Divergent transcriptomic responses underlying the ranaviruses-amphibian interaction processes on interspecies infection of Chinese giant salamander.

BACKGROUND: Ranaviruses (family Iridoviridae, nucleocytoplasmic large DNA viruses) have been reported as promiscuous pathogens of cold-blooded vertebrates. Rana grylio virus (RGV, a ranavirus), from diseased frog Rana grylio with a genome of 105.79 kb and Andrias davidianus ranavirus (ADRV), from diseased Chinese giant salamander (CGS) with a genome of 106.73 kb, contains 99% homologous genes. RESULTS: To uncover the differences in virus replication and host responses under interspecies infection, we analyzed transcriptomes of CGS challenged with RGV and ADRV in different time points (1d, 7d) for the first time. A total of 128,533 unigenes were obtained from 820,858,128 clean reads. Transcriptome analysis revealed stronger gene expression of RGV than ADRV at 1 d post infection (dpi), which was supported by infection in vitro. RGV replicated faster and had higher titers than ADRV in cultured CGS cell line. RT-qPCR revealed the RGV genes including the immediate early gene (RGV-89R) had higher expression level than that of ADRV at 1 dpi. It further verified the acute infection of RGV in interspecies infection. The number of differentially expressed genes and enriched pathways from RGV were lower than that from ADRV, which reflected the variant host responses at transcriptional level. No obvious changes of key components in pathway "Antigen processing and presentation" were detected for RGV at 1 dpi. Contrarily, ADRV infection down-regulated the expression levels of MHC I and CD8. The divergent host immune responses revealed the differences between interspecies and natural infection, which may resulted in different fates of the two viruses. Altogether, these results revealed the differences in transcriptome responses among ranavirus interspecies infection of amphibian and new insights in DNA virus-host interactions in interspecies infection. CONCLUSION: The DNA virus (RGV) not only expressed self-genes and replicated quickly after entry into host under interspecies infection, but also avoided the over-activation of host responses. The strategy could gain time for the survival of interspecies pathogen, and may provide opportunity for its adaptive evolution and interspecies transmission.

Fish reovirus GCReV-109 VP33 protein elicits protective immunity in rare minnows.

Grass carp reovirus strain 109 (GCReV-109) was previously isolated from a grass carp (Ctenopharyngodon idellus) with hemorrhagic disease, and its complete genome has been sequenced. However, the infectivity of GCReV-109 has not been studied, and the viral protein VP33, encoded on genome segment S11, had no detectable sequence homology to other known reovirus proteins. In this study, we characterized GCReV-109 infections in vivo and in vitro, as well as the VP33 protein. Infectivity analysis showed that GCReV-109 caused severe hemorrhagic disease and 100% mortality at dilutions up to 10(-4) in rare minnows (Gobiocypris rarus) by 8 days postinfection, but no visible cytopathic effect was observed in GCReV-109-infected subcultured grass carp muscle (GCM) cells. To confirm that GCReV-109 could be propagated in GCM cells, three virus genome segments were detected by RT-PCR, and large numbers of virus particles were observed by transmission electron microscopy in samples from the infected GCM cells. The suspension of GCReV-109-infected GCM cells was pathogenic to rare minnows. VP33 protein was expressed and purified for generation of an anti-VP33 antiserum. In western blot analysis of purified GCReV-109 particles, the antiserum specifically recognized a protein band (approximately 33 kDa). This revealed that VP33 is a major structural protein of GCReV-109 that might have immunogenic properties. The protective efficacy of the anti-VP33 antiserum against GCReV-109 infection was tested. The death of infected fish was delayed and the mortality fell to 10% when fish were treated with the anti-VP33 antiserum, suggesting that it might be useful for the prevention and control of fish reoviral disease.

Complete genome sequence and architecture of crucian carp Carassius auratus herpesvirus (CaHV).

Crucian carp Carassius auratus herpesvirus (CaHV) was isolated from diseased crucian carp with acute gill hemorrhages and high mortality. The CaHV genome was sequenced and analyzed. The data showed that it consists of 275,348 bp and contains 150 predicted ORFs. The architecture of the CaHV genome differs from those of four cyprinid herpesviruses (CyHV1, CyHV2, SY-C1, CyHV3), with insertions, deletions and the absence of a terminal direct repeat. Phylogenetic analysis of the DNA polymerase sequences of 17 strains of Herpesvirales members, and the concatenated 12 core ORFs from 10 strains of alloherpesviruses showed that CaHV clustered together with members of the genus Cyprinivirus, family Alloherpesviridae.

Development and application of monoclonal antibodies for detection and analysis of aquareoviruses.

Monoclonal antibodies (mAbs) play an important role in detection of aquareoviruses. Three mAbs against grass carp reovirus (GCRV) were prepared. Isotyping revealed that all three mAbs were of subclass IgG2b. Western blot assay showed that all three mAbs reacted with GCRV 69 kDa protein (the putative VP5). In addition to the 69 kDa protein of GCRV, mAb 4B6 also recognize a 54 kDa protein. All three mAbs were used for detecting aquareovirus by Western blot assay and indirect immunofluorescence assay (IFA). All of them reacted with GCRV, and mAb 4A3 could also react with turbot Scophthalmus maximus reovirus (SMReV) and largemouth bass Microptererus salmonides reovirus (MsReV). Viral antigens were only observed in the cytoplasm of infected cells. Finally, syncytia formation was observed with light microscopy and fluorescence microscopy using fluorescein labelled 4A3 mAb at various times post-infection. Syncytia were observed at 36 hr post-infection (hpi) by light microscopy and at 12 hpi by fluorescence microscopy. The immunofluorescence based assay allowed earlier detection of virus than observation of virus-induced cytopathic effect (CPE) assay in inoculated cell cultures. The sensitivity and specificity of these mAbs may be useful for diagnosis and monitoring of aquareoviruses.

Establishment of three cell lines from Chinese giant salamander and their sensitivities to the wild-type and recombinant ranavirus.

Known as lethal pathogens, Ranaviruses have been identified in diseased fish, amphibians (including Chinese giant salamander Andrias davidianus, the world's largest amphibian) and reptiles, causing organ necrosis and systemic hemorrhage. Here, three Chinese giant salamander cell lines, thymus cell line (GSTC), spleen cell line (GSSC) and kidney cell line (GSKC) were initially established. Their sensitivities to ranaviruses, wild-type Andrias davidianus ranavirus (ADRV) and recombinant Rana grylio virus carrying EGFP gene (rRGV-EGFP) were tested. Temporal transcription pattern of ranavirus major capsid protein (MCP), fluorescence and electron microscopy observations showed that both the wild-type and recombinant ranavirus could replicate in the cell lines.

Complete genome sequence and comparative analysis of grass carp reovirus strain 109 (GCReV-109) with other grass carp reovirus strains reveals no significant correlation with regional distribution.

A new grass carp reovirus strain, tentatively named GCReV-109, was isolated in Hubei, China, and its complete genome sequence was determined. The genome contained 11 double-stranded RNA segments (S1-S11) covering 24,620 base pairs. All of the segments had conserved terminal nucleotides, with GUAA(U)/CU at the 5' end and UCAUC at the 3' end. Protein sequence comparison showed that GCReV-109 was most closely related to GCRV-GD108 and shared 96.6-99.5 % protein sequence identity but only shared 16.7-46.1 and 15.1-45.4 % identity with GCRV-873 and HGDRV, respectively. Phylogenetic analysis showed that grass carp reovirus strains in China can be divided into three genotypes. Further analysis revealed homology between the GCRV-109 VP56 and HGDRV VP55 proteins, as well as GCReV-109 NS38, GCRV-873 NS38, and HGDRV VP39. The results of these comparisons also indicated that the homology between viruses was not necessarily linked to their geographical distribution. Our study will help in recognizing and understanding the genome structure and genetic diversity of grass carp reovirus.

Human-like adrenal features in Chinese tree shrews revealed by multi-omics analysis of adrenal cell populations and steroid synthesis.

The Chinese tree shrew ( Tupaia belangeri chinensis) has emerged as a promising model for investigating adrenal steroid synthesis, but it is unclear whether the same cells produce steroid hormones and whether their production is regulated in the same way as in humans. Here, we comprehensively mapped the cell types and pathways of steroid metabolism in the adrenal gland of Chinese tree shrews using single-cell RNA sequencing, spatial transcriptome analysis, mass spectrometry, and immunohistochemistry. We compared the transcriptomes of various adrenal cell types across tree shrews, humans, macaques, and mice. Results showed that tree shrew adrenal glands expressed many of the same key enzymes for steroid synthesis as humans, including CYP11B2, CYP11B1, CYB5A, and CHGA. Biochemical analysis confirmed the production of aldosterone, cortisol, and dehydroepiandrosterone but not dehydroepiandrosterone sulfate in the tree shrew adrenal glands. Furthermore, genes in adrenal cell types in tree shrews were correlated with genetic risk factors for polycystic ovary syndrome, primary aldosteronism, hypertension, and related disorders in humans based on genome-wide association studies. Overall, this study suggests that the adrenal glands of Chinese tree shrews may consist of closely related cell populations with functional similarity to those of the human adrenal gland. Our comprehensive results (publicly available at http://gxmujyzmolab.cn:16245/scAGMap/) should facilitate the advancement of this animal model for the investigation of adrenal gland disorders.

Isolation and identification of a lethal rhabdovirus from farmed rice field eels Monopterus albus.

We provide the first description of a virus responsible for a systemic hemorrhagic disease causing high mortality in farmed rice field eels Monopterus albus in China. Typical signs exhibited by the diseased fish were extensive hemorrhages in the skin and viscera and some neurological signs, such as loss of equilibrium and disorganized swimming. Histopathological examination revealed various degrees of necrosis within the spleen and liver. Virus isolation was attempted from visceral tissues of diseased fish by inoculation on 6 fish cell lines. Typical cytopathic effects (CPE) were produced in bluegill fry (BF2) cells, so this cell line was chosen for further isolation and propagation of the virus. Electron microscopy observation showed that the negative stained viral particles had the characteristic bullet shape of rhabdoviruses and an estimated size of 60 × 120 nm. We therefore tentatively refer to this virus as Monopterus albus rhabdovirus (MoARV). Molecular characterization of MoARV, including sequence analysis of the nucleoprotein (N), phosphoprotein (P), and glycoprotein (G) genes, revealed 94.5 to 97.3% amino acid similarity to that of Siniperca chuatsi rhabdovirus. Phylogenetic analysis based on the amino acid sequences of N and G proteins indicated that MoARV should be a member of the genus Vesiculovirus. Koch's postulates were fulfilled by infecting healthy rice field eels with MoARV, which produced an acute infection. RT-PCR analysis demonstrated that MoARV RNA could be detected in both naturally and experimentally infected fish. The data suggest that MoARV was the causative pathogen of the disease.

Characterization and virus susceptibility of a skin cell line from red-spotted grouper (Epinephelus akaara).

A red-spotted grouper Epinephelus akaara skin (RGS) cell line was established and characterized. RGS cells had a normal diploid chromosome number of 2n = 48, the morphology of which was fibroblastic-like in 3 days and epithelial-like over 5 after 16 passages. The cells multiplied well in Dulbecco's modified Eagle's medium supplemented with 10% of fetal bovine serum at 25°C. Susceptibilities of RGS and grass carp ovary (GCO) cells to two viruses were tested, and the results showed that the titer of an iridovirus Rana grylio virus (RGV) in RGS cells was 10³·5 TCID50 ml⁻¹, which was much higher than a rhabdovirus spring viremia of carp virus (SVCV) in the cells (10°·5 TCID50 ml⁻¹). The titers of RGV and SVCV in GCO were 106·° TCID50 ml⁻¹ and 108·° TCID50 ml⁻¹, respectively, which were higher than those in RGS cells. The data may imply that RGS cells could be selectively resistible to some viruses during infection. RT-PCR analysis of RGV-infected RGS cells showed that RGV could replicate in RGS cells. Further study of virus replications in RGS cells was conducted by electron microscopy and immunofluorescence microscopy has shown that virus particles scattered in the cytoplasm and virus protein appeared in both the cytoplasm and nucleus. The results suggested that RGS cells could be used as a potential in vitro model to study the cutaneous barrier function against virus infection.

Mitochondrial phylogenomics of Acanthocephala: nucleotide alignments produce long-branch attraction artefacts.

BACKGROUND: Classification of the Acanthocephala, a clade of obligate endoparasites, remains unresolved because of insufficiently strong resolution of morphological characters and scarcity of molecular data with a sufficient resolution. Mitochondrial genomes may be a suitable candidate, but they are available for a small number of species and their suitability for the task has not been tested thoroughly. METHODS: Herein, we sequenced the first mitogenome for the large family Rhadinorhynchidae: Micracanthorhynchina dakusuiensis. These are also the first molecular data generated for this entire genus. We conducted a series of phylogenetic analyses using concatenated nucleotides (NUC) and amino acids (AAs) of all 12 protein-coding genes, three different algorithms, and the entire available acanthocephalan mitogenomic dataset. RESULTS: We found evidence for strong compositional heterogeneity in the dataset, and Micracanthorhynchina dakusuiensis exhibited a disproportionately long branch in all analyses. This caused a long-branch attraction artefact (LBA) of M. dakusuiensis resolved at the base of the Echinorhynchida clade when the NUC dataset was used in combination with standard phylogenetic algorithms, maximum likelihood (ML) and Bayesian inference (BI). Both the use of the AA dataset (BI-AAs and ML-AAs) and the CAT-GTR model designed for suppression of LBA (CAT-GTR-AAs and CAT-GTR-NUC) at least partially attenuated this LBA artefact. The results support Illiosentidae as the basal radiation of Echinorhynchida and Rhadinorhynchidae forming a clade with Echinorhynchidae and Pomporhynchidae. The questions of the monophyly of Rhadinorhynchidae and its sister lineage remain unresolved. The order Echinorhynchida was paraphyletic in all of our analyses. CONCLUSIONS: Future studies should take care to attenuate compositional heterogeneity-driven LBA artefacts when applying mitogenomic data to resolve the phylogeny of Acanthocephala.

Real-World Survival Outcomes Based on EGFR Mutation Status in Chinese Patients With Lung Adenocarcinoma After Complete Resection: Results From the ICAN Study.

INTRODUCTION: The adjuvant treatment of patients with resected lung adenocarcinoma (LUAD) remains unstandardized. We analyzed the survival outcomes of these patients based on EGFR mutation status and adjuvant chemotherapy treatment. METHODS: This noninterventional real-world study (ICAN) enrolled Chinese patients with resected stages I to III LUAD from April 8, 2010, to December 31, 2010. Tumor EGFR mutation status and 3-year disease-free survival (DFS) were determined. The extension phase provided long-term follow-up with overall survival (OS) as the primary end point. Secondary end points included DFS and prognostic factors of survival. Survival outcomes based on adjuvant chemotherapy treatment, EGFR mutation status, and postoperative stage were analyzed post hoc. RESULTS: Among 568 patients in the ICAN cohort, 472 continued to the extension phase and remained eligible. The 3-year DFS rate was 58.8%. In the extension cohort, 260 patients (55.1%) had EGFR-mutant disease and 207 (43.9%) received adjuvant chemotherapy. At a median follow-up of 109.0 (95% confidence interval [CI]: 106.6-111.4) months, median OS and DFS were 103.3 (95% CI: 101.7-104.9) and 67.4 (95% CI: 49.7-85.2) months, respectively. The 5-year OS and DFS rates were 68.9% (95% CI: 64.3-73.6) and 52.9% (95% CI: 48.2-57.7), respectively. EGFR wild-type disease was a significant independent predictor of worse OS (HR = 1.24, 95% CI: 1.07-1.44, p= 0.004) based on the Cox regression analysis of common factors. Post hoc subgroup analysis revealed that survival outcomes were not significantly different with adjuvant chemotherapy regardless of EGFR mutation status across all postoperative stages. CONCLUSIONS: EGFR mutations are common in operable LUAD, and recurrence and mortality after resection were considerable. Adjuvant chemotherapy did not improve survival outcomes, regardless of EGFR mutation status and postoperative stage.

Development and characterization of monoclonal antibodies to spring viraemia of carp virus.

Five monoclonal antibodies (mAbs) against spring viraemia of carp (SVCV0504, isolated from common carp in China) were produced from mice immunized with purified virus preparations. The virion of SVCV contains five structural proteins, representing the nucleoprotein (N), phosphoprotein (P), matrix protein (M), glycoprotein (G) and RNA-dependent RNA polymerase (L). Western blotting analysis revealed that three mAbs (1H5, 1E10, and 1H7) recognized specifically to a single protein of 47kDa (N), the mAb 3G4 reacted with two SVCV0504 proteins of 69kDa (G) and 47kDa (N), while the mAb 1A9 reacted with three SVCV0504 proteins of 69kDa (G), 50kDa (P), and 47kDa (N). By indirect ELISA, two mAbs (1H5 and 1H7) showed cross-reactivity with pike fry rhabdovirus (PFRV), but no cross-reactions with the Siniperca chuatsi rhabdovirus (SCRV), Scophthalmus maximus rhabdovirus (SMRV), Paralichthys olivaceus rhabdovirus (PoRV) were demonstrated with the five mAbs. Indirect immunofluorescence showed intense fluorescence in the cytoplasm of the SVCV0504-infected epithelioma papulosum cyprini (EPC) cells in areas corresponding to the location of granular structures. The sucrose gradient-purified SVCV0504 particles could be detected successfully by these mAbs using immunodot blotting. mAb 1A9 could completely neutralize 100 TCID(50) (50% tissue culture infective dose) of SVCV0504 at a dilution of 1:8. This is the first report of development of the neutralizing mAbs against SVCV. The mAb 1A9 was analyzed further and could be used to successfully detect viral antigens in the infected-EPC cell cultures or in cryosections from experimentally infected crucian carp (Carassius auratus) by immunohistochemistry assay. Furthermore, a flow cytometry procedure for the detection and quantification of cytoplasmic SVCV0504 in cell cultures was developed with mAb 1A9. At 28h after inoculation with the virus (0.01PFU/cell), 10.12% of infected cells could be distinguished from the uninfected cells. These mAbs will be useful in diagnostic test development and pathogenesis studies for fish rhabdovirus.

Protective Immunity Induced by DNA Vaccination against Ranavirus Infection in Chinese Giant Salamander Andrias davidianus.

Andrias davidianus ranavirus (ADRV) is an emerging viral pathogen that causes severe systemic hemorrhagic disease in Chinese giant salamanders. There is an urgent need for developing an effective vaccine against this fatal disease. In this study, DNA vaccines containing the ADRV 2L gene (pcDNA-2L) and the 58L gene (pcDNA-58L) were respectively constructed, and their immune protective effects were evaluated in Chinese giant salamanders. In vitro and in vivo expression of the vaccine plasmids were confirmed in transfected cells and muscle tissues of vaccinated Chinese giant salamanders by using immunoblot analysis or RT-PCR. Following ADRV challenge, the Chinese giant salamanders vaccinated with pcDNA-2L showed a relative percent survival (RPS) of 66.7%, which was significant higher than that in Chinese giant salamanders immunized with pcDNA-58L (RPS of 3.3%). Moreover, the specific antibody against ADRV was detected in Chinese giant salamanders vaccinated with pcDNA-2L at 14 and 21 days post-vaccination by indirect enzyme-linked immunosorbent assay (ELISA). Transcriptional analysis revealed that the expression levels of immune-related genes including type I interferon (IFN), myxovirus resistance (Mx), major histocompatibility complex class IA (MHCIA), and immunoglobulin M (IgM) were strongly up-regulated after vaccination with pcDNA-2L. Furthermore, vaccination with pcDNA-2L significantly suppressed the virus replication, which was seen by a low viral load in the spleen of Chinese giant salamander survivals after ADRV challenge. These results indicated that pcDNA-2L could induce a significant innate immune response and an adaptive immune response involving both humoral and cell-mediated immunity that conferred effective protection against ADRV infection, and might be a potential vaccine candidate for controlling ADRV disease in Chinese giant salamanders.

[Phase I/II clinical trial of weekly administration of docetaxel plus cisplatin for advanced non-small cell lung cancer].

OBJECTIVE: The purpose of this phase I/II study is to investigate the safety/toxicity profile of weekly administration of docetaxel in combination with cisplatin for the chemo-naive patients with advanced non-small cell lung cancer (NSCLC), and to evaluate the efficacy of this regime. METHODS: In phase I trial, 15 patients were included. IV infusion of escalating doses of docetaxel consisting of four levels from 25 to 40 mg/m2 (25, 30, 35, 40 mg/m2) on D1, 8, 15 and cisplatin of 75 mg/m2 on D1 was administered. The regime was repeated every 4 weeks. Blood samples were obtained on D1, 15 in the first cycle to measure the PK. Dose limiting toxicity (DLT) was determined in cycle 1 and defined as any grade 3 non-hematologic toxicity which could not be reverted into grade less than grade 2 within 4 days or any grade 4 hematologic toxicity. Eighty-three patients completed their phase II study with administration of docetaxel at a dose of 35 mg/m2 based on the data of phase I trial. RESULTS: In the phase I trial, grade 3/4 neutropenia was mainly observed in patients who received docetaxel of 40 mg/m2 (level 4) with one patient suffering from an infection signifying dose limiting toxicity (DLT). Non-hematological toxicities including nausea/vomiting, alopecia, fluid retension and asthenia were tolerable. Based on these data, the maximum tolerence dose (MTD) did not reach the level of weekly giving docetaxel at a dose of 40 mg/m2 in combination with cisplatin 75 mg/m2 every 4 weeks. The pharmacokinetic/dynamics results There was no statistically significant difference between clearance value among the 4 dose levels of docetaxel from 25 to 40 mg/m2 when measured by Cmax and AUC. The pharmacokinetics of docetaxel was not influenced by the presence of co-administration of cisplatin when compared D1 with D15 as based on CmaxN, AUCN and CL. In the phase II trial, totally 83 patients received 216 cycles of chemotherapy. One CR (complete response) and 22 PR (partial response) were achieved with an objective response rate of 27.7% in this series and 30.7% in the evaluable patients. The 1-year survival was 48.6% with a median survival of 10.7 months (range: 3-34 months). Hematologic toxicities were the major side effects, though most were mild; grade III/IV neutropenia developed in 15%. The common non-hematologic toxicities were nausea, vomiting and asthenia. CONCLUSION: Weekly consecutive administration of docetaxel on D1, 8, 15 for 3 weeks plus cisplatin on D1 is tolerable and effective with minimal myelosuppression in chemo-naive patients with advanced NSCLC.

[Pulmonary sclerosing hemangioma: report of 15 cases and review of the literature].

OBJECTIVE: To improve the understanding of pulmonary sclerosing hemangioma (PSH). METHODS: The clinical data of 15 cases of PSH were analyzed, and the literature was reviewed. The etiology, clinical manifestations, differential diagnosis, treatment and outcome of PSH were described. RESULTS: The etiology and histological origin of PSH were unclear. Most cases were asymptomatic or only with mild symptoms. The radiology of PSH often showed isolated nodule with distinct margin in the lung field. The characteristics of its pathological manifestation were as follows: (1) background of cell gathering or mucin matrix with scattered white blood cells; (2) proliferation of hemangioma with sclerosis of vessel wall; (3) papillary proliferation of small vessels intruding into the air space; (4) existence of hemorrhage or sclerosis zone. Immunohistological studies had not defined the correct histological origin of PSH. A pre-operation diagnosis of PSH was difficult. Thirteen cases had been misdiagnosed as malignancy. The outcome of the disease was good when early surgical resection was performed. CONCLUSIONS: PSH is an uncommon disease, and can be easily misdiagnosed. More attention should be paid to its clinical features and management.

Whole-Genome Analysis of a Novel Fish Reovirus (MsReV) Discloses Aquareovirus Genomic Structure Relationship with Host in Saline Environments.

Aquareoviruses are serious pathogens of aquatic animals. Here, genome characterization and functional gene analysis of a novel aquareovirus, largemouth bass Micropterus salmoides reovirus (MsReV), was described. It comprises 11 dsRNA segments (S1-S11) covering 24,024 bp, and encodes 12 putative proteins including the inclusion forming-related protein NS87 and the fusion-associated small transmembrane (FAST) protein NS22. The function of NS22 was confirmed by expression in fish cells. Subsequently, MsReV was compared with two representative aquareoviruses, saltwater fish turbot Scophthalmus maximus reovirus (SMReV) and freshwater fish grass carp reovirus strain 109 (GCReV-109). MsReV NS87 and NS22 genes have the same structure and function with those of SMReV, whereas GCReV-109 is either missing the coiled-coil region in NS79 or the gene-encoding NS22. Significant similarities are also revealed among equivalent genome segments between MsReV and SMReV, but a difference is found between MsReV and GCReV-109. Furthermore, phylogenetic analysis showed that 13 aquareoviruses could be divided into freshwater and saline environments subgroups, and MsReV was closely related to SMReV in saline environments. Consequently, these viruses from hosts in saline environments have more genomic structural similarities than the viruses from hosts in freshwater. This is the first study of the relationships between aquareovirus genomic structure and their host environments.

Thymus cDNA library survey uncovers novel features of immune molecules in Chinese giant salamander Andrias davidianus.

A ranavirus-induced thymus cDNA library was constructed from Chinese giant salamander, the largest extant amphibian species. Among the 137 putative immune-related genes derived from this library, these molecules received particular focus: immunoglobulin heavy chains (IgM, IgD, and IgY), IFN-inducible protein 6 (IFI6), and T cell receptor beta chain (TCRß). Several unusual features were uncovered: IgD displays a structure pattern distinct from those described for other amphibians by having only four constant domains plus a hinge region. A unique IgY form (IgY(ΔFc)), previously undescribed in amphibians, is present in serum. Alternative splicing is observed to generate IgH diversification. IFI6 is newly-identified in amphibians, which occurs in two forms divergent in subcelluar distribution and antiviral activity. TCRß immunoscope profile follows the typical vertebrate pattern, implying a polyclonal T cell repertoire. Collectively, the pioneering survey of ranavirus-induced thymus cDNA library from Chinese giant salamander reveals immune components and characteristics in this primitive amphibian.

Extensive diversification of MHC in Chinese giant salamanders Andrias davidianus (Anda-MHC) reveals novel splice variants.

A series of MHC alleles (including 26 class IA, 27 class IIA, and 17 class IIB) were identified from Chinese giant salamander Andrias davidianus (Anda-MHC). These genes are similar to classical MHC molecules in terms of characteristic domains, functional residues, deduced tertiary structures and genetic diversity. The majority of variation between alleles is found in the putative peptide-binding region (PBR), which is driven by positive Darwinian selection. The coexistence of two isoforms in MHC IA, IIA, and IIB alleles are shown: one full-length transcript and one novel splice variant. Despite lake of the external domains, these variants exhibit similar subcellular localization with the full-length transcripts. Moreover, the expression of MHC isoforms are up-regulated upon in vivo and in vitro stimulation with Andrias davidianus ranavirus (ADRV), suggesting their potential roles in the immune response. The results provide insights into understanding MHC variation and function in this ancient and endangered urodele amphibian.

The antiviral defense mechanisms in mandarin fish induced by DNA vaccination against a rhabdovirus.

Plasmid DNAs containing Siniperca chuatsi rhabdovirus (SCRV) glycoprotein gene (pcDNA-G) and nucleoprotein gene (pcDNA-N) were constructed, and used to determine the antiviral immune response elicited by DNA vaccination in mandarin fish. In vitro and in vivo expression of the plasmid constructs was confirmed in transfected cells and muscle tissues of vaccinated fish by Western blot, indirect immunofluorescence or RT-PCR analysis. Fish injected with pcDNA-G exhibited protective effect against SCRV challenge with a relative percent survival (RPS) of 77.5%, but no significant protection (RPS of 2.5%) was observed in fish vaccinated with pcDNA-N. Immunohistochemical analysis showed that vaccination with pcDNA-G decreased histological lesions and suppressed the virus replication in fish target organs, e.g. kidney, liver, spleen, gill and heart. Transcriptional analysis further revealed that the expression levels of type I IFN system genes including interferon regulation factor-7 (IRF-7) gene, myxovirus resistance (Mx) gene and virus inhibitory protein (Viperin) gene were strongly up-regulated after injection with pcDNA-G, whereas the level of transcription of immunoglobulin M (IgM) gene did not show a statistically significant change. These results reveal that type I IFN antiviral immune response is rapidly triggered by the plasmid DNA containing rhabdovirus glycoprotein gene in fish, which offers an explanation of molecular mechanisms for DNA vaccination inducing mandarin fish resist to SCRV disease.

Mutations of the epidermal growth factor receptor gene in NSCLC patients.

Mutations of the epidermal growth factor receptor (EGFR) in patients with non-small cell lung cancer (NSCLC) were identified by re-sequencing all exons of this gene to evaluate the frequencies of EGFR gene mutation and identify rare or novel EGFR mutations. A total of 55 NSCLC samples from 55 patients were included in the study. Genomic DNA was extracted and exons 1-28 of the EGFR gene were sequenced to identify mutations. The cDNA of the EGFR gene with P848L and T790M double mutants was constructed by introducing point mutations into the wild-type EGFR vector using a site-directed mutagenesis kit. Among the 55 patients with NSCLC, 8 patients carried mutations of the EGFR gene. Notably, of the mutation-harboring patients with a pathological type of adenocarcinoma, 6 were non-smokers. The in vitro study demonstrated that the P848L mutant had a similar response to that of the wild-type EGFR after gefitinib treatment, and the P848L and T790M double mutant exhibited high resistance to gefitinib. These EGFR mutations preferentially occurred in lung adenocarcinoma patients, most of whom were non-smokers. In the in vitro study, P848L mutant EGFR had a similar response as the wild-type EGFR to gefitinib treatment, suggesting that lung cancer patients with a rare mutation of EGFR, such as the P848L mutation, do not respond to gefitinib treatment.