Routine detection of equine arteritis virus (EAV) can be achieved by virus isolation (VI) in cell culture, or by the amplification of viral genome by molecular methods. To simplify molecular ...diagnosis, a number of different Reverse Transcriptase Polymerase Chain Reaction (RT-PCR) and RT-nested PCR (RT-nPCR) assays were compared, and a one-tube method was developed and optimised utilizing a fluorogenic probe (TaqMan). An artificial RNA template (Mimic) and associated probe were also constructed to provide in-tube validation of the RT-PCR system. To assess the utility of the RT-PCR TaqMan assay, 28 different isolates of EAV representing different genetic groups of American and European strains were tested. Furthermore, the ability of VI and RT-PCR TaqMan assay to detect EAV in different biological matrices such as semen, nasal and faecal swabs and blood was compared. All 28 EAV strains were detected by the RT-PCR TaqMan assay. The results of TaqMan and VI testing were in agreement for 30 of the 33 semen samples and all of the 50 other clinical specimens examined: the RT-PCR TaqMan assay detected 18 positive semen samples, three more than VI. In conclusion, the one-tube RT-PCR TaqMan assay is a rapid, reliable method for the detection of EAV.
ABSTRACT
We show that cells infected with the pestivirus classical swine fever virus (CSFV) fail to produce alpha/beta interferon not only following treatment with double-stranded RNA but also after ...superinfection with a heterologous virus, the alphavirus Sindbis virus, a virus shown to normally induce interferon. We investigated whether the inhibition of interferon synthesis by CSFV involved a block in interferon regulatory factor 3 (IRF3) activity. Cells infected with CSFV exhibited a lack of translocation of green fluorescent protein-IRF3 to the nucleus; however, constitutive shuttling of IRF3 was not blocked, since it could still accumulate in the nucleus in the presence of leptomycin B. Interestingly subcellular fractionation analysis showed that IRF3 was lost from the cytoplasm of infected cells from 18 h postinfection onwards. Using IRF3 promoter-luciferase reporter constructs, we demonstrate that loss of IRF3 was due to an inhibition of transcription of the IRF3 gene in CSFV-infected cells. Further, we investigated which viral protein may be responsible for the inhibition of interferon and loss of IRF3. We used cell lines expressing the CSFV N-terminal protease (N
pro
) to show that this single viral protein, unique to pestiviruses, inhibited interferon production in response to Sindbis virus. In addition to being lost from CSFV-infected cells, IRF3 was lost from N
pro
-expressing cells. The results demonstrate a novel viral evasion of innate host defenses, where interferon synthesis is prevented by inhibiting transcription of IRF3 in CSFV-infected cells.
Accounts of field disease and experimental studies involving porcine reproductive and respiratory syndrome (PRRS) are reviewed for evidence of immunomodulation or immunosuppression by the causative ...virus. The conclusion is that immunomodulation through infection of alveolar macrophages is likely to occur, but that it is transient and at a local level, in the lung. There is some evidence for more subtle effects via more disseminated replication or induction of apoptosis with some isolates, but more definitive studies are needed. There is some emerging evidence of interaction between PRRSV and different cells of the immune system, but its significance for the course of disease or pig health are unclear. Likewise, the current experimental evidence for any interaction of PRRSV with other pathogens is ambiguous and therefore no firm conclusions can yet be drawn. Strains of PRRSV do vary in pathogenicity, which may be related to their degree of ability to cause overt respiratory disease in the absence of other agents. Experimentally, varying degrees of interstitial pneumonia are a common histological finding. There is, as yet, no firm evidence of general immunosuppression--in fact, some contrary evidence exists in the form of observations of a transient enhancement of humoral response, possibly through polyclonal B cell activation. The basis of pathogenicity of PRRSV and of any interaction with other agents is still unknown and is likely to remain unclear. Virus interaction with the pig's immune system must be addressed before any assessment of virulence of any known or emergent strains of PRRSV can be made.
A single-step, multiplex, real-time polymerase chain reaction (RT-PCR) was developed for the simultaneous and differential laboratory diagnosis of Classical swine fever virus (CSFV) and African swine ...fever virus (ASFV) alongside an exogenous internal control RNA (IC-RNA). Combining a single extraction methodology and primer and probe sets for detection of the three target nucleic acids CSFV, ASFV and IC-RNA, had no effect on the analytical sensitivity of the assay and the new triplex RT-PCR was comparable to standard PCR techniques for CSFV and ASFV diagnosis. After optimisation the assay had a detection limit of 5 CSFV genome copies and 22 ASFV genome copies. Analytical specificity of the triplex assay was validated using a panel of viruses representing 9 of the 11 CSFV subgenotypes, at least 8 of the 22 ASFV genotypes as well as non-CSFV pestiviruses. Positive and negative clinical samples from animals infected experimentally, due to field exposure or collected from the UK which is free from both swine diseases, were used to evaluate the diagnostic sensitivity and specificity for detection of both viruses. The diagnostic sensitivity was 100% for both viruses whilst diagnostic specificity estimates were 100% for CSFV detection and 97.3% for ASFV detection. The inclusion of a heterologous internal control allowed identification of false negative results, which occurred at a higher level than expected. The triplex assay described here offers a valuable new tool for the differential detection of the causative viruses of two clinically indistinguishable porcine diseases, whose geographical occurrence is increasingly overlapping.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
1 CVL (Weybridge), New Haw, Addlestone, KT15 3NB, UK
and 2 ID-DLO, Houtribweg 39, PO Box 365, NL-8200 AB Lelystad, The Netherlands
This report describes the preparation of six monoclonal antibodies ...(MAbs) raised against a British isolate of porcine reproductive and respiratory syndrome virus (PRRSV), their characterization in terms of protein specificity and their reactivity with different PRRS viruses from Europe and the USA. Radioimmunoprecipitation and Western blotting studies of MAb reactivity with proteins from cell lysates of infected cells and purified virus revealed that four of the six MAbs (WBE1 and WBE46) precipitated a 15 kDa viral protein. Further studies using in vitro translated products of the Lelystad virus genome showed that this protein was the product of ORF7, the putative nucleocapsid protein. The specificity of another MAb, WBE2, was found to be for a 45 kDa protein, determined to be the product of ORF3 and demonstrated to be present in purified virion preparations. The protein specificity of the sixth MAb, WBE3 could not be determined. Thirty-three PRRSV isolates from Europe and the USA were grown in alveolar macrophages and examined by immunoperoxidase staining, using the panel of six MAbs. All European isolates were recognized by the four MAbs specific for the putative nucleocapsid, but the viruses showed different patterns of reactivity with WBE2 and WBE3. Furthermore, these two MAbs stained only a small proportion of the cells infected with certain isolates, suggesting that a single isolate may be antigenically heterogeneous. No MAbs bound to US isolates, indicating a consistent antigenic difference between the putative nucleocapsid of US and European isolates. Detergent extraction of cell lysate antigen abrogated the binding of WBE13, suggesting that the epitopes are conformation dependent.
* Author for correspondence. Fax +44 1932 347046.
Received 16 September 1994;
accepted 27 January 1995.
We show that cells infected with the pestivirus classical swine fever virus (CSFV) fail to produce alpha/beta interferon not only following treatment with double-stranded RNA but also after ...superinfection with a heterologous virus, the alphavirus Sindbis virus, a virus shown to normally induce interferon. We investigated whether the inhibition of interferon synthesis by CSFV involved a block in interferon regulatory factor 3 (IRF3) activity. Cells infected with CSFV exhibited a lack of translocation of green fluorescent protein-IRF3 to the nucleus; however, constitutive shuttling of IRF3 was not blocked, since it could still accumulate in the nucleus in the presence of leptomycin B. Interestingly subcellular fractionation analysis showed that IRF3 was lost from the cytoplasm of infected cells from 18 h postinfection onwards. Using IRF3 promoter-luciferase reporter constructs, we demonstrate that loss of IRF3 was due to an inhibition of transcription of the IRF3 gene in CSFV-infected cells. Further, we investigated which viral protein may be responsible for the inhibition of interferon and loss of IRF3. We used cell lines expressing the CSFV N-terminal protease (N super(pro)) to show that this single viral protein, unique to pestiviruses, inhibited interferon production in response to Sindbis virus. In addition to being lost from CSFV-infected cells, IRF3 was lost from N super(pro)-expressing cells. The results demonstrate a novel viral evasion of innate host defenses, where interferon synthesis is prevented by inhibiting transcription of IRF3 in CSFV-infected cells.
Previous studies using monoclonal antibodies (mAbs) have revealed antigenic variation among UK isolates of porcine reproductive and respiratory syndrome viruses (PRRSV) and the use of in vitro ...translation products has shown that this variation lies in the protein encoded by open reading frame (ORF) 3. This protein has been shown to be present in purified virion preparations, suggesting that it is a structural protein. The original objective was to investigate the degree of variation of ORF3 among a number of UK isolates of different mAb reactivity and diverse chronology by sequencing and to correlate this with the mAb reactivity, in an attempt to define conserved and variable antigenic sites. A number of PRRSV isolates, from different outbreaks in the UK between 1991 and 1994, were propagated in pig alveolar macrophages and RNA extracted. The ORF3 and ORF7 regions of the individual viruses were amplified by the polymerase chain reaction (PCR) and their sequences were determined using internal sense and antisense primers. A number of differences among the sequences were noted within specific regions of the ORF3, with a hypervariable area detected at the carboxyterminal end, in the area of overlap with ORF4. With the most divergent isolate, 9.5% of the 84 translated amino acids encoded by the area of overlap were different from Lelystad isolate, translating the sequence in both reading frames. In view of consistent changes elsewhere in the ORF that suggest a common ancestry among the isolates studied, we conclude that this region may be subject to rapid change in comparison to other regions studied, and therefore may be an area subjected to immunoselective pressure.
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