291 research outputs found
Constitutive expression of Atlantic salmon Mx1 protein in CHSE-214 cells confers resistance to Infectious Salmon Anaemia virus
No full textAbstract Infectious salmon anaemia (ISA) is a highly fatal viral disease affecting marine-farmed Atlantic salmon which is caused by ISA virus (ISAV), a fish orthomyxovirus that has recently been assigned to the new genus Isavirus within the family Orthomyxoviridae. Mx proteins are among the interferon (IFN)-induced proteins responsible for the development of an antiviral state in vertebrate cells. We used real-time reverse transcription-polymerase chain reaction (RT-PCR) and Chinook salmon embryo (CHSE-214) cells constitutively expressing Atlantic salmon Mx1 protein (ASMx1) to examine the antiviral properties of ASMx1 against two ISAV strains, NBISA01 and HKS-36, having phenotypically different growth properties (cytopathic vs non-cytopathic) in the CHSE-214 cell line. We present evidence that ISAV is sensitive to ASMx1. CHSE-214 cells constitutively expressing ASMx1 showed increased resistance to infection with the cytopathic ISAV strain NBISA01, manifested as delayed development of cytopathic effects (CPE) and significant reduction in the severity of CPE, as well as a 10-fold reduction in virus yield. However, by real-time RT-PCR we observed no significant difference in the mean threshold cycle (Ct) values of ISAV RNA levels, suggesting that the ASMx1 activity on ISAV occurs at the post-transcription steps of virus replication, possibly in the cytoplasm.</p
Identification of the 3' and 5' terminal sequences of the 8 rna genome segments of european and north american genotypes of infectious salmon anemia virus (an orthomyxovirus) and evidence for quasispecies based on the non-coding sequences of transcripts
No full textAbstract Background Infectious salmon anemia (ISA) virus (ISAV) is a pathogen of marine-farmed Atlantic salmon (Salmo salar); a disease first diagnosed in Norway in 1984. This virus, which was first characterized following its isolation in cell culture in 1995, belongs to the family Orthomyxoviridae, genus, Isavirus. The Isavirus genome consists of eight single-stranded RNA segments of negative sense, each with one to three open reading frames flanked by 3' and 5' non-coding regions (NCRs). Although the terminal sequences of other members of the family Orthomyxoviridae such as Influenzavirus A have been extensively analyzed, those of Isavirus remain largely unknown, and the few reported are from different ISAV strains and on different ends of the different RNA segments. This paper describes a comprehensive analysis of the 3' and 5' end sequences of the eight RNA segments of ISAV of both European and North American genotypes, and evidence of quasispecies of ISAV based on sequence variation in the untranslated regions (UTRs) of transcripts. Results Two different ISAV strains and two different RNA preparations were used in this study. ISAV strain ADL-PM 3205 ISAV-07 (ADL-ISAV-07) of European genotype was the source of total RNA extracted from ISAV-infected TO cells, which contained both viral mRNA and cRNA. ISAV strain NBISA01 of North American genotype was the source of vRNA extracted from purified virus. The NCRs of each segment were identified by sequencing cDNA prepared by three different methods, 5' RACE (Rapid amplification of cDNA ends), 3' RACE, and RNA ligation mediated PCR. Sequence analysis of five clones each derived from one RT-PCR product from each NCR of ISAV transcripts of segments 1 to 8 revealed significant heterogeneity among the clones of the same segment end, providing unequivocal evidence for presence of intra-segment ISAV quasispecies. Both RNA preparations (mRNA/cRNA and vRNA) yielded complementary sequence information, allowing the simultaneous identification and confirmation of the 3' and 5' NCR sequences of the 8 RNA genome segments of both genotypes of ISAV. The 3' sequences of the mRNA transcripts of ADL-ISAV-07 terminated 13-18 nucleotides from the full 3' terminus of cRNA, continuing as a poly(A) tail, which corresponded with the location of the polyadenylation signal. The lengths of the 3' and 5' NCRs of the vRNA were variable in the different genome segments, but the terminal 7 and 11 nucleotides of the 3' and 5' ends, respectively, were highly conserved among the eight genomic segments of ISAV. The first three nucleotides at the 3' end are GCU-3' (except in segment 5 with ACU-3'), whereas at the 5' end are 5'-AGU with the polyadenylation signal of 3-5 uridines 13-15 nucleotides downstream of the 5' end terminus of the vRNA. Exactly the same features were found in the respective complementary 5' and 3' end NCR sequences of the cRNA transcripts of ADL-ISAV-07, indicating that the terminal sequences of the 8 RNA genome segments are highly conserved among the two ISAV genotypes. The 5' NCR sequences of segments 1, 2, 3, 5, and 7, and the 3' NCR sequences of segments 3 and 4 cRNA were 100% identical in the two genotypes, and the 3' NCR sequences of segment 5 cRNA was the most divergent, with a sequence identity of 77.2%. Conclusions We report for the first time, the presence of intra-segment ISAV quasispecies, based on sequence variation in the NCR sequences of transcripts. In addition, this is the first report of a comprehensive unambiguous analysis of the 3' and 5' NCR sequences of all 8 RNA genome segments from two strains of ISAV representing the two genotypes of ISAV. Because most ISAV sequences are of cDNA to mRNA, they do not contain the 3' end sequences, which are removed during polyadenylation of the mRNA transcripts. We report for the first time the ISAV consensus sequence CAT/ATTTTTACT-3' (in the message sense 5'-3') in all segments of both ISAV genotypes.</p
Characterization of gene expression on genomic segment 7 of infectious salmon anaemia virus
No full textAbstract Background Infectious salmon anaemia (ISA) virus (ISAV), an important pathogen of fish that causes disease accompanied by high mortality in marine-farmed Atlantic salmon, is the only species in the genus Isavirus, one of the five genera of the Orthomyxoviridae family. The Isavirus genome consists of eight single-stranded RNA species, and the virions have two surface glycoproteins; haemagglutinin-esterase (HE) protein encoded on segment 6 and fusion (F) protein encoded on segment 5. Based on the initial demonstration of two 5'-coterminal mRNA transcripts by RT-PCR, ISAV genomic segment 7 was suggested to share a similar coding strategy with segment 7 of influenza A virus, encoding two proteins. However, there appears to be confusion as to the protein sizes predicted from the two open reading frames (ORFs) of ISAV segment 7 which has in turn led to confusion of the predicted protein functions. The primary goal of the present work was to clone and express these two ORFs in order to assess whether the predicted protein sizes match those of the expressed proteins so as to clarify the coding assignments, and thereby identify any additional structural proteins of ISAV. Results In the present study we show that ISAV segment 7 encodes 3 proteins with estimated molecular masses of 32, 18, and 9.5 kDa. The 18-kDa and 9.5-kDa products are based on removal of an intron each from the primary transcript (7-ORF1) so that the translation continues in the +2 and +3 reading frames, respectively. The segment 7-ORF1/3 product is variably truncated in the sequence of ISAV isolates of the European genotype. All three proteins are recognized by rabbit antiserum against the 32-kDa product of the primary transcript, as they all share the N-terminal 22 amino acids. This antiserum detected a single 35-kDa protein in Western blots of purified virus, and immunoprecipitated a 32-kDa protein in ISAV-infected TO cells. Immunofluorescence staining of infected cells with the same antiserum revealed the protein(s) to be localized in the cytoplasm. Vaccination of farmed Atlantic salmon with the 32-kDa protein resulted in a higher survival rate than what was attainable with the HE protein, albeit a moderate protection against the low ISAV challenge. Conclusion Collectively, our observations suggest that the product of ISAV segment 7 primary transcript (7-ORF1) is a structural protein. The 18-kDa (7-ORF1/2) protein is identified as the putative ISAV nuclear export protein based on the presence of nuclear export signals. The function of the 9.5-kDa (7-ORF1/3) protein is not presently known.</p
Rapid passage of avian reovirus in one-day-old chicks: clinical and virological findings
No full textTwo avian reoviruses, strain Reo-25 and isolate W3-492 were given by mouth to eight one-day-old chicks. 3-7 days after inoculation, the liver, spleen, pancreas, caecal tonsil and duodenum were collected, weighed and titrated in cell culture for viral content. Tissue homogenates were passaged several times in day-old chicks. Reo-25 virus was passaged at 3-day and W3-492 virus at 3- and 7- or 14-day intervals. For both Reo-25 and W3-492 viruses, pathological effects and virus yields in tissues decreased with continued passages. In direct comparisons of reovirus W3-492 before chick passage (PO) and after four passages at 7-day intervals (P4) using standardised amounts of virus for inoculation of chickens, no major differences in pathological effects were observed. P4 virus could be recovered from duodenal tissue at 28 days and from liver tissue at 14 days. PO virus could be recovered from duodenal tissue at 14 days and from liver tissue at 10 days..RE: 25 ref.; SC: ZA; CA; VE; 0V; 7A; 0ISource type: Electronic(1) http://upei-resolver.asin-risa.ca?sid=SP:CABI&id=pmid:&id=&issn=0307-9457&isbn=&volume=16&issue=2&spage=213&pages=213-225&date=1987&title=Avian%20Pathology&atitle=Rapid%20passage%20of%20avian%20reovirus%20in%20one-day-old%20chicks%3a%20clinical%20and%20virological%20findings.&aulast=Kibenge&pid=%3Cauthor%3EKibenge%2c%20F%20S%20B%3bDhillon%2c%20A%20S%3C%2Fauthor%3E%3CAN%3E19872296778%3C%2FAN%3E%3CDT%3EJournal%20article%3C%2FDT%3
Bulletin of the Aquaculture Association of Canada 104 2 71 78 St. Andrews, Canada: Aquaculture Association of Canada.
No full textInfectious salmon anaemia (ISA) virus (ISAV) is currently one of the most important viral pathogens threatening commercial aquaculture in the northern hemisphere. The virus is classified in the family Orthomyxoviridae, genus Isavirus. The virus offers a significant challenge to fish biologists interested in diagnosis and control of ISA, and to molecular virologists because of its evolutionary relationship with influenza viruses. Being an orthomyxovirus, ISAV is expected to be prone to genetic and antigenic variation. However, while the ISAV genome, like the influenza A virus genome, encodes at least nine structural proteins, the gene order and putative functional identities of the ISAV proteins are significantly different from those of influenza viruses. Although the virulence of influenza A viruses is a polygenic trait, one virulence factor is correlated with the haemagglutinin cleavage site. It remains to be shown which genetic variations in ISAV are associated with the virulence of the virus. The ability of ISAV to kill rainbow trout is a correlate of ISAV pathogenicity that might facilitate the identification of ISAV virulence genes.
Identification of serotype II infectious bursal disease virus proteins
No full textPolyacrylamide gel electrophoresis (PAGE) of serotype II IBDV (OH and MO strains) purified from infected Vero cells resolved a previously undetected major viral polypeptide, VP2. The molecular weight (MW) of VP2 was different between the two strains of serotype II. It was 43.5 kDa in strain OH and 44 kDa in strain MO. This was higher than the MW of VP2 in SAL strain of serotype I IBDV which was 41 kDa. VPX (50 kDa), VP3 (33 kDa) and VP4 (30.5 kDa) were similar in both serotype II virus strains but were also of higher MW than VPX (48 kDa), VP3 (32 kDa) and VP4 (30 kDa) of SAL virus. VP1 (80 kDa) had the same MW in both serotypes..RE: 25 ref.; SC: ZA; CA; VE; 0V; 7A; 0ISource type: Electronic(1) http://upei-resolver.asin-risa.ca?sid=SP:CABI&id=pmid:&id=&issn=0307-9457&isbn=&volume=17&issue=3&spage=679&pages=679-687&date=1988&title=Avian%20Pathology&atitle=Identification%20of%20serotype%20II%20infectious%20bursal%20disease%20virus%20proteins.&aulast=Kibenge&pid=%3Cauthor%3EKibenge%2c%20F%20S%20B%3bDhillon%2c%20A%20S%3bRussell%2c%20R%20G%3C%2Fauthor%3E%3CAN%3E19882212161%3C%2FAN%3E%3CDT%3EJournal%20article%3C%2FDT%3
Pathogenicity of four strains of staphylococci isolated from chickens with clinical tenosynovitis
No full textFour strains of staphylococci of different phage patterns (two principal and two minor phage types) associated with naturally occurring tenosynovitis were studied for virulence in chickens and tropism for tendon tissue. There were significant differences in virulence between the principal and minor phage types of Staphylococcus aureus. Infection with the principal phage types produced a generalized septicaemic disease and no specific tropism of the bacteria for tendon tissue although there were gross and histopathological changes in tendons and tendon sheaths indistinguishable from those in natural tenosynovitis. However, the pathogenesis of the experimental infections was different from that considered to occur in natural cases and provided further indirect evidence for the secondary nature of the staphylococcal infection in tenosynovitis..RE: 26 ref.; SC: ZA; CA; VE; 0V; 7A; 0ISource type: Electronic(1) http://upei-resolver.asin-risa.ca?sid=SP:CABI&id=pmid:&id=&issn=0307-9457&isbn=&volume=12&issue=2&spage=213&pages=213-220&date=1983&title=Avian%20Pathology&atitle=Pathogenicity%20of%20four%20strains%20of%20staphylococci%20isolated%20from%20chickens%20with%20clinical%20tenosynovitis.&aulast=Kibenge&pid=%3Cauthor%3EKibenge%2c%20F%20S%20B%3bWilcox%2c%20G%20E%3bPass%2c%20D%20A%3C%2Fauthor%3E%3CAN%3E19842232875%3C%2FAN%3E%3CDT%3EJournal%20article%3C%2FDT%3
Avian Diseases 31 3 654 657 UNITED STATES
No full textThe lesions and etiologic agents associated with 13 outbreaks of respiratory disease in commercial chickens were investigated. Adenoviruses were isolated from tracheal and lung tissues of affected chickens in all 13 outbreaks. Escherichia coli was isolated from the lung of an occasional bird. The tracheal specimens were consistently negative for Bordetella avium, but E. coli and occasionally Staphylococcus aureus were isolated. There was also serological evidence in one outbreak, and pathological evidence in another, of a concurrent infectious bursal disease virus (IBDV) infection of chickens affected with the disease. Gross and microscopic alterations in the tracheas and lungs of affected chickens were similar in all outbreaks and consisted of catarrhal tracheitis and occasionally multifocal pneumonia with mononuclear cell infiltrates. Hepatitis and splenitis with heterophil infiltrates occasionally were seen in birds with coliform septicemia. The tracheal and lung lesions in the present investigation were considered primarily of adenovirus etiology, complicated by secondary bacterial infection
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