Determination and Characterization of Novel Papillomavirus and Parvovirus Associated with Mass Mortality of Chinese Tongue Sole (Cynoglossus semilaevis) in China.

A massive mortality event concerning farmed Chinese tongue soles occurred in Tianjin, China, and the causative agent remains unknown. Here, a novel Cynoglossus semilaevis papillomavirus (CsPaV) and parvovirus (CsPV) were simultaneously isolated and identified from diseased fish via electron microscopy, virus isolation, genome sequencing, experimental challenges, and fluorescence in situ hybridization (FISH). Electron microscopy showed large numbers of virus particles present in the tissues of diseased fish. Viruses that were isolated and propagated in flounder gill cells (FG) induced typical cytopathic effects (CPE). The cumulative mortality of fish given intraperitoneal injections reached 100% at 7 dpi. The complete genomes of CsPaV and CsPV comprised 5939 bp and 3663 bp, respectively, and the genomes shared no nucleotide sequence similarities with other viruses. Phylogenetic analysis based on the L1 and NS1 protein sequences revealed that CsPaV and CsPV were novel members of the Papillomaviridae and Parvoviridae families. The FISH results showed positive signals in the spleen tissues of infected fish, and both viruses could co-infect single cells. This study represents the first report where novel papillomavirus and parvovirus are identified in farmed marine cultured fish, and it provides a basis for further studies on the prevention and treatment of emerging viral diseases.

Rapid Detection of SARS-CoV-2 Virus Using Dual Reverse Transcriptional Colorimetric Loop-Mediated Isothermal Amplification.

The outbreak and pandemic of COVID-19, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has developed into a public health emergency of international concern. The rapid and accurate detection of the virus is a critical means to prevent and control the disease. Herein, we provide a novel, rapid, and simple approach, named dual reverse transcriptional colorimetric loop-mediated isothermal amplification (dRT-cLAMP) assay, to accelerate the detection of the SARS-CoV-2 virus without using expensive equipment. The result of this assay is shown by color change and is easily detected by the naked eye. To improve the detection accuracy, we included two primer sets that specifically target the viral orf1ab and N genes in the same reaction mixture. Our assay can detect the synthesized SARS-CoV-2 N and orf1ab genes at a low level of 100 copies/µL. Sequence alignment analysis of the two synthesized genes and those of 9968 published SARS-CoV-2 genomes and 17 genomes of other pathogens from the same infection site or similar symptoms as COVID-19 revealed that the primers for the dRT-cLAMP assay are highly specific. Our assay of 27 clinical samples of SARS-CoV-2 virus and 27 standard-added environmental simulation samples demonstrated that compared to the commercial kits, the consistency of the positive, negative, and probable clinical samples was 100, 92.31, and 44.44%, respectively. Moreover, our results showed that the positive, but not negative, standard-added samples displayed a naked-eye-detectable color change. Together, our results demonstrate that the dRT-cLAMP assay is a feasible detection assay for SARS-CoV-2 virus and is of great significance since rapid onsite detection of the virus is urgently needed at the ports of entry, health care centers, and for internationally traded goods.

Impact of environmental bacterial communities on fish health in marine recirculating aquaculture systems.

Marine cultured fish diseases caused by bacteria in recirculating aquaculture systems (RASs) greatly threaten fish aquaculture. To date, the dynamics of bacterial populations in RAS and their impacts to fish health remain largely unknown. In the present study, the bacterial communities in the water from two different marine RASs were analyzed using pyrosequencing technique. Fish disease syndromes and mortality had been reported from one RAS (RAS-d) while the fish in the other RAS remained healthy (RAS-h). The diversity of bacteria in each RAS and the abundance of each bacterium were identified based on sequencing the V4 hypervariable region of the 16S rRNA gene. A total number of 107,476 effective sequences were obtained from the pyrosequencing results. 640 and 844 operational taxonomic units (OTUs) were identified in RAS-d and RAS-h, respectively. In order level, tags annotation showed that Vibrionales and Flavobacteriales were the predominant strains in RAS-d with a relative abundance 50.5% and 36.5%, respectively. In contrast, the bacterial community in RAS-h contained 35.8% Vibrionales, 17.3% Alteromonadales, 10.7% Rhodobacterales, 7.43% Kordiimonadales, and 6.26% Oceanospirillales. In addition, the Vibrionaceae in the RAS-d represented 6.98% of the population which was significantly higher than that in RAS-h (0.40%). More potential pathogenic bacteria in fish, such as Vibrio harveyi, Vibrio rotiferianus were also found in the bacterial population in RAS-d. The results also showed that the bacteria community in RAS-h was more diverse and balanced than in RAS-d. These findings of this study suggested a potential correlation between fish diseases and environmental bacterial populations.

Facile synthesis of multifunctional multi-walled carbon nanotube for pathogen Vibrio alginolyticus detection in fishery and environmental samples.

Interest in carbon nanotubes for detecting the presence of pathogens arises because of developments in chemical vapor deposition synthesis and progresses in biomolecular modification. Here we reported the facile synthesis of multi-walled carbon nanotubes (MWCNTs), which functioned as immuno-, magnetic, fluorescent sensors in detecting Vibrio alginolyticus (Va). The structures and properties of functionalized MWCNTs were characterized by ultraviolet (UV), Fourier transform infrared spectra (FT-IR), X-ray diffraction (XRD), vibrating sample magnetometer (VSM), magnetic property measurement system (MPMS) and fluorescent spectra (FL). It was found that the functionalized MWCNTs showed: (1) low nonspecific adsorption for antibody-antigen, (2) strong interaction with antibody, and (3) high immune-magnetic activity for pathogenic cells. Further investigations revealed a strong positive linear relationship (R=0.9912) between the fluorescence intensity and the concentration of Va in the range of 9.0 × 10(2) to 1.5 × 10(6) cfum L(-1). Moreover, the relative standard deviation for 11 replicate detections of 1.0 × 10(4) cfum L(-1) Va was 2.4%, and no cross-reaction with the other four strains was found, indicating a good specificity for Va detection. These results demonstrated the remarkable advantages of the multifunctional MWCNTs, which offer great potential for the rapid, sensitive and quantitative detection of Va in fishery and environmental samples.

Changes in the organics metabolism in the hepatopancreas induced by eyestalk ablation of the Chinese mitten crab Eriocheir sinensis determined via transcriptome and DGE analysis.

BACKGROUND: To understand the regulation mechanism of eyestalk ablation on the activities of hepatopancreas, Illumina RNA-Seq and digital gene expression (DGE) analyses were performed to investigate the transcriptome of the eyestalk, Y-organ, and hepatopancreas of E. sinensis and to identify the genes associated with the hepatopancreas metabolism that are differentially expressed under eyestalk ablation conditions. RESULTS: A total of 58,582 unigenes were constructed from 157,168 contigs with SOAPdenovo. A BlastX search against the NCBI Nr database identified 21,678 unigenes with an E-value higher than 10⁻5. Using the BLAST2Go and BlastAll software programs, 6,883 unigenes (11.75% of the total) were annotated to the Gene Ontology (GO) database, 7,386 (12.6%) unigenes were classified into 25 Clusters of Orthologous Groups of Proteins (COGs), 16,200 (27.7%) unigenes were assigned to 242 Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways, and 1,846 unigenes were matched to "metabolism pathways". The DGE analysis revealed that 1,416 unigenes were significantly differentially expressed in the hepatopancreas, of which 890 unigenes were up-regulated and 526 unigenes were down-regulated. Of the differentially expressed genes, 382 unigenes were annotated and 63 were classified into metabolism pathways. The results of the real-time polymerase chain reaction (PCR) analysis of four unigenes related to carbohydrate metabolism were consistent with those obtained from the DGE analysis, which demonstrates that the sequencing data were satisfactory for further gene expression analyses. CONCLUSION: This paper reported the transcriptom of the eyestalk, Y-organ, and hepatopancreas from E. sinensis. DGE analysis provided the different expressed genes of the metabolism processes in hepatopancreas that are affected by eyestalk ablation. These findings will facilitate further investigations on the mechanisms of the metabolism of organic substances during development and reproduction in crustaceans.

Sequencing and de novo analysis of the hemocytes transcriptome in Litopenaeus vannamei response to white spot syndrome virus infection.

BACKGROUND: White spot syndrome virus (WSSV) is a causative pathogen found in most shrimp farming areas of the world and causes large economic losses to the shrimp aquaculture. The mechanism underlying the molecular pathogenesis of the highly virulent WSSV remains unknown. To better understand the virus-host interactions at the molecular level, the transcriptome profiles in hemocytes of unchallenged and WSSV-challenged shrimp (Litopenaeus vannamei) were compared using a short-read deep sequencing method (Illumina). RESULTS: RNA-seq analysis generated more than 25.81 million clean pair end (PE) reads, which were assembled into 52,073 unigenes (mean size = 520 bp). Based on sequence similarity searches, 23,568 (45.3%) genes were identified, among which 6,562 and 7,822 unigenes were assigned to gene ontology (GO) categories and clusters of orthologous groups (COG), respectively. Searches in the Kyoto Encyclopedia of Genes and Genomes Pathway database (KEGG) mapped 14,941 (63.4%) unigenes to 240 KEGG pathways. Among all the annotated unigenes, 1,179 were associated with immune-related genes. Digital gene expression (DGE) analysis revealed that the host transcriptome profile was slightly changed in the early infection (5 hours post injection) of the virus, while large transcriptional differences were identified in the late infection (48 hpi) of WSSV. The differentially expressed genes mainly involved in pattern recognition genes and some immune response factors. The results indicated that antiviral immune mechanisms were probably involved in the recognition of pathogen-associated molecular patterns. CONCLUSIONS: This study provided a global survey of host gene activities against virus infection in a non-model organism, pacific white shrimp. Results can contribute to the in-depth study of candidate genes in white shrimp, and help to improve the current understanding of host-pathogen interactions.

Role of chymotrypsin-like serine proteinase in white spot syndrome virus infection in Fenneropenaeus chinensis.

White spot syndrome virus (WSSV) caused a great economic loss in shrimp aquaculture. Although great efforts have been undertaken to characterize the virus disease during the last two decades, there are still lack of effective methods to prevent or cure it. In this study, we investigated the transcriptional expression profiles of 18 key immune-related genes in the Chinese shrimp Fenneropenaeus chinensis which was severely infected by WSSV. We found that the expression levels of 6 genes including chymotrypsin-like serine proteinase (CH-SPase), heat shock protein 70 cognate (HSP70), penaeidin (PEN), peroxinectin (PO), proliferating cell nuclear antigen (PCNA) and argonaute (AGO) changed significantly, while the expression of the other 12 genes had no significant changes compared to the control group. Among the 6 screened genes, CH-SPase showed significantly up-regulation, while the other 5 ones were significantly down-regulated. Knockdown of the expression of CH-SPase in WSSV-infected Chinese shrimp reduced the copy number of WSSV and delayed cumulative mortalities, suggesting that CH-SPase is important for WSSV infection. This study will be helpful to control the disease in shrimp caused by WSSV.