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During the workshop, the short lectures of several research groups were presented. The report covers both the presentations and subsequent discussion session.
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The negative stranded RNA virus viral haemorrhagic septicaemia virus (VHSV) is an important disease-causing agent in aquacultured fish and internationally harmonized diagnostic procedures are continuously under development. The pr...
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The negative stranded RNA virus viral haemorrhagic septicaemia virus (VHSV) is an important disease-causing agent in aquacultured fish and internationally harmonized diagnostic procedures are continuously under development. The present study concerns the suitability of genotyping by sequencing of RT-PCR products for epidemiological analysis. Focus was put on a specific case story involving an acute outbreak of VHS in a Danish rainbow trout farm which otherwise had been free of VHSV during the previous 5 years. Tissue materials from individual fish were collected during routine inspection and the initial diagnosis was based on isolation of the virus by cell cultivation and subsequent identification by ELISA. Additional tissue samples were collected 25 days after the initial sampling. RT-PCR amplification and sequencing of the entire glycoprotein-gene (1524 nt) was performed on RNA purified from collected tissue material as well as from inoculated cell culture. No nucleotide substitutions where identified when aligning the obtained sequence data for the two sample types. The presented data indicate that the overall consensus sequence of the virus outbreak was stable during the survey, and that initial passage of the virus on BF-2 cells did not result in changes within the G-gene at a detectable level. The results suggest that genotyping of VHSV isolates based on RT-PCR products amplified from infected primary tissues material is a reliable tool for epidemiological studies.
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The study involved 30-135 g carp more than one year old, that had been cultured since the stage of summer fry at the Fisheries Research Station operated by the Agricultural University's Department of Aquaculture, University of Szc...
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The study involved 30-135 g carp more than one year old, that had been cultured since the stage of summer fry at the Fisheries Research Station operated by the Agricultural University's Department of Aquaculture, University of Szczecin and located in a Dolna Odra power station cooling water canal. The fish selected for analyses showed the following clinical signs in summer: apathy; strong necrotic patches on gills; lustreless and rough skin with numerous deep necrotic spots extending down to the muscles; deposits of thick mucus under the gill covers. On the 4th of June 2004, three carp samples of 15 individuals each were delivered live to the German National Reference Laboratory Insel Riems for analyses. Koi herpesvirus was detected in two out of the three samples using different PCR assays. The PCR results were confirmed by nested PCR and in situ hybridization. Assays were performed on gills, brain, and kidney tissues. Samples were also taken from outbreak survivors showing no clinical signs of disease in autumn 2004 and tested by PCR and nested PCR. These results were also confirmed by in situ hybridization using different probes. This is the first detection, virus isolation and confirmation of KHV in Poland.
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In order to establish a rapid and sensitive LAMP visual detection method for Potato virus Y on-site, this study used the conserved fragment of PVY CP gene sequence as a template to design primers, and the rapid visual LAMP detecti...
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In order to establish a rapid and sensitive LAMP visual detection method for Potato virus Y on-site, this study used the conserved fragment of PVY CP gene sequence as a template to design primers, and the rapid visual LAMP detection method of Potato virus Y was successfully established by optimizing the reaction system components (concentration ratio of internal and external primers, and concentrations of loop primers, Bst DNA, Mg2+, dNTP and betaine) and reaction conditions (temperature). The established rapid visual LAMP detection method for Potato virus Y can specifically identify Potato virus Y, providing complete visual detection within 40 min, and the detection limit can reach 4.34 pg & mu;L-1 (cDNA) concentration, which is 100 times more sensitive than PCR. An analysis of 50 potato field samples showed that the results of rapid visualization LAMP and RT-PCR were basically the same. This method is suitable for the rapid detection of Potato virus Y on-site, and this study can also provide theoretical reference for the early diagnosis of other potato viruses.
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In spite of the establishment of modern techniques like PCR for virus detection, virus isolations in susceptible fish cell lines are still the basis for international certification of most notifiable viral fish diseases mentioned ...
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In spite of the establishment of modern techniques like PCR for virus detection, virus isolations in susceptible fish cell lines are still the basis for international certification of most notifiable viral fish diseases mentioned by the OIE and the European Union (OIE, 2000; European Commission, 1996). Cell cultures may change in susceptibility (Lorenzen et al., 1999, unpublished own findings,). If no regular susceptibility checks are done in the laboratory, using an alternative sensitive cell culture might therefore increase the chance of isolating a certain virus. The CCB (common carp brain) cell line, developed by and cultured according to Neukirch et al., 1999, was originally developed for the diagnosis of koi herpesvirus (KHV). Moreover, we isolated other viruses from diseased koi in CCB cells. Besides KHV, myxoviruses, birna- and/or reoviruses (not all isolates were identified) and rhabdoviruses (spring viraemia of carp virus, SVCV) were obtained (Neukirch Kunz, 2001). The susceptibility of CCB cells was also investigated for viruses isolated from golden ide, European eel and salmonids. CCB cells and the standard cell lines used in our laboratory: Epithelioma papulosum cyprinid, EPC (Tomasec and Fijan, 1971), Chinook Salmon Embryo, CHSE (Fryer et al., 1965) and Eel kidney, EK-1 (Chen et al., 1982) for the different viruses were inoculated with the following viruses: infectious pancreatic necrosis virus (IPNV, strains Sp, Ab and Ni), viral haemorrhagic septicaemia virus (VHSV, serotype I, II and III), infectious haematopoietic necrosis virus (IHNV), golden ide reovirus (GiRV), chum salmon reovirus (CSV), herpesvirus anguillae (HVA), and another eel virus designated A sub(1)B virus. Non-infected cell lines were incubated as negative controls. Two passages were carried out in the cell lines before quantification of virus infectivity by titration of supernatants in 10-fold dilution steps (Mayr et al., 1977). Table 1 summarizes the inoculated viruses used, and results concerning titration of virus infectivity obtained in both the routine cell lines and the CCB cell line.
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Clustered Regularly Interspaced Short Palindromic Repeats (CRISPRs) together with CRISPR-associated (Cas) proteins have catalysed a revolution in genetic engineering. Native CRISPR-Cas systems exist in many bacteria and archaea wh...
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Clustered Regularly Interspaced Short Palindromic Repeats (CRISPRs) together with CRISPR-associated (Cas) proteins have catalysed a revolution in genetic engineering. Native CRISPR-Cas systems exist in many bacteria and archaea where they provide an adaptive immune response through sequence-specific degradation of an invading pathogen's genome. This system has been reconfigured for use in genome editing, drug development, gene expression regulation, diagnostics, the prevention and treatment of cancers, and the treatment of genetic and infectious diseases. In recent years, CRISPR-Cas systems have been used in the diagnosis and control of viral diseases, for example, CRISPR-Cas12/13 coupled with new amplification techniques to improve the specificity of sequence-specific fluorescent probe detection. Importantly, CRISPR applications are both sensitive and specific and usually only require commonly available lab equipment. Unlike the canonical Cas9 which is guided to double-stranded DNA sites of interest, Cas13 systems target RNA sequences and thus can be employed in strategies directed against RNA viruses or for transcriptional silencing. Many challenges remain for these approach, including issues with specificity and the requirement for better mammalian delivery systems. In this review, we summarize the applications of CRISPR-Cas systems in controlling mammalian viral infections. Following necessary improvements, it is expected that CRISPR-Cas systems will be used effectively for such applications in the future.
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A dot-ELISA test kit, developed for detecting Newcastle disease virus, was composed for specificity and sensitivity with haemagglutination and haemagglutination inhibition tests; agreement with these standard procedures was 98.28%...
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A dot-ELISA test kit, developed for detecting Newcastle disease virus, was composed for specificity and sensitivity with haemagglutination and haemagglutination inhibition tests; agreement with these standard procedures was 98.28%. Of the 258 samples of tissues (brain, spleen, trachea, proventriculus, ileocaecal tonsil) and intestinal contents tested from birds suspected of infection, 174 (67.4%) were positive by the dot-ELISA. This rapid, inexpensive test kit was considered suitable for field diagnosis of Newcastle disease.
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The objectives were to vaccinate peri-pubertal bulls with a modified-live vaccine consisting of cytopathic BVDV strains Singer and 296 and evaluate the resulting: (a) transient shed of modified-live, cytopathic BVDV in semen; (b) ...
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The objectives were to vaccinate peri-pubertal bulls with a modified-live vaccine consisting of cytopathic BVDV strains Singer and 296 and evaluate the resulting: (a) transient shed of modified-live, cytopathic BVDV in semen; (b) risk of prolonged testicular infection; and (c) protection against subsequent testicular infection due to viral challenge. Seronegative, peri-pubertal bulls were vaccinated subcutaneously with a standard dose of vaccine (n =11) or were maintained as unvaccinated controls (n =11). Forty-nine days after vaccination, all bulls were intranasally inoculated with a noncytopathic field strain of BVDV. Semen and testicular biopsies collected after vaccination and challenge were assayed for BVDV using virus isolation, reverse transcription-nested PCR, or immunohistochemistry, and the identity of viral strains was determined by nucleotide sequencing of PCR products. Vaccination of peri-pubertal bulls with this vaccine caused a short-term, transient shed of only the type 1a strain of modified-live, cytopathic BVDV in semen for up to 10d after vaccination. The vaccine did not cause prolonged testicular infection. Vaccination with this product prevented development of prolonged testicular infections after subsequent exposure to a field strain of BVDV.
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The objectives were to vaccinate peri-pubertal bulls with a modified-live vaccine consisting of cytopathic BVDV strains Singer and 296 and evaluate the resulting: (a) transient shed of modified-live, cytopathic BVDV in semen; (b) ...
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The objectives were to vaccinate peri-pubertal bulls with a modified-live vaccine consisting of cytopathic BVDV strains Singer and 296 and evaluate the resulting: (a) transient shed of modified-live, cytopathic BVDV in semen; (b) risk of prolonged testicular infection; and (c) protection against subsequent testicular infection due to viral challenge. Seronegative, peri-pubertal bulls were vaccinated subcutaneously with a standard dose of vaccine (n =11) or were maintained as unvaccinated controls (n =11). Forty-nine days after vaccination, all bulls were intranasally inoculated with a noncytopathic field strain of BVDV. Semen and testicular biopsies collected after vaccination and challenge were assayed for BVDV using virus isolation, reverse transcription-nested PCR, or immunohistochemistry, and the identity of viral strains was determined by nucleotide sequencing of PCR products. Vaccination of peri-pubertal bulls with this vaccine caused a short-term, transient shed of only the type 1a strain of modified-live, cytopathic BVDV in semen for up to 10d after vaccination. The vaccine did not cause prolonged testicular infection. Vaccination with this product prevented development of prolonged testicular infections after subsequent exposure to a field strain of BVDV.
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