression and up-regulation of EsMDA5 and the IPS-1 genes in different tissues indicate that EsMDA5 may play an important role in sensing viral PAMPs in conjunction with IPS-1.

Identification, ontogeny and expression analysis of a novel laboratory of genetics and physiology 2 (LGP2) transcript in Asian seabass, Lates calcarifer

ABSTRACT LGP2 (laboratory of genetics and physiology 2) is an important member of the retinoic acid‐inducible gene I (RIG‐I)‐like receptors (RLRs), which plays a significant role in antiviral innate immunity. In this study, we have cloned the full‐length cDNA sequence of LGP2 from Asian seabass, Lates calcarifer (AsLGP2). The complete AsLGP2 cDNA sequence consisted of 2586 nucleotides encoding a putative protein of 681 amino acids with a molecular mass of 77.6 kDa. From the AsLGP2 protein, four different conserved domains were predicted: a DExDc (DEAD/DEAH box helicase domain), a bacterial type III restriction enzyme domain (RES III), a HELICc (Helicase superfamily c‐terminal domain and a RIG‐I_C‐RD (RIG‐I C‐terminal regulatory domain). The transcript of AsLGP2 could be detected in all the 11 tissues tested in healthy animals with high expression noticed in tissues facing external environment such as gill, hindgut and skin. The ontogenic expression profile of AsLGP2 implies a possible maternal transfer of this gene as it has been detected in all early embryonic developmental stages along with unfertilized eggs. Viral analogue, poly I:C, injection resulted in rapid up‐regulated expression in different tissues with the highest modulation of expression observed in kidney followed by liver and gill. A rapid response of AsLGP2 expression was also observed in the different tissues of Vibrio alginolyticus‐injected L. calcarifer, while significant change in expression was noticed following Staphylococcus aureus infection. Similarly, exposure to different pathogen‐mimicking microbial analogues such as poly I:C, LPS and PGN resulted in enhanced expression of AsLGP2 in SISK cell‐line. Taking together, these observations suggest that AsLGP2 can act as both antiviral and antibacterial cytosolic receptor and may play a significant role in embryonic and larval development in marine euryhaline teleosts like Asian seabass. HighlightsA novel LGP2 transcript identified, cloned and characterized from Asian seabass, Lates calcarifer (AsLGP2).AsLGP2 constitutively expressed in embryonic and larval developmental stages.Poly I:C positively modulates the AsLGP2 expression in vivo and in vitro.AsLGP2 responds towards bacterial infection and different bacterial ligands.AsLGP2 may act as a positive regulatory molecule with both antiviral and antibacterial functions in marine euryhaline teleost.

Molecular characterisation, ontogeny and expression analysis of melanoma differentiation-associated factor 5 (MDA5) from Asian seabass, Lates calcarifer

ABSTRACT MDA5 is the pivotal member of the retinoic acid‐inducible gene I (RIG‐I)‐like receptors (RLRs) and is reported to play a crucial role in type I IFN‐mediated responses against pathogen‐associated molecular patterns (PAMPs), especially nucleic acids. In this study, we have identified and cloned the full‐length cDNA sequence of MDA5, which comprises 3398 nucleotides and encodes for a putative protein of 978 AA length, in Asian seabass, Lates calcarifer. From the putative amino acid sequence of AsMDA5, four different conserved domains could be predicted: two N‐terminal CARD domains, a DExDc domain, a HELICc domain and a C‐terminal RIG‐1_C‐RD domain. The mRNA transcript of AsMDA5 could be detected in all the 11 tissues tested in healthy animals with the highest expression in heart followed by gill and skin. The ontogenetic expression profile showed constitutive expression in developmental stages starting from unfertilized eggs, which implies the possibility of maternally acquired immunity of RLRs in offspring. The viral analogue poly I:C could modulate the AsMDA5 expression both in vivo and in vitro. In all the tissues, AsMDA5 expression was found to be highly regulated following injection with poly I:C with the highest expression observed in kidney. The expression level of AsMDA5 was found to be modulated at different time‐points following challenge with Gram‐negative bacterium, Vibrio alginolyticus, and Gram‐positive bacterium, Staphylococcus aureus. Similarly, noticeable change in AsMDA5 expression was detected in SISK cell line induced with either LPS or PGN. The observations made in this study suggest that in euryhaline marine teleosts like Asian seabass, MDA5 gene serves as one of the pivotal receptor for the detection of viral and bacterial PAMP, and might play an important antimicrobial role during early embryonic development. HighlightsFull‐length cDNA sequence of MDA5 was identified, cloned and characterized from Asian seabass, Lates calcarifer (AsMDA5).AsMDA5 constitutively expressed in embryonic and larval developmental stages.Viral analogue, poly I:C positively modulates the AsMDA5 expression in vivo and in vitro.AsMDA5 responds towards bacterial pathogens and different bacterial ligands.AsMDA5 may be the pivotal RLR molecule with both antiviral and antibacterial functions in marine euryhaline teleost.

Toll-like receptor (TLR) 22, a non-mammalian TLR in Asian seabass, Lates calcarifer: Characterisation, ontogeny and inductive expression upon exposure with bacteria and ligands

&NA; Toll‐like receptor (TLR) 22 is a non‐mammalian TLR found mostly in teleosts and characterized initially as a cell surface surveillance receptor for detecting extracellular long dsRNA. In the current study, the full‐length cDNA sequence consisting of 3312 nucleotides encoding for 960 amino acids in Asian seabass (Lates calcarifer) TLR22 (AsTLR22) was identified. From the putative protein sequence, signature TLR domains such as 18 LRR domains, two transmembrane domains, a single LRR_CT domain and an intracellular TIR domain could be predicted. Phylogenetic analysis showed that AsTLR22 is clustered with other teleost TLR22 and is distinctly different from the other TLR groups. The transcript of AsTLR22 was ubiquitously expressed in all the tissues tested of healthy juveniles with the highest expression in gill followed by hindgut, spleen and skin. The AsTLR22 mRNA transcript was also detected in all the developmental stages as early as unfertilized eggs with higher expression in later stages such as neurula and early embryo. The dsRNA viral analogue, poly (I:C) and Gram‐negative bacterium, Vibrio alginolyticus, were found to modulate the AsTLR22 expression in different tissues with the highest expression in kidney and liver. Gram‐positive bacterium, Staphylococcus aureus, was also found to regulate the AsTLR22 expression at certain time‐points with the highest expression in gill. Similarly, noticeable change in AsTLR22 expression was detected in SISK cell line induced with different ligands such as poly (I:C), LPS and PGN. The findings indicate that AsTLR22 responds in transcript level towards bacteria‐borne PAMPs and extracellular dsRNA in the euryhaline teleost Asian seabass. Further, this might act as an important pathogen surveillance receptor during early developmental stages. HighlightsThe TLR22 transcript was identified and characterized in Asian seabass, Lates calcarifer (AsTLR22).AsTLR22 constitutively expressed in embryonic and larval developmental stages.Viral analogue, poly I:C positively modulates the AsTLR22 expression in vivo and in vitro.AsTLR22 responds towards bacterial challenge and different bacterial ligands.

Development of SYBR Green and TaqMan quantitative real-time PCR assays for hepatopancreatic parvovirus (HPV) infecting Penaeus monodon in India.

Hepatopancreatic parvovirus (HPV) infects Penaeus monodon and causes mortality in the larval stages. Further, it has been implicated in the growth retardation in cultured P. monodon. Though different geographical isolates of HPV show large sequence variations, a sensitive PCR assay specific to Indian isolate has not yet been reported. Here, we developed a sensitive SYBR Green-based and TaqMan real-time PCR for the detection and quantification of the virus. A 441-bp PCR amplicon was cloned in pTZ57 R/T vector and the plasmid copy number was estimated. A 10-fold serial dilution of the plasmid DNA from 1 × 10(9) copies to 1 copy was prepared and used as the standard. The primers were tested initially using the standard on a conventional PCR format to determine the linearity of detection. The standards were further tested on real-time PCR format using SYBR Green and TaqMan chemistry and standard curves were generated based on the Ct values from three well replicates for each dilution. The assays were found to be sensitive, specific and reproducible with a wide dynamic range (1 × 10(9) to 10 copies) with coefficient of regression (R(2)) > 0.99, calculated average slope -3.196 for SYBR Green assay whereas, for TaqMan assay it was >0.99 and -3.367, respectively. The intra- and inter-assay variance of the Ct values ranged from 0.26% to 0.94% and 0.12% to 0.81%, respectively, for SYBR Green assay, and the inter-assay variance of the Ct values for TaqMan assay ranged from 0.07% to 1.93%. The specificity of the assays was proved by testing other DNA viruses of shrimp such as WSSV, IHHNV and MBV. Standardized assays were further tested to detect and quantify HPV in the post-larvae of P. monodon. The result was further compared with conventional PCR to test the reproducibility of the test. The assay was also used to screen Litopeneaus vannamei, Macrobrachium rosenbergii and Scylla serrata for HPV.

Nucleotide-binding oligomerization domain-containing protein 1 (NOD1) in Asian seabass, Lates calcarifer: Cloning, ontogeny and expression analysis following bacterial infection or ligand stimulation

ABSTRACT NOD1 (Nucleotide‐binding oligomerization domain‐containing protein 1) is one of the most prominent intracellular Nod‐like receptors (NLRs), responsible for detecting different microbial components and products arising from tissue injury. Here, we have identified and cloned NOD1 transcript in the Asian seabass, Lates calcarifer (AsNOD1), which consists of 3749 nucleotides and encodes for a predicted putative protein of 900 AA. The AsNOD1 possesses the typical structure of NLR family, consisting of N‐terminal CARD domain, centrally located NACHT domain and C‐terminal LRRs. The AsNOD1 showed ubiquitous tissue expression in 11 different tissues of healthy animals tested with high levels of expression in hindgut and gill. From the ontogenetic expression profile of AsNOD1, it is quite evident that this gene might follow a maternally‐transferred trend in euryhaline teleosts, as it is highly abundant in embryonic developmental stages. The constitutive immunomodulation of AsNOD1 in terms of expression level was clearly evident in the different tissues of Asian seabass‐injected either with Vibrio alginolyticus or poly I:C. However, injection with Staphylococcus aureus did not elicit similar immunomodulation except for the up‐regulation noticed at few time‐points in some tissues. SISK‐cell line induced with different ligands such as poly I:C, LPS and PGN also showed up‐regulation of AsNOD1 in certain time‐points in vitro. Based on the results obtained in the present study, it can be inferred that the AsNOD1 might play an immunoregulatory role upon exposure to different bacterial as well as viral PAMPs and also might be an important component of innate immune element during embryonic and larval development in the euryhaline teleost Asian seabass. HIGHLIGHTSAsian seabass NOD1 (AsNOD1) was identified, cloned and characterized.AsNOD1 was constitutively expressed in developmental stages.In healthy animals, AsNOD1 was highly expressed in hindgut and gill.V. aliginolyticus, poly I:C and bacterial ligands modulate the AsNOD1 expression both in vivo and in vitro.AsNOD1 might have both antiviral and antibacterial functions in marine euryhaline teleost.