Polymerase chain reaction-mediated cloning and in vitro translation of the genes coding for the structural proteins of hog cholera virus

Muyldermans, Gaëtan; Gabriel, M C San; Caij, Ann Brigitte; Smet, A. De; Hamers, R.

doi:10.1007/bf01309551

Cited by 10 publications

(6 citation statements)

References 15 publications

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“…In the present work, single nucleotide dif- (11,39), it cannot be excluded that some of the differences found between the clones analyzed were due to sequence heterogeneity in the original virus stock. Previous partial sequencing of the Alfort/ 187 genome in at least two laboratories had suggested that the nucleotide sequence published for Alfort Tübingen (25) was only distantly related to the sequence of Alfort/187 (14,32,37,38), a clone of the original Alfort strain (1,6). This is clearly confirmed by the sequence data presented in this work, which revealed an overall nucleotide sequence identity of 86.2% between the Alfort Tübingen sequence (25) and the Alfort/187 sequence.…”

Section: Discussionmentioning

confidence: 99%

“…On the basis of animal experiments performed at our institute and according to the criteria of van Oirschot (46), Alfort/187 must be classified as a moderate-virulent strain (our unpublished data). Data obtained previously from partial sequencing of the Alfort/187 genome (14,32,37,38) indicated that Alfort Tübingen and Alfort/187 were only distantly related strains.…”

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confidence: 95%

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Nucleotide sequence of classical swine fever virus strain Alfort/187 and transcription of infectious RNA from stably cloned full-length cDNA

Ruggli¹,

Tratschin²,

Mittelholzer³

et al. 1996

J Virol

192

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The complete nucleotide sequence of the genome of classical swine fever virus (CSFV) strain Alfort/187 was determined from three cDNA libraries constructed by cloning of DNA fragments obtained from independent sets of reverse transcription and PCR. The cDNA fragments were then assembled and inserted downstream of a T7 promoter in a P15A-derived plasmid vector to obtain the full-length cDNA clone pA187-1. The first nucleotide of the CSFV genome was positioned at the transcription start site of the T7 promoter. Cleavage at an SrfI restriction site introduced at the exact 3 end of the cloned viral cDNA allowed the in vitro synthesis of full-length viral RNA by runoff transcription. This RNA proved to be infectious after transfection into porcine kidney cells. Infectivity was not increased after capping of the synthetic RNA. Virus recovered from transfected cells was titrated in porcine kidney cells by endpoint dilution using indirect immunofluorescence and a CSFV-specific monoclonal antibody. RNA transcripts generated from plasmid DNA isolated from bacteria which had been cultured and cloned 10 times remained infectious, indicating that the full-length clone is stable in bacterial cells. A silent point mutation introduced at position 11842 of the genome was retained in the recombinant virus recovered from transfected cells. An infectious chimeric construct was obtained by replacing a 696-bp fragment in pA187-1 with the corresponding cDNA fragment from the CSFV strain CAP. The stably cloned full-length CSFV cDNA allows site-specific mutagenesis of the viral genome and thus will be useful for detailed molecular characterization of the virus as well as for studies of viral pathogenesis. MATERIALS AND METHODS Cells and viruses. Porcine kidney cell line PK-41 (IFFA-Mérieux, Lyon, France) was grown in Dulbecco's modified Eagle medium (DMEM) (Gibco BRL) containing 5% horse serum (Sebak GmbH, Aidenbach, Germany). The swine kidney cell line SK-6 (17), kindly provided by M.

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Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 95%

Nucleotide sequence of classical swine fever virus strain Alfort/187 and transcription of infectious RNA from stably cloned full-length cDNA

Ruggli¹,

Tratschin²,

Mittelholzer³

et al. 1996

J Virol

192

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“…In addition to the aforementioned published nucleic acid sequences the multiple alignment for the gp fragments included sequence from the Alfort 187 (Muyldermans et al, 1993) and the Weybridge (Yu et al, 1993) strains of CSFV. All the sequences showed a high level of conservation with variation occurring mainly at the wobble bases.…”

Section: Relationships Between Italian Csfvsmentioning

confidence: 99%

Classical swine fever: genetic detection and analysis of differences between virus isolates

Lowings

Paton

Sands

et al. 1994

Journal of General Virology

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“…Several PCRs for the detection of CSFV have been reported. 8,12,15 Of these, no PCR assay was applied to test semen samples for the presence of CSFV. The amount of CSFV nucleic acid present in semen appears to be low because it could only be detected after the seminested RT-PCR.…”

Section: Discussionmentioning

confidence: 99%

Detection of Classical Swine Fever Virus in Boar Semen by Reverse Transcription–Polymerase Chain Reaction

Choi

Chae

2003

J VET Diagn Invest

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Abstract.A seminested reverse transcription-polymerase chain reaction (RT-PCR) was developed for the detection of classical swine fever virus (CSFV) in semen. Five boars were inoculated intranasally with CSFV isolate propagated in PK15 cells. Two boars inoculated with the supernatant of noninfected PK15 cells were kept as controls. Semen and serum samples were collected twice weekly for 63 days postinoculation (dpi). Samples were tested for the presence of antibodies to CSFV by an enzyme-linked immunosorbent assay and for the presence of CSFV nucleic acid by seminested RT-PCR. Antibodies to CSFV could be detected as early as 7 dpi in 1 boar, and all 5 infected boars were found positive by 14 dpi. CSFV from boar semen was infrequently identified by virus isolation compared with seminested RT-PCR. CSFV nucleic acid was detected in semen by seminested RT-PCR as early as 7 dpi in 3 infected boars and persistently thereafter in all 5 infected boars until 63 dpi. When separated fractions of CSFV-contaminated semen were analyzed by the seminested RT-PCR, the CSFV nucleic acid was detected mainly in seminal fluid and occasionally in nonsperm cells. CSFV antigen was also detected in nonsperm cells from semen smear by immunohistochemistry. Thus, infection via semen, specially through CSFV-infected seminal fluid, seems to be a major route of transmission of CSFV.Classical swine fever virus (CSFV) is the causative agent of classical swine fever (CSF), an important disease of swine, causing significant economical losses in the swine industry all over the world. Classical swine fever virus, together with the structurally and serologically related bovine viral diarrhea virus (BVDV) of cattle and the Border disease virus (BDV) of sheep, belongs to the Pestivirus genus within the family Flaviviridae. 7 The disease is characterized by a peracute, acute, subacute, chronic, atypical, or inapparent course. 19,21,22 The peracute form is characterized by a rapid course without typical clinical signs suspect for CSF followed by sudden death. 22 Pathologic changes in acute and subacute CSF, which is a disease with high mortality, include multiple hemorrhages of various sizes (caused by necrosis of endothelial cells) in conjunction with defects in the blood coagulation mechanism. 18 Chronic CSF is a lethal disease with a duration of at least 30 days. 14 There is often little evidence of CSF in terms of petechial hemorrhages in most organ systems, but a single organ system (lung, gastrointestinal tract, and central nervous system) may predominantly be affected. 21 Although the primary route of CSFV transmission may be through contact spread, 22 recent investigations 3,6 indicated that infected boars can transmit CSFV in semen. Venereal transmission of viral diseases is a major concern in human and veterinary med- icine. In the swine industry in which artificial insemination (AI) is a common practice to improve the genetic pool, attention has been focused on disease, such as porcine reproductive and respiratory syndrome virus (PRRSV), 1 ps...

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Polymerase chain reaction-mediated cloning and in vitro translation of the genes coding for the structural proteins of hog cholera virus

Cited by 10 publications

References 15 publications

Nucleotide sequence of classical swine fever virus strain Alfort/187 and transcription of infectious RNA from stably cloned full-length cDNA

Nucleotide sequence of classical swine fever virus strain Alfort/187 and transcription of infectious RNA from stably cloned full-length cDNA

Classical swine fever: genetic detection and analysis of differences between virus isolates

Detection of Classical Swine Fever Virus in Boar Semen by Reverse Transcription–Polymerase Chain Reaction

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