Wenzhi Liu

Identification of Type I IFN in Chinese giant salamander (Andrias davidianus) and the response to an iridovirus infection

The type I IFNs play a major role in the first line of defense against virus infections. In this study, the type I IFN gene designated gsIFN was identified and characterized in the Chinese giant salamander (Andrias davidianus). The genomic DNA of gsIFN contains 5 exons and 4 introns and has a total length of 5622 bp. The full-length cDNA sequence of gsIFN is 1113 bp and encodes a putative protein of 186 amino acids that has a 43% identity to type I IFN of Xenopus tropicalis. The deduced amino acid sequence has the C-terminal CAWE motif, that is mostly conserved in the higher vertebrate type I IFNs. Real-time fluorescence quantitative RT-PCR analysis revealed broad expression of gsIFN in vivo and the highest level expression in blood, kidney and spleen. Additionally, the expression of gsIFN at the mRNA level was significantly induced in peripheral blood leucocytes after stimulation with poly I:C and after infection with the Chinese giant salamander iridovirus (GSIV). A plasmid expressing gsIFN was constructed and transfected into the Chinese giant salamander muscle cell line. Expression of the IFN-inducible gene Mx was up-regulated in the gsIFN-overexpressing cells after GSIV infection. The virus load and titer were significantly reduced compared with that in control cells. Additionally, a lower level of virus major capsid protein synthesis was confirmed by immunofluorescence assay compared to the control cells. These results suggest that the gsIFN gene plays an important role in the antiviral innate immune response.

Immunological responses and protection in Chinese giant salamander Andrias davidianus immunized with inactivated iridovirus

Chinese giant salamander hemorrhage is a newly emerged infectious disease in China and has caused huge economic losses. The causative pathogen has been identified as the giant salamander iridovirus (GSIV). In this study, the immunological responses and protection in Chinese giant salamander immunized with β-propiolactone inactivated GSIV are reported. Red and white blood cell counting and classification, phagocytic activity, neutralizing antibody titration, immune-related gene expression and determination of the relative percent survival were evaluated after vaccination. The red and white blood cell counts showed that the numbers of erythrocytes and leukocytes in the peripheral blood of immunized Chinese giant salamanders increased significantly on days 4 and 7 post-injection (P<0.01). Additionally, the differential leukocyte count of monocytes and neutrophils were significantly different compared to the control group (P<0.01); the percentage of lymphocytes was 70.45±7.52% at day 21. The phagocytic percentage and phagocytic index was 38.78±4.33% and 3.75±0.52, respectively, at day 4 post-immunization which were both significantly different compared to the control group (P<0.01). The serum neutralizing antibody titer increased at day 14 post-immunization and reached the highest titer (341±9.52) at day 21. The quantitative PCR analysis revealed that the immunization significantly up-regulated the expression of immune related genes TLR-9 and MyD88 the first two weeks after immunization. The challenge test conducted at day 30 post-injection demonstrated that the immunized group produced a relative survival of 72%. These results indicate that the inactivated GSIV could elicit significant non-specific and specific immunological responses in Chinese giant salamander that resulted in significant protection against GSIV induced disease.

Cloning, sequence analysis and expression profiles of Toll-like receptor 7 from Chinese giant salamander Andrias davidianus.

The Chinese giant salamander, Andrias davidianus, is the largest extant amphibian species in the world, which is of significance due to its specific position in the evolutionary history of vertebrates. Currently, limited information about the innate immune system of this animal is known. In this study, the toll-like receptor 7 (TLR7), designated CgsTLR7, was cloned from Chinese giant salamander, A. davidianus. The full-length cDNA of CgsTLR7 is 3747 bp, with an open reading frame of 3150 bp, encoding 1049 amino acids. The TLR family motifs, including the leucine-rich repeat (LRR) and Toll/interleukin (IL)-1 receptor (TIR) domain are conserved in CgsTLR7, which includes 19 LRRs and a TIR domain. The predicted amino acid sequence of CgsTLR7 has 71%, 65%, 63% and 55% identity with turtle, chicken, human and fugu TLR7 homologues, respectively. Phylogenetic analysis showed that CgsTLR7 is closest to that of frog TLR7 among the examined species. Quantitative real-time PCR analysis revealed broad expression of CgsTLR7 in tissues from apparently healthy Chinese giant salamanders with the highest expression in the liver and the lowest expression in the intestine. The mRNA expression was up-regulated and reached a peak level in the kidney, liver and spleen at 12 h, 24 h and 48 h after infecting the animals with the giant salamander iridovirus (GSIV), respectively. These results suggest that CgsTLR7 has a conserved gene structure and might play an important role in immune regulation against viral infections in the Chinese giant salamander.

Characterization of Chinese giant salamander iridovirus tissue tropism and inflammatory response after infection.

The Chinese giant salamander iridovirus (GSIV), belonging to the genus Ranavirus in the family Iridoviridae, causes severe hemorrhagic lesions and nearly 100% mortality in naturally infected Chinese giant salamanders Andrias davidiamus. However, the replication and distribution of the virus has not been well characterized in vivo. Using in situ hybridization, the expression of the GSIV major capsid protein (MCP) was detected in the cytoplasm of cells of the spleen, kidney, liver and gut tissues. MCP expression in the spleen and kidney appeared to fluctuate significantly during the acute phase of infection. Using an immunofluorescence assay, GSIV antigens were abundant in the spleen and kidney tissues but appeared to be at relatively low levels in the liver and gut. Additionally, there were significant changes in the expression of the pro-inflammatory cytokines macrophage migration inhibitory factor (MIF), tumor necrosis factor α (TNF-α) and interleukin-1β (IL-1β) in different tissues in response to infection with GSIV. The expression of MIF, TNF-α and IL-1β had significantly increased in the spleen at 3 d post-infection; this correlated with a decrease in virus replication in the spleen. These results suggest that the spleen and kidney are the major target tissues of GSIV, and the increased expression of MIF, TNF‑α and IL-1β may contribute to a reduction of virus replication in the spleen.

Efficient resistance to grass carp reovirus infection in JAM-A knockout cells using CRISPR/Cas9

&NA; The hemorrhagic disease of grass carp (Ctenopharyngodon idellus) induced by grass carp reovirus (GCRV) leads to huge economic losses in China and currently, there are no effective methods available for prevention and treatment. The various GCRV genotypes may be one of the major obstacles in the pursuit of an effective antiviral treatment. In this study, we exploited CRISPR/Cas9 gene editing to specifically knockout the DNA sequence of the grass carp Junctional Adhesion Molecule‐A (gcJAM‐A) and evaluated in vitro resistance against various GCRV genotypes. Our results show that CRISPR/Cas9 effectively knocked out gcJAM‐A and reduced GCRV infection for two different genotypes in permissive grass carp kidney cells (CIK), as evidenced by suppressed cytopathic effect (CPE) and GCRV progeny production in infected cells. In addition, with ectopic expression of gcJAM‐A in cells, non‐permissive cells derived from Chinese giant salamander (Andrias davidianus) muscle (GSM) could be highly infected by both GCRV‐JX0901 and Hubei grass carp disease reovirus (HGDRV) strains that have different genotypes. Taken together, the results demonstrate that gcJAM‐A is necessary for GCRV infection, implying a potential approach for viral control in aquaculture. HighlightsgcJAM‐A is necessary for GCRV infection.The antiviral activity of CRISPR/Cas9 to target gcJAM‐A efficiently suppress multiple‐genotypes GCRV infection.With ectopic expression of gcJAM‐A in cells, the non‐permissive cells could be highly infected by GCRV.The first application of CRISPR/Cas9 system to suppress viral infections in aquatic animals.

Molecular characteristics and virulence analysis of eight Aeromonas hydrophila isolates obtained from diseased Amur sturgeon Acipenser schrenckii Brandt, 1869

Aeromonas hydrophila is an opportunistic pathogen of a variety of aquatic animals that displays extreme diversity in drug resistance, phenotypes, virulence genes, and virulence. In this study, eight pathogenic A. hydrophila strains were isolated from diseased Amur sturgeons and investigated for their sensitivity to select antibiotics, their phenotype, virulence genes, and virulence. According to the phylogenetic analysis of the DNA gyrase subunit B protein, the eight isolates formed a single branch in the A. hydrophila group. The antibiotics ceftazidime, cefuroxime, cefoperazone, cefotaxime, ceftriaxone, aztreonam, and cefepime appeared effective against them. All of the isolates possessed the virulence genes for aerolysin, flagellin, heat-stable cytotonic enterotoxin, heat-labile cytotonic enterotoxin, hemolysin, and elastase, while only one isolate, HZ8, possessed the gene for lateral flagella. The cytolytic enterotoxin and lipase genes were present in all isolates, except in ZJ10 and ZJ12. Enterobacterial repetitive intergenic consensus sequence PCR indicated that the eight A. hydrophila isolates could be divided into four types. Isolates YW2, TR3, HZ8 and ZJ10, each representing a different type, were selected for challenge experiments. The challenge tests revealed that isolate HZ8 had the lowest lethal dose, causing 50% mortality at 2.30 × 104 colony forming units (cfu)/ml. The isolate ZJ10 had the highest LD50, 1.25 × 106 cfu/ml. Knowledge of the characteristics of the A. hydrophila isolates obtained from Amur sturgeon will be beneficial in developing potential disease control strategies.

Histopathology and ultrastructural pathology of cyprinid herpesvirus II (CyHV-2) infection in gibel carp, Carassius auratus gibelio

Cyprinid herpesvirus II (CyHV-2) infection is identified in cultured gibel carp, Carassius auratus gibelio, with high mortality in China in recent years. Histological pathology includes acute hepatocellular necrosis, splenic necrosis, kidney necrosis, hyperplasia of the secondary lamellae with focal necrosis. Acute necrotic myocarditis and granulocytes are prominent within the cardiac lumen in infected fish. In addition, necrosis is observed in the submucosa and mucosa epithelium of intestinal tract. Edemas are observed in renal glomerulus, submucosa and mucosa epithelium of intestinal tract, myocardial cells and neurons. Transmission electron microscopy indicates the cytoplasmic inclusions in splenocytes, glomerulus cells and hematopoietic tissue cells of kidney, epithelial cells of gills and brain cells. The histopathology and ultrastructural pathology in CyHV-2 infected gibel carp are characterized with extensive necrosis and cytoplasmic inclusions in spleen, kidney, gill and brain, which suggests that CyHV-2 may mainly infect the spleen, kidney, gill and brain of fish.

Development of cross-priming amplification coupled with vertical flow visualization for rapid detection of infectious spleen and kidney necrosis virus (ISKNV) in mandarin fish, Siniperca chuatsi

Infectious spleen and kidney necrosis virus (ISKNV) has been recognized as the causative agent of the most serious disease in cultured mandarin fish, Siniperca chuatsi, in China. Disease outbreaks have resulted in substantial losses to the aquaculture industry. Currently, reliable laboratory detection and identification methods are available for this virus. However, rapid detection methods applicable for on-site diagnosis of this infectious agent are unavailable. To address this need, a nearly instrument-free, cost-effective and simple detection method was developed and optimized and incorporates cross priming amplification coupled with vertical flow visualization for rapid identification of ISKNV (ISKNV-CPA-VF). Results show that cross circulation amplification targeting the conserved region of the major capsid protein (MCP) regiment of the ISKNV genome had a sensitivity 10 times greater than traditional PCR at 64 °C within 60 min. The optimized concentration of dNTPs and the concentration for Mg2+ were 1.0 mmol/L and 10 mmol/L, respectively. No cross-reactions with other viruses or bacteria were observed. When combined with the nucleic acid strip detection technology, visual detection of ISKNV amplified products was realized within 3-5 min following amplification. The simplicity and nearly instrument-free method for this ISKNV-CPA-VF assay shows great potential for on-site diagnostics of ISKNV infection in Siniperca chuatsi.

Identification, structural characterization, and expression analysis of toll-like receptors 2 and 3 from gibel carp (Carassius auratus gibelio)

ABSTRACT Toll‐like receptors (TLRs) are important components of innate immunity. TLRs recognize pathogen‐associated molecular patterns (PAMPs) and initiate downstream signaling pathways in response. In present study, we report the identification of two TLRs from gibel carp (Carassius auratus gibelio), TLR2 and TLR3 (designated CagTLR2 and CagTLR3, respectively). We report on the genomic structures and mRNA expression patterns of CagTLR2 and CagTLR3. Five exons and four introns were identified from the genomic DNA sequence of CagTLR3 (4749 bp in total length); this genomic organization is similar to that of TLR3 in zebrafish and human. However, only one intron was identified from the CagTLR2 genomic locus (3166 bp in total length); this unique genomic organization of CagTLR2 is different from that of TLR2 in fish and humans. The cDNAs of CagTLR2 and CagTLR3 encoded 791 and 904 amino acid residues, respectively. CagTLR2 and CagTLR3 contained two distinct structural/functional motifs of the TLR family: a leucine‐rich repeat (LRR) domain in the extracellular portion and a toll/interleukin‐1 receptor (TIR) domain in the intracellular portion. The positions of critical amino acid residues involed in PAMP recognition and signaling pathway transduction in mammalian TLRs were conserved in CagTLR2 and CagTLR3. Phylogenetic analysis revealed a closer clustering of CagTLR2 and CagTLR3 with TLRs from freshwater fish than with marine fish species. In healthy gibel carp, transcripts of these genes were detected in all examined tissues, and high expression levels of CagTLR2 and CagTLR3 were observed in liver and brain, respectively. Following injection with CyHV‐2, expression levels of CagTLR2 and CagTLR3 were significantly upregulated in the spleens of gibel carp after three days, and CagTLR3 transcript levels were rapidly increased in head kidney after 12 h. These results suggest that CagTLR2 and CagTLR3 are functionally involved in the induction of antiviral immune response. HighlightsMolecular cloning and identification of TLR2 and TLR3 in gibel carp.Unique genomic organization of TLR2 in gibel carp was carefully characterized and discussed.TLR2 and TLR3 showed different expression patterns in response to DNA virus (CyHV‐2) infection in gibel carp.

Rag1 and rag2 gene expressions identify lymphopoietic tissues in juvenile and adult Chinese giant salamander (Andrias davidianus)

ABSTRACT Rag1 and rag2 are two closely linked recombination activating genes required for V(D)J recombination of antigen receptors in immature lymphocytes, whose expression can serve as marker to identify the lymphopoietic tissues. To study the development of lymphopoietic tissues in Chinese giant salamander (Andrias davidianus), the Chinese giant salamander rag1 and rag2 coding sequences were cloned and determined. High transcript levels of rag1 and rag2 were co‐detected in the thymus before 14 months of age, whereas levels were lower in spleen, liver and kidney at all stage of development. The spatial expression patterns of rag1 and rag2 were studied in combination with igY and tcr&bgr; gene expression using in situ hybridization. Significant transcript signals for rag1, rag2, tcr&bgr; and igY were detected not only in the thymus and spleen but also the liver and kidney of juvenile and adult Chinese giant salamanders, which suggests that cells of lymphocyte lineage are present in multiple tissues of the Chinese giant salamander. This implies that lymphopoiesis may take place in these tissues. The tissue morphology of thymus suggested that the branched thymic primordium developed into mature organ with the development of thymocyte from juvenile to adult. These results not only confirm that as expected the thymus and spleen are primordial lymphopoietic tissues but also suggest that the liver and kidney provide site of lymphocyte differentiation in Chinese giant salamander. HighlightsA. davidianus rag1 and 2 gene homologs are closely related to X. laevis rag1 and 2, respectively.The high expression levels of rags were used to detect lymphocyte differentiation in the thymus as early as 14 months.Besides the thymus and spleen, liver and kidney are suggested to serve as lymphopoietic tissues in A. davidianus.