Andrias davidianus ranavirus 1R encoding a delayed-early protein promotes cell proliferation by driving cell cycle progression into S phase.

Andrias davidianus ranavirus 1R (ADRV-1R), a core gene of the family Iridoviridae, is predicted to encode a viral transcription factor (vTF) since the protein contains a virus late transcription factor-3 like (VLTF3 like) domain. However, its characteristics and function are still unclear. In this study, the transcription and expression of ADRV-1R were investigated in Chinese giant salamander thymus cells (GSTCs). ADRV-1R transcription starts 6 hours post-infection (hpi), while the protein expression starts 8 hpi. Drug inhibition assay showed that the transcripts are inhibited by cycloheximide (CHX), a de novo protein synthesis inhibitor, indicating that ADRV-1R is a viral delayed-early (DE) gene. Subcellular localization showed that ADRV-1R is distributed in the cell nucleus and cytoplasm. The effect of ADRV-1R overexpression on cell proliferation and virus titer was analyzed. ADRV-1R overexpression significantly promoted the cell proliferation starting at day 2. Flow cytometry analysis further indicated that the protein promotes the GSTC cell cycle progression from G1 phase into S phase (G1/S transition). Moreover, ADRV-1R overexperession significantly increased ADRV titer in GSTCs. The virus titer was 6.3-6.9-fold higher at 36 hpi and further after than the control GSTC lines. These data showed that ADRV-1R is a delayed-early protein promoting cell proliferation and virus titers. Keywords: ranavirus; Andrias davidianus ranavirus; core gene; cell cycle; cell proliferations.

Characterization, Expression Pattern and Antiviral Activities of Mx Gene in Chinese Giant Salamander, Andrias davidianus.

Mx, Myxovirus resistance is an important interferon-stimulated protein that mediates antiviral responses. In this study, the expression and activities of Chinese giant salamander, Andrias davidianus Mx gene, AdMx, were investigated. The AdMx cDNA sequence contains an open reading frame (ORF) of 2112 nucleotides, encoding a putative protein of 703 aa. Meanwhile, AdMx possesses the conserved tripartite GTP binding motif and a dynamin family signature. qRT-PCR analysis revealed a broad expression of AdMx in vivo, with the highest expression levels in brain, kidney and spleen. The AdMx expression level in kidney, spleen and muscle significantly increased at 6 h after Chinese giant salamander iridovirus (GSIV) infection and peaked at 48 h, while that in muscle cell line (GSM) was not noticeably up-regulated until 72 h post infection. Additionally, a plasmid expressing AdMx was constructed and transfected into the Chinese giant salamander GSM cells. The virus load and gene copies in AdMx over-expressed cells were significantly reduced compared with those in the control cells. Moreover, compared to the control cells, a lower level of virus major capsid protein (MCP) synthesis in AdMx over-expressed cells was confirmed by Western blot. These results collectively suggest that Mx plays an important antiviral role in the immune responses against GSIV in Chinese giant salamander.

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.

Pathological and microbiological findings from mortality of the Chinese giant salamander (Andrias davidianus).

The Chinese giant salamander, Andrias davidianus, is a nationally protected and cultured species in China. Recently, a severe epizootic occurred in cultured Chinese giant salamanders in Hubei, Hunan, Sichuan, Shaanxi, and Zhejiang provinces of China, causing substantial economic losses. The typical clinical signs of diseased larval animals were jaw and abdominal swelling and subcutaneous hemorrhaging. Diseased adult animals exhibited skin hemorrhages, ulceration of the hind limbs, and multiple hemorrhagic spots in the visceral organs. Histopathological observation indicated tissue necrosis and cytoplasmic inclusions in the spleen, liver and kidney, suggestive of viral disease. A viral agent was isolated from affected tissues in cell culture. The virus was determined to be pathogenic after experimental infection. Electron microscopy revealed iridovirus-like virions with a size of 140-180 nm in diameter inside the kidney of naturally infected animals and in cell culture. The major capsid protein (MCP) of the virus exhibited 98-99 % sequence identity to ranaviruses. Additionally, phylogenetic analysis indicated that the virus belonged to the genus Ranavirus. Comparative analysis of the MCP gene sequence with those of other viruses previously isolated from Chinese giant salamanders revealed that these isolates were highly similar, although a few variations were observed. The virus was preliminarily named Chinese giant salamander iridovirus (GSIV).

First Report of Endemic Frog Virus 3 (FV3)-like Ranaviruses in the Korean Clawed Salamander (Onychodactylus koreanus) in Asia.

Frog virus 3 (FV3) in the genus Ranavirus of the family Iridoviridae causes mass mortality in both anurans and urodeles worldwide; however, the phylogenetic origin of FV3-like ranaviruses is not well established. In Asia, three FV3-like ranaviruses have been reported in farmed populations of amphibians and reptiles. Here, we report the first case of endemic FV3-like ranavirus infections in the Korean clawed salamander Onychodactylus koreanus, caught in wild mountain streams in the Republic of Korea (ROK), through whole-genome sequencing and phylogenetic analysis. Two isolated FV3-like ranaviruses (Onychodactylus koreanus ranavirus, OKRV1 and 2) showed high similarity with the Rana grylio virus (RGV, 91.5%) and Rana nigromaculata ranavirus (RNRV, 92.2%) but relatively low similarity with the soft-shelled turtle iridovirus (STIV, 84.2%) in open reading frame (ORF) comparisons. OKRV1 and 2 formed a monophyletic clade with previously known Asian FV3-like ranaviruses, a sister group of the New World FV3-like ranavirus clade. Our results suggest that OKRV1 and 2 are FV3-like ranaviruses endemic to the ROK, and RGV and RNRV might also be endemic strains in China, unlike previous speculation. Our data have great implications for the study of the phylogeny and spreading routes of FV3-like ranaviruses and suggest the need for additional detection and analysis of FV3-like ranaviruses in wild populations in Asian countries.

Evidence for multiple recent host species shifts among the Ranaviruses (family Iridoviridae).

Members of the genus Ranavirus (family Iridoviridae) have been recognized as major viral pathogens of cold-blooded vertebrates. Ranaviruses have been associated with amphibians, fish, and reptiles. At this time, the relationships between ranavirus species are still unclear. Previous studies suggested that ranaviruses from salamanders are more closely related to ranaviruses from fish than they are to ranaviruses from other amphibians, such as frogs. Therefore, to gain a better understanding of the relationships among ranavirus isolates, the genome of epizootic hematopoietic necrosis virus (EHNV), an Australian fish pathogen, was sequenced. Our findings suggest that the ancestral ranavirus was a fish virus and that several recent host shifts have taken place, with subsequent speciation of viruses in their new hosts. The data suggesting several recent host shifts among ranavirus species increase concern that these pathogens of cold-blooded vertebrates may have the capacity to cross numerous poikilothermic species barriers and the potential to cause devastating disease in their new hosts.

The Immune System and the Antiviral Responses in Chinese Giant Salamander, Andrias davidianus.

The Chinese giant salamander, belonging to an ancient amphibian lineage, is the largest amphibian existing in the world, and is also an important animal for artificial cultivation in China. However, some aspects of the innate and adaptive immune system of the Chinese giant salamander are still unknown. The Chinese giant salamander iridovirus (GSIV), a member of the Ranavirus genus (family Iridoviridae), is a prominent pathogen causing high mortality and severe economic losses in Chinese giant salamander aquaculture. As a serious threat to amphibians worldwide, the etiology of ranaviruses has been mainly studied in model organisms, such as the Ambystoma tigrinum and Xenopus. Nevertheless, the immunity to ranavirus in Chinese giant salamander is distinct from other amphibians and less known. We review the unique immune system and antiviral responses of the Chinese giant salamander, in order to establish effective management of virus disease in Chinese giant salamander artificial cultivation.

Bid is involved in apoptosis induced by Chinese giant salamander iridovirus and contributes to the viral replication in an amphibian cell line.

Bid is a pro-apoptotic BH3-only member of the Bcl-2 superfamily that functions to link the extrinsic apoptotic pathway and the mitochondrial amplification loop of the intrinsic pathway. In this study, the expression and functions of Chinese giant salamander (Andrias davidianus) Bid (AdBid) were investigated. The AdBid cDNA sequence contains an open reading frame (ORF) of 576 nucleotides, encoding a putative protein of 191 aa. AdBid possesses the conserved BH3 interacting domain and shared 34-52% sequence identities with other amphibian Bid. mRNA expression of AdBid was most abundant in muscle. The expression level of AdBid in Chinese giant salamander muscle, kidney and spleen significantly increased after Chinese giant salamander iridovirus (GSIV) infection. Additionally, a plasmid expressing AdBid was constructed and transfected into the Chinese giant salamander muscle cell line (GSM cells). The morphology and cytopathic effect (CPE) and apoptotic process in AdBid over-expressed GSM cells was significantly enhanced during GSIV infection compared with that in control cells. Moreover, a higher level of the virus major capsid protein (MCP) gene copies and protein synthesis was confirmed in the AdBid over-expressed cells. These results indicated that AdBid played a positive role in GSIV induced apoptosis and the viral replication. This study may contribute to the better understanding on the infection mechanism of iridovirus-induced apoptosis.

Functional differences in the products of two TRAF3 genes in antiviral responses in the Chinese giant salamander, Andrias davidianus.

Tumour necrosis factor receptor associated factor 3 (TRAF3) is a crucial transducing protein for linking upstream receptor signals and downstream antiviral signalling pathways. Previous studies mostly clarified the functions of TRAF3 in mammals, birds and fish, but little is known about the characterization and function of TRAF3 in amphibians. In this study, the molecular and functional identification of two TRAF3 genes, AdTRAF3A and AdTRAF3B, were investigated in the Chinese giant salamander Andrias davidianus. The complete open reading frames (ORFs) of AdTRAF3A and AdTRAF3B were 1698 bp and 1743 bp in length, encoding 565 and 580 amino acids, respectively. Both AdTRAF3A and AdTRAF3B deduced proteins contained a RING finger, two TRAF-type zinc fingers, a coiled-coil and a MATH domain. Phylogenetic analysis showed that the AdTRAF3 protein clustered together with other known TRAF3 proteins. Gene expression analysis showed that AdTRAF3s were broadly distributed in all examined tissues with similar distribution patterns. AdTRAF3s in the blood or spleen positively responded to Giant salamander iridovirus (GSIV) and poly (I:C) induction but exhibited distinct response patterns. Silencing AdTRAF3A/B remarkably suppressed the expression of IFN signalling pathway-related genes when leukocytes were treated with DNA virus and the viral RNA analogue. Moreover, overexpression of AdTRAF3A may induce the activation of the IFN-ß promoter, and the zinc finger, coiled coil and MATH domains of AdTRAF3A were essential for IFN-ß promoter activation. However, the overexpression of AdTRAF3B significantly suppressed IFN-ß promoter activity, and its inhibitory effect was enhanced when the RING finger or MATH domain was deleted. Furthermore, AdTRAF3A rather than AdTRAF3B significantly induced NF-κB activation, implying that AdTRAF3A may function as an enhancer in both the IFN and NF-κB signalling pathways. Taken together, our results suggest that the two TRAF3 genes play different crucial regulatory roles in innate antiviral immunity in Chinese giant salamanders.

GEOGRAPHIC AND INDIVIDUAL DETERMINANTS OF IMPORTANT AMPHIBIAN PATHOGENS IN HELLBENDERS (CRYPTOBRANCHUS ALLEGANIENSIS) IN TENNESSEE AND ARKANSAS, USA.

Wildlife diseases are a major threat for species conservation and there is a growing need to implement disease surveillance programs to protect species of concern. Globally, amphibian populations have suffered considerable losses from disease, particularly from chytrid fungi (Batrachochytrium dendrobatidis [Bd] and Batrachochytrium salamandrivorans [Bsal]) and ranavirus. Hellbenders (Cryptobranchus alleganiensis) are large riverine salamanders historically found throughout several watersheds of the eastern and midwestern US. Populations of both subspecies (Ozark hellbender, Cryptobranchus alleganiensis bishopi; eastern hellbender, Cryptobranchus alleganiensis alleganiensis) have experienced precipitous declines over at least the past five decades, and emerging pathogens are hypothesized to play a role. We surveyed Ozark hellbender populations in Arkansas (AR) and eastern hellbender populations in Middle Tennessee (MTN) and East Tennessee (ETN) for both chytrid fungi and ranavirus from swabs and tail tissue, respectively, from 2011 to 2017. Overall, we detected Bd on hellbenders from nine out of 15 rivers, with total prevalence of 26.7% (54/ 202) that varied regionally (AR: 33%, 28/86; MTN: 11%, 4/36; ETN: 28%, 22/80). Ranavirus prevalence (9.0%, 18/200) was comparatively lower than Bd, with less regional variation in prevalence (AR: 6%, 5/ 85; MTN: 11%, 4/36; ETN: 10%, 8/79). We did not detect Bsal in any hellbender populations. We detected a significant negative correlation between body condition score and probability of ranavirus infection (ß=-0.13, SE=0.06, 95% confidence interval: -0.24, -0.02). Evaluation of infection load of positive individuals revealed different trends than prevalence alone for both ranavirus and Bd, with MTN having a significantly greater average ranaviral load than both other regions. We documented a variety of lesions that likely have multiple etiologies on hellbenders located within all geographic regions. Our data represent a multiyear pathogen dataset across several regions of C. alleganiensis, and we emphasize the need for continued pathogen surveillance.

Spatial and temporal patterns of Ambystoma tigrinum virus (ATV) prevalence in tiger salamanders Ambystoma tigrinum nebulosum.

Amphibian ranaviruses have been documented as causes of mass mortality in amphibian populations throughout the world. The temporal and spatial dynamics of ranavirus infections when epidemics are not apparent remains unclear. To address this question, we collected tissue samples from 2003 to 2006 in 4 geographically separated tiger salamander Ambystoma tigrinum nebulosum host populations on the Kaibab Plateau in northern Arizona. We tested for Ambystoma tigrinum virus (ATV), a lethal ranavirus of tiger salamanders, calculated ATV prevalence for each sampling date, and examined temporal and spatial patterns by quantifying the annual level of ATV synchrony among populations using the intraclass correlation coefficient. Salamander populations were commonly infected with ATV. We observed no morbidity or mortality in these populations even as ATV prevalence values varied from 0 to 57%. Infection prevalence across the landscape was more similar within a given year than between years. There was no statistically significant spatial pattern in prevalence across the landscape. Our findings highlight the need to explore new hypotheses regarding the population level impact of these pathogens on amphibian communities.

Rapid detection of giant salamander iridovirus by cross-priming amplification.

Giant salamander iridovirus (GSIV) belongs to the epizootic genus Ranavirus, and is the cause of epidemic diseases associated with high mortality and great losses to artificial breeding and farming. Here, we established a simple, accurate, and reliable cross-priming amplification (CPA) method to detect GSIV. The CPA assay targets the major caspid protein gene of the GSIV genome to design crossing primer pairs, and the reaction conditions were optimized, including optimal concentrations of the primers, betaine, dNTPs, Mg2+, and Bst DNA polymerase, and reaction conditions. The sensitivity was shown to be 10 times higher than that of conventional polymerase chain reaction (PCR), and the specificity was 100%. The results were identified on nucleic acid strips within 3-5 min. Application of the CPA and PCR to 54 samples of giant salamander showed a positive rate of 72.22% and 74.07%, respectively, demonstrating high coincidence (94.44%, kappa = 8.7, P < 0.0001). The sensitivity of the CPA assay was 97.50% and the specificity was 92.86%. Thus, the CPA assay is as effective as conventional PCR, but with added practical advantages of simplicity and an almost instrument-free platform, which will be useful for both laboratories and giant salamander farms.

Amphibian commerce as a likely source of pathogen pollution.

The commercial trade of wildlife occurs on a global scale. In addition to removing animals from their native populations, this trade may lead to the release and subsequent introduction of nonindigenous species and the pathogens they carry. Emerging infectious diseases, such as chytridiomycosis caused by the chytrid fungus Batrachochytrium dendrobatidis (Bd), and ranaviral disease have spread with global trade in amphibians and are linked to amphibian declines and die-offs worldwide, which suggests that the commercial trade in amphibians may be a source of pathogen pollution. We screened tiger salamanders involved in the bait trade in the western United States for both ranaviruses and Bd with polymerase chain reaction and used oral reports from bait shops and ranavirus DNA sequences from infected bait salamanders to determine how these animals and their pathogens are moved geographically by commerce. In addition, we conducted 2 surveys of anglers to determine how often tiger salamanders are used as bait and how often they are released into fishing waters by anglers, and organized bait-shop surveys to determine whether tiger salamanders are released back into the wild after being housed in bait shops. Ranaviruses were detected in the tiger salamander bait trade in Arizona, Colorado, and New Mexico, and Bd was detected in Arizona bait shops. Ranaviruses were spread geographically through the bait trade. All tiger salamanders in the bait trade were collected from the wild, and in general they moved east to west and north to south, bringing with them their multiple ranavirus strains. Finally, 26-73% of anglers used tiger salamanders as fishing bait, 26-67% of anglers released tiger salamanders bought as bait into fishing waters, and 4% of bait shops released tiger salamanders back into the wild after they were housed in shops with infected animals. The tiger salamander bait trade in the western United States is a useful model for understanding the consequences of the unregulated anthropogenic movement of amphibians and their pathogens through trade.

Frog virus 3-like infections in aquatic amphibian communities.

Frog virus 3 (FV3) and FV3-like viruses, are members of the genus Ranavirus (family Iridoviridae), and they have been associated with infectious diseases that may be contributing to amphibian population declines. We examined the mode of transmission of an FV3-like virus, and potential hosts and reservoirs of the virus in a local amphibian community. Using the polymerase chain reaction to detect infected animals, we found an FV3-like virus in south-central Ontario, Canada, amphibian communities, where it infects sympatric amphibian species, including ranid and hylid tadpoles (Rana sylvatica, Hyla versicolor, and Pseudacris spp.), larval salamanders (Ambystoma spp.), and adult eastern-spotted newts (Notophthalmus viridescens). The high prevalence of FV3-like infections in caudate larvae suggests that salamanders are likely to be both hosts and reservoirs. In laboratory FV3 challenges of R. sylvatica, the rate of infection was dependent on the amount of virus to which the animals were exposed. In addition, although vertical transmission was suspected, horizontal transmission through exposure to infected pond water is the most likely route of infection in tadpoles. Based on our observations, a simple model of FV3/FV3-like virus transmission postulates that, in aquatic amphibian communities, transmission of the virus occurs between anuran and urodele species, with ambystomatid salamanders the most likely reservoir for the ranavirus in our study.

Iridoviral infection can be reduced by UCHL1-loaded exosomes from the testis of Chinese giant salamanders (Andrias davidianus).

Chinese giant salamander iridovirus (CGSIV) is a large double-stranded DNA virus that infects Chinese giant salamanders (CGSs) and is responsible for a high mortality rate of CGSs under certain conditions. It is generally believed that CGSIV is a horizontally transmitting virus that affects lower vertebrates. Exosomes from tissues and cells affect the mechanism of viral infections. UCHL1, a deubiquitinating enzyme, is indirectly involved in virus propagation via cytokine and chemokine suppression. In our study, a few CGSIVs were detected in the testis of the special symptom CGSs using PCR and immunofluorescence analysis. The exosomes originating in the testicular fluid was isolated and identified using the Nanosight NS300 system and scanning electron microscopy. The UCHL1-loaded exosomes may resist CGSIV entry by fusing with and remodeling CGSIV. UCHL1 in the primary testicular fibroblasts was maintained at a stable level to inhibit the infection and replication of CGSIV by secreting and sorting exosomes. These data provided a new insight into CGSIV being a type of horizontally transmitting virus.

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.

MicroRNA repertoire and comparative analysis of Andrias davidianus infected with ranavirus using deep sequencing.

Andrias davidianus is a large and economically important amphibian in China. Ranavirus infection causes serious losses in A. davidianus farming industry. MicroRNA mediated host-pathogen interactions are important in antiviral defense. In this study, five small-RNA libraries from ranavirus infected and non-infected A. davidianus spleens were sequenced using high throughput sequencing. The miRNA expression pattern, potential functions, and target genes were investigated. In total, 1356 known and 431 novel miRNAs were discovered. GO and KEGG analysis revealed that certain miRNA target genes are associated with apoptotic, signal pathway, and immune response categories. Analysis identified 82 downregulated and 9 upregulated differentially expressed miRNAs, whose putative target genes are involved in pattern-recognition receptor signaling pathways and immune response. These findings suggested miRNAs play key roles in A. davidianus's response to ranavirus and could provide a reference for further miRNA functional identification, leading to novel approaches to improve A. davidianus ranavirus resistance.

Phylogenetic concordance analysis shows an emerging pathogen is novel and endemic.

Distinguishing whether pathogens are novel or endemic is critical for controlling emerging infectious diseases, an increasing threat to wildlife and human health. To test the endemic vs. novel pathogen hypothesis, we present a unique analysis of intraspecific host-pathogen phylogenetic concordance of tiger salamanders and an emerging Ranavirus throughout Western North America. There is significant non-concordance of host and virus gene trees, suggesting pathogen novelty. However, non-concordance has likely resulted from virus introductions by human movement of infected salamanders. When human-associated viral introductions are excluded, host and virus gene trees are identical, strongly supporting coevolution and endemism. A laboratory experiment showed an introduced virus strain is significantly more virulent than endemic strains, likely due to artificial selection for high virulence. Thus, our analysis of intraspecific phylogenetic concordance revealed that human introduction of viruses is the mechanism underlying tree non-concordance and possibly disease emergence via artificial selection.

Characterization of galectin-1 from Chinese giant salamanders Andrias davidianus and its involvements during immune response.

Galectins are considered as a multifunctional protein which play essential roles in cell adhesion and apoptosis, inflammation, tumor progression and immune response. In spite of extensive studies of galectin importance in immune system among different animals, few studies have been devoted to their functions in amphibian. In the present study, we characterized one proto type of galectin (named AdGal1) from Chinese giant salamander Andrias davidianus and studied its function in immune response. AdGal1 cDNA possesses an open reading frame of 598 bp, which encodes a putative galectin of 134 amino acids containing one carbohydrate recognition domains (CRDs). The constitutive expression of mRNA transcripts was detected in a wide range of tissues, with the highest expression in kidney. Immune challenges with Aeromonas hydrophila and Chinese giant salamander iridovirus (GSIV), the transcript level of AdGal1 in kidney was significantly upregulated. The mature protein of AdGal1 was successfully expressed and purified in Escherichia coli BL21 (DE3). The recombinant AdGal1 (rAdGal1) could show bind activity to different Gram negative and Gram positive bacteria. It could also strongly agglutinate different kinds of bacteria at different concentrations. Collectively, these data from the present study indicate that AdGal1 is a vital pattern recognition receptor to recognize different microbes in the innate immune system of Andrias davidianus.