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Fisheries Research Services Virus Diagnostics at FRS Marine Laboratory Introduction Fisheries Research Services (FRS) staff carry out diagnostic identified by an immunofluorescent antibody test (IFAT) and surveillance tests for five types of virus: infectious using a specific antibody (Fig. 2). Negative screening salmon anaemia (ISA), viral haemorrhagic septicaemia normally takes 28 days. (VHS), infectious haematopoietic necrosis (IHN), infectious pancreatic necrosis (IPN) and spring viraemia of carp (SVC). The group also responds to requests for testing for other viral pathogens, for example, nodavirus, alphaviruses and aquareoviruses. Virus Testing Methods Used Infectious Salmon Anaemia (ISA) FRS staff sample heart, kidney, liver and spleen tissues from all fish for virus testing. ISA virus (ISAV) is isolated by cell culture using the established fish cell lines salmon Figure 2 ISAV infected TO cells showing positive staining by IFAT. head kidney (SHK-1) or a leucocyte line (TO). Cell cultures Other techniques are also used to provide evidence of are observed for cytopathic effect (CPE). Since ISAV may ISAV infection which include staining for viral antigen in not give rise to CPE in infected cells, an additional kidney imprints (Fig. 3) and identification of virus specific detection method for intra-cellular virus is also used. ISAV nucleic acid sequences. is a virus which possesses a surface glyco-protein haemagglutinin to which red blood cells will adhere. This property can be exploited by overlaying the test cell cultures with salmon or trout red blood cells. Red cell clusters are observed which reveal centres of virus infection (Fig. 1). Virus associated with infected cells is Figure 3 Highly stained kidney imprint showing severe ISAV infection in salmon. Viral Haemorrhagic Septicaemia (VHS) and Infectious Heamatopoietic Necrosis (IHN) Brain or heart, spleen and kidney from immature fish, Figure 1 Red blood cells cluster overlying ISAV infected SHK-1 cells. and gonadal fluids from mature fish are sampled in the course of statutory surveillance for VHS virus (VHSV) and Fisheries Research Services is an agency of the Scottish Executive FRS Marine Laboratory PO Box 101 375 Victoria Road Aberdeen tel +44 (0)1224 876544 fax +44 (0)1224 295511 [email protected] http://www.frs-scotland.gov.uk AB11 9DB UK Fisheries Research Services IHN virus (IHNV). Tests are carried out by cell culture Spring Viraemia of Carp (SVC) isolation on bluegill fibroblast (BF-2) or rainbow trout Brain, spleen and kidney tissues are sampled from all gonad (RTG-2) cells for VHSV and epithelioma papulosum sizes of fish for testing. SVC virus is detected by cell cyprini (EPC) or fathead minnow (FHM) cells for IHNV. culture isolation using EPC or FHM cells. Cell cultures are Microscopic examination of cell cultures for CPE is carried observed microscopically for CPE and virus identification out. is made by ELISA. The test normally takes 14 days for negative screening. Identification of both viruses is made by an enzyme-linked immunosorbent assay (ELISA). The test normally takes 14 days for negative screening. Viral Nervous Necrosis (VNN) Brain, spinal cord, spleen and kidney tissues are sampled from all sizes of fish for testing. The causal nodavirus Infectious Pancreatic Necrosis (IPN) Diagnostics may affect many different fish species including Atlantic In diagnostic testing, mid-sections are sampled from sac salmon, halibut and turbot. Nodavirus can be detected fry and small fry, for larger fish kidney tissue is sampledand by cell culture isolation on striped snakehead cells (SSN- for mature fish gonadal fluids have to be sampled. 1) or a clone of the SSN-1 cell line, named E-11. Cultures Detection of IPNV is carried out by cell culture isolation are observed microscopically for CPE over 28 days. on chinook salmon embryo (CHSE-214) cells. Cell cultures Nodavirus leads to partial CPE which is recognised by are observed microscopically for CPE (Fig. 4). Identification trained readers. Virus identification is carried out by IFAT of IPNV from the positive cultures is carried out by ELISA. using a specific antiserum. Negative screening takes Negative screening normally takes 14 days. approximately 28 days. Sleeping Disease Virus (SDV) and Salmon Pancreas Disease Virus (SPDV) The two salmonid alphaviruses SDV and SPDV are genetically very closely related and regarded as types of the same genus. Kidney, heart, pancreas and serum are sampled for testing. Both viruses can be detected by cell culture isolation on CHSE-214 cells; however isolation is relatively difficult and slow. Careful microscopic observation of cultures is required to detect CPE which may only become evident after several passages. Virus Figure 4 IPNV infected CHSE-214 cells showing CPE. identification is carried out using PCR. Negative screening takes a minimum of 42 days. AAAH10|04|06 Fisheries Research Services is an agency of the Scottish Executive FRS Marine Laboratory PO Box 101 375 Victoria Road Aberdeen AB11 9DB UK tel +44 (0)1224 876544 fax +44 (0)1224 295511 [email protected] http://www.frs-scotland.gov.uk © Crown copyright Printed on Revive Silk, a recycled paper