Establishment of a real-time Recombinase Polymerase Amplification (RPA) for the detection of decapod iridescent virus 1 (DIV1).

A rapid and simple real-time recombinase polymerase amplification (RPA) assay was developed to detect decapod iridescent virus 1 (DIV1). The assay was developed using optimized primers and probes designed from the conserved sequence of the DIV1 major capsid protein (MCP) gene. Using the optimized RPA assay, the DIV1 test was completed within 20 min at 39 ℃. The RPA assay was specific to DIV1 with a detection limit of 2.3 × 101 copies/reaction and there was no cross-reactivity with the other aquatic pathogens (WSSV, IHHNV, NHPB, VpAHPND, EHP, IMNV, YHV-1 and GAV) tested. Four out of 45 field-collected shrimp samples tested positive for DIV1 by real-time RPA. The same assay results were obtained by both methods. Thus, the real-time RPA assay developed could be a simple, rapid, sensitive, reliable and affordable method for the on-site diagnosis of DIV1 infection and has significant potential in helping to control DIV1 infections and reduce economic losses to the shrimp industry.

Fraudulent antibiotic products on the market for aquaculture use.

Antibiotics in aquaculture are used to treat bacterial infections. In order for these products to work effectively fish need to be properly dosed. One of the emerging issues in aquaculture is under-dosing large populations of fish with antibiotics. This happens inadvertently for a number of reasons including the use of fraudulent medications. In this study we evaluated 17 antibiotic products (8 florfenicol and 9 oxytetracycline brands purchased in Asia) by HPLC to determine if the product labels accurately reflected the active pharmaceutical ingredient (API) in the package. We determined authenticity scores for different batches of products at two separate laboratories by comparing the observed API to the label API concentration. We found that 48 % of the antibiotic batches had authenticity scores below 80 % (i.e. observed API in package was at least 20 % less than the label API concentration). Further, there were 9 or the 31 batches of drugs tested had no measureable API. Some products had variation in their authenticity scores between batches making it difficult to rely on a brand. The price of florfenicol products may help identify products with low authenticity scores, but in the case of oxytetracycline, the price of all the products tested was relatively similar. The findings in this study suggest that not all florfenicol and oxytetracycline antibiotic products on the market in Asia have API concentrations indicated on their labels. This could be problematic for medicating fish on aquaculture farms.

Comparison of insight and clinical variables in homeless and non-homeless psychiatric inpatients in China.

There are no published data on insight in homeless patients with psychiatric disorders in China. This study examined insight in homeless and non-homeless Chinese psychiatric inpatients in relation to demographic and clinical variables. A total of 278 homeless and 222 non-homeless inpatients matched in age and gender were included in the study. Demographic and clinical characteristics were collected based on a review of medical charts and a clinical interview with standardized instruments. Insight was evaluated with the Insight and Treatment Attitudes Questionnaire. Altogether 20.5% of homeless inpatients and 43.7% of the non-homeless controls had good insight. Compared with homeless inpatients with impaired insight, homeless inpatients with good insight had higher physical quality of life, longer duration of illness and less severe positive and negative symptoms. Impaired insight appeared more common in homeless psychiatric inpatients in China. Further studies should address the need for effective therapeutic interventions that promote homeless patients' insight.

Analytical validation of a reverse transcriptase droplet digital PCR (RT-ddPCR) for quantitative detection of infectious hematopoietic necrosis virus.

Infectious hematopoietic necrosis virus (IHNV) is an important pathogen of salmonid fishes. A validated universal reverse transcriptase quantitative PCR (RT-qPCR) assay that can quantify levels of IHNV in fish tissues has been previously reported. In the present study, we adapted the published set of IHNV primers and probe for use in a reverse-transcriptase droplet digital PCR (RT-ddPCR) assay for quantification of the virus in fish tissue samples. The RT-ddPCR and RT-qPCR assays detected 13 phylogenetically diverse IHNV strains, but neither assay produced detectable amplification when RNA from other fish viruses was used. The RT-ddPCR assay had a limit of detection (LOD) equating to 2.2 plaque forming units (PFU)/µl while the LOD for the RT-qPCR was 0.2 PFU/µl. Good agreement (69.4-100%) between assays was observed when used to detect IHNV RNA in cell culture supernatant and tissues from IHNV infected rainbow trout (Oncorhynchus mykiss) and arctic char (Salvelinus alpinus). Estimates of RNA copy number produced by the two assays were significantly correlated but the RT-qPCR consistently produced higher estimates than the RT-ddPCR. The analytical properties of the N gene RT-ddPCR test indicated that this method may be useful to assess IHNV RNA copy number for research and diagnostic purposes. Future work is needed to establish the within and between laboratory diagnostic performance of the RT-ddPCR assay.

Identification of Infectious Salmon Anaemia Virus from Imported Iced Atlantic Salmons.

Infectious Salmon anaemia virus (ISAV) has become a threat to the salmon industry worldwide and has caused considerable economic loss. In the present study, 9 suspect cases of ISAV infection were identified from iced Atlantic salmons imported from Norway in 2014 through Shenzhen port (Shenzhen, China) using methods recommended by the World Organization for Animal Health. However, the results of virus isolation were negative., Based on the sequence analysis of ISAV segment 6, the 9 ISAV isolates belonged to the HPRO type, had high homology (98.3%~100.0%) and closest relationship with Norway strains. We identified the 9 positive HPRO ISAVs from 491 iced Atlantic salmons (1. 8%). Therefore, we should strengthen the quarantine of iced Atlantic salmons from Norway in case of HPRO ISAV into China.

Toll-like receptors and interferon associated immune factors responses to spring viraemia of carp virus infection in common carp (Cyprinus carpio).

Pattern recognition receptor (PRR) toll-like receptors (TLRs), antiviral agent interferon (IFN) and the effector IFN stimulated genes (ISGs) play a fundamental role in the innate immune response against viruses among all vertebrate classes. Common carp is a host for spring viraemia of carp virus (Rhabdovirus carpio, SVCV), which belong to Rhabdoviridae family. The present in-vivo experiment was conducted to investigate the expression of these innate immune factors in early phase as well as during recovery of SVCV infection by real-time quantitative reverse transcriptase polymerase chain reaction. A less lethal SVCV infection was generated in common carp (Cyprinus carpio) and was sampled at 3, 6, 12 h post infection (hpi), 1, 3, 5, 7 and 10 days post infection (dpi). At 3 hpi, the SVCV N gene was detected in all three fish and all three fish showed a relative fold increase of TLR2, TLR3 and TLR7, IFNa1, ISG15 and Vig1. Viral copies rapidly increased at 12 hpi then remained high until 5 dpi. When viral copy numbers were high, a higher expression of immune genes TLR2, TLR3, TLR7, IFNa1, IFNa2, IFNa1S, IFN regulatory factor 3 (IRF3), IRF7, interleukin 1ß (IL1ß), IL6, IL10, ADAR, ISG15, Mx1, PKR and Vig1 were observed. Viral copies were gradually reduced in 5 to 10 dpi fish, and also the immune response was considerably reduced but remained elevated. A high degree of correlation was observed between immune genes and viral copy number in each of the sampled fish at 12 hpi. The quick and prolonged elevated expression of the immune genes indicates their crucial role in survival of host against SVCV.

Determination of the complete genome sequence of infectious hematopoietic necrosis virus (IHNV) Ch20101008 and viral molecular evolution in China.

This study determined the complete genomic sequence of the infectious hematopoietic necrosis virus (IHNV) strain Ch20101008 isolated from farmed brook trout (Salvelinus fontinalis) that died from a disease caused by the virus in northeast China. The sequence was determined from 10 overlapping clones obtained through RT-PCR amplification. The whole genome length of Ch20101008 comprised 11,129 nucleotides (nt), and the overall organization was typical of that observed for all other IHNV strains. The phylogenetic analysis results of the 65 IHNV glycoprotein genes and 47 IHNV partial nucleoprotein genes presented five major genogroups (J, U, L, E and M). The J genogroup included the J Nagano and J Shizuoka subgroups. The IHNV Ch20101008 strain belonged to the J Nagano subgroup of the J genogroup and was significantly related to other Chinese IHNV strains. All Chinese IHNV isolates are monophyletic, with a recent common ancestor, except for the BjLL strain. The N, P, M, G, NV and L genes of Ch20101008 were compared with the available IHNV sequences in GenBank. The results indicated that 198 nt were substituted, 53 of which exhibited amino acid change in the Ch20101008 genome. An adenine nucleotide deletion was found at position 4959 of the 5' UTR of the L gene. In the G gene, specific nucleotides and amino acid variations of the Chinese IHNV strains were observed when compared with 23 isolates from other countries. Of the 15 nucleotide sites that changed, seven resulted in amino acid substitution. The data further demonstrated that the J genogroup IHNV was introduced to and evolved in salmon farm environments in China.

Selection and characterization of single-chain recombinant antibodies against infectious haematopoietic necrosis virus from mouse phage display library.

Six single-chain fragment variable (scFv) antibodies against infectious haematopoietic necrosis virus (IHNV) were selected from an antibody phage display library by phage display technology. The soluble scFv antibodies showed a molecular weight 32kDa by Western blot. Dot blot analysis revealed that the six scFv antibodies could recognize IHNV. For enzyme linked immunosorbent assay (ELISA), four scFv antibodies (P1A4, P1A12, P1D5 and P3E2) showed cross-reactivity with spring viraemia of carp virus (SVCV). However, none of the six scFv antibodies had cross-reaction with Pike fry rhabdovirus (PFRV), Soft-shelled turtle iridovirus (STIV), viral haemorrhagic septicemia virus (VHSV), or viral nervous necrosis virus (VNNV). Indirect immunofluorescence results showed that all of these scFv antibodies reacted positively with virus in the IHNV-infected cells. These scFv antibodies will be useful in diagnostic test development and pathogenesis studies for IHNV.

Selection and characterization of single-chain recombinant antibodies against spring viraemia of carp virus from mouse phage display library.

Antibody-displaying phage library was selected after three rounds of panning against spring viraemia of carp virus (SVCV) by phage display technology. Eight positive clones which could produce soluble single-chain fragment variable (scFv) antibody induced by isopropyl-beta-d-thiogalactopyranoside (IPTG) were obtained. Dot blot results showed that the eight scFv antibodies could recognize SVCV. The soluble scFv antibodies showed a molecular weight 29 kD by Western blot. All scFv antibodies could recognize SVCV proteins specifically without cross-reaction with other virus proteins by ELISA. Indirect immunofluorescence results showed that all of these scFv antibodies reacted positively with virus in the SVCV-infected cells. These scFv antibodies will be useful tools to establish immunological detection methods for SVCV.

Development and evaluation of a loop-mediated isothermal amplification assay for diagnosis of Cyprinid herpesvirus 2.

Goldfish Haematopoietic Necrosis caused by Cyprinid herpesvirus 2 (CyHV-2) is a severe fish disease with high level of mortality. This is the first study on detection of this disease by loop-mediated isothermal amplification (LAMP). A set of six primers targeting terminase gene (accession no. EU349285.1) was determined after a serial of tests. Detection limit was 1.09 × 10(-4)µg/µL, which was superior to conventional PCR and real-time PCR. No cross reaction with 28 other viruses or bacteria commonly found in fish was observed. The application of commercial kit and instrument for the LAMP assay could reduce the risk of cross contamination, which is suitable for detection of infection under field conditions.

Complete sequence of a viral nervous necrosis virus (NNV) isolated from red-spotted grouper (Epinephelus akaara) in China.

A nodavirus isolated from red-spotted grouper (Epinephelus akaara) larvae in China has been subjected to genome analysis. The full-length genome sequences of RNA1 and RNA2 were determined, and the 5'-non-coding region (NCR) and 3'NCR sequences were determined by 5' rapid amplification of cDNA ends (RACE) and 3'RACE. RNA1 is 3,103 nt in length and contains a 982-amino-acid open reading frame (ORF) encoding protein A with a calculated molecular mass of 110.74 kDa. RNA2 is 1,433 nt long and contains a 338-amino-acid major ORF encoding coat protein with a calculated molecular mass of 37.059 kDa. Multiple alignment and phylogenetic analysis clearly supported including this virus in the species Redspotted grouper nervous necrosis virus, genus Betanodavirus, family Nodaviridae.

The ClpP protease homologue is required for the transmission traits and cell division of the pathogen Legionella pneumophila.

BACKGROUND: Legionella pneumophila, the intracellular bacterial pathogen that causes Legionnaires' disease, exhibit characteristic transmission traits such as elevated stress tolerance, shortened length and virulence during the transition from the replication phase to the transmission phase. ClpP, the catalytic core of the Clp proteolytic complex, is widely involved in many cellular processes via the regulation of intracellular protein quality. RESULTS: In this study, we showed that ClpP was required for optimal growth of L. pneumophila at high temperatures and under several other stress conditions. We also observed that cells devoid of clpP exhibited cell elongation, incomplete cell division and compromised colony formation. Furthermore, we found that the clpP-deleted mutant was more resistant to sodium stress and failed to proliferate in the amoebae host Acanthamoeba castellanii. CONCLUSIONS: The data present in this study illustrate that the ClpP protease homologue plays an important role in the expression of transmission traits and cell division of L. pneumophila, and further suggest a putative role of ClpP in virulence regulation.

Identification and characterization of novel ColE1-type, high-copy number plasmid mutants in Legionella pneumophila.

ColE1-type plasmids are commonly used in bacterial genetics research, and replication of these plasmids is regulated by interaction of RNA I and RNA II. Although these plasmids are narrow-host-range, they can be maintained in Legionella pneumophila under antibiotic selection, with low-copy number and instability. Here, we have described the isolation of two novel spontaneous mutants of pBC(gfp)Pmip, pBG307 and pBG309, which are able to mark the L. pneumophila with strong green fluorescence when exposed to visible light. One of the mutants, pBG307, has a single CG-->TA mutation in RNA II promoter located 2-bases upstream the - 10 region. Another one, pBG309, has the same mutation, as well as an additional CG-->AT mutation in the 76th nucleotide of RNA I, or in the 6th nucleotide of RNA II. A plasmid with the single mutation in RNA I, pBG308, was also constructed. Characterization of these plasmids carrying the enhanced green fluorescent protein (gfpmut2) gene revealed that the green fluorescence intensities of these plasmids were 2- to 30-fold higher than that of the wild type and both of the mutations contribute to increase the plasmid copy number and/or plasmid stability. The mutation located in RNA II promoter played a more dominant role in elevating the copy number, compared to the mutation in RNA I. We also tested the mutant plasmids for replication in Escherichia coli, and found that their copy number and stability were dramatically decreased, except pBG307. Our data suggest that these plasmids might be useful and convenient in genetic studies in L. pneumophila.