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PubMed2003

Safety and protective efficacy of porcine reproductive and respiratory syndrome recombinant virus vaccines in young pigs.

Three porcine reproductive and respiratory syndrome virus (PRRSV) recombinants, generated by mutagenesis of an infectious cDNA clone of the Lelystad virus (LV) isolate, were tested for their safety and protective efficacy as potential PRRSV vaccines in pigs. Recombinant vABV688 contains two amino acid substitutions in the minor structural protein GP(2) resulting in improved growth on cell line CL2621; in recombinant vABV707 the region encoding the ectodomain of the major unglycosylated membrane protein M has been replaced by that of the murine lactate dehydrogenase-elevating arterivirus; recombinant vABV746 lacks the six C-terminal amino acids of the nucleocapsid protein N. First, we determined the safety of these recombinant viruses by monitoring the stability of the introduced mutations in 8-week-old pigs. We showed that the introduced genomic mutations were maintained throughout the viraemic period. Second, the protective efficacy of immunization with the recombinant viruses against challenge with a homologous and a heterologous PRRSV strain was determined in two pigs and compared with the efficacy of vABV437, a virus derived from the parental LV cDNA. The viraemia in pigs immunized with the recombinant viruses was reduced compared to pigs immunized with vABV437. In addition, the length of viraemia was reduced in the sentinel pigs that were introduced into the groups immunized with vABV746, vABV688, and vABV707, however, all of the sentinel pigs became infected. Pigs immunized with vABV707 and vABV437 were protected against challenge with homologous virus LV-Ter Huurne and transmission of the latter virus. None of the immunized pigs were protected against heterologous challenge with the virulent US isolate SDSU#73, but the vABV707- and vABV746-immunized pigs were protected against transmission of this virus from challenged pigs. In conclusion, the obtained viral recombinants are interesting candidates to be further explored for their use as vaccines against PRRSV.

Amino Acid Substitution↗

PubMed1988

Strategy for construction of live picornavirus vaccines.

A number of recombinant viruses between the virulent Mahoney and the attenuated Sabin 1 strains of type 1 poliovirus were constructed in vitro using their infectious cDNA clones. Monkey neurovirulence tests on these recombinant viruses revealed that the surface structure of the virion particle had a little correlation with the neurovirulent phenotype, and that the strong neurovirulence determinant(s) resided in the 5' noncoding sequence. These results in turn led to two possible strategies for constructing live attenuated picornavirus strains. One is to use the Sabin 1 strain as a vector carrying foreign antigenicities. The other is the construction of the attenuated picornaviruses by introducing mutations into the 5' noncoding sequences. The antigenicity of the Sabin 1 strain was successfully changed to those of other poliovirus serotypes without loss of vaccine quality. Furthermore, it was proved that introduction of deletion mutation into the 5' noncoding sequence of genomes of the Sabin 1 and Mahoney strains resulted in construction of viruses with a less neurovirulent phenotype.

Chromosome Deletion↗

PubMed1996

[New hepatitis viruses "E, GB, etc."].

Some cases of hepatitis were non A, non B, non C, non D. Hepatitis E virus (Calicivirus) transmission is fecal-oral similar to that of the hepatitis A virus. Viral hepatitis E is endemic and frequently epidemic in many developing countries, but exceptionally observed in France. A high mortality rate was observed in pregnant women. Recently, ELISA assays for IgM anti-HCV are available. Many hepatitis cases (acute and chronic) are not caused by known viruses (non A-non E). Four Flavivirus like have recently been cloned from infectious tamarin serum, derived from human viral hepatitis. Three viruses GB Virus A (GBV-A), GB Virus B (GBV-B) and GB Virus C (GBV-C) were identified. The GB viruses and HG Virus are not genotypes of hepatitis C Virus (HCV) and perhaps that GBV-A/GBV-C and HGV are closely related. GBV-A could be an indigenous tamarin virus. The new hepatitis (non A-non E) viruses were associated with blood transmission and with chronic diseases. The development of specific diagnostic reagents using polymerase chain reaction (PCR) and ELISA assays are essential to answer many questions on the epidemiology (and the incidence of post-transfusion hepatitis), and the pathogenesis of GBV and HGV viruses.

Enzyme-Linked Immunosorbent Assay↗

PubMed1999

Cell-dependent replication potentials of HIV-1 gag mutants.

An infectious molecular clone of human immunodeficiency virus type 1 (HIV-1), designated pNLaiKH, which is tropic for both lymphocytic and monocytic cells, was constructed. To study the early function of HIV-1 Gag proteins in two types of cells, the mutations known to give host cell-dependent early defects were introduced into pNLaiKH, and the replication potentials and defective replication sites in the cells of the resultant mutants were monitored. All mutants grew in some lymphocytic cells, but not at all in monocytic cells. A nucleocapsid mutant was found to be defective at an early replication phase in all the cell lines to various extent, as expected. In contrast, a matrix mutant and a capsid mutant displayed a replication defect in a producer-cell-dependent manner. These results demonstrated that complex interactions of cell factors and Gag proteins are involved in an early process of HIV-1 replication.

Cell Line↗

PubMed1997

Plant-derived vaccine protects target animals against a viral disease.

The successful expression of animal or human virus epitopes on the surface of plant viruses has recently been demonstrated. These chimeric virus particles (CVPs) could represent a cost-effective and safe alternative to conventional animal cell-based vaccines. We report the insertion of oligonucleotides coding for a short linear epitope from the VP2 capsid protein of mink enteritis virus (MEV) into an infectious cDNA clone of cowpea mosaic virus and the successful expression of the epitope on the surface of CVPs when propagated in the black-eyed bean, Vigna unguiculata. The efficacy of the CVPs was established by the demonstration that one subcutaneous injection of 1 mg of the CVPs in mink conferred protection against clinical disease and virtually abolished shedding of virus after challenge with virulent MEV, demonstrating the potential utility of plant CVPs as the basis for vaccine development. The epitope used occurs in three different virus species-MEV, canine parvovirus, and feline panleukopenia virus- and thus the same vaccine could be used in three economically important viral hosts-mink, dogs, and cats, respectively.

Amino Acid Sequence↗

PubMed2000

Cooperative induction of mammary tumorigenesis by TGFalpha and Wnts.

We previously reported that multiparous WAP-TGFalpha transgenic mice develop mammary gland carcinomas with complete incidence. TGFalpha-induced tumors appear stochastically and with relatively long latency, indicating an additional requirement for other genetic alterations. To identify genes that cooperate with TGFalpha in mammary tumorigenesis, we used a retroviral insertion approach featuring a cloned and infectious hybrid MMTV (C3H/Mtv-1; (Shackleford and Varmus, 1988)). Tumor latency was decreased approximately 30% in MMTV-infected WAP-TGFalpha transgenic animals compared to noninfected transgenic controls, and > 30% of the corresponding tumors displayed evidence of integrated C3H/Mtv-1 DNA. PCR-based analyses of DNAs from two virus-infected, transgenic tumors revealed integration of hybrid MMTV in 3' untranslated exons of the Wnt-1 or Wnt-3 oncogenes. Moreover, Northern blots confirmed dramatic induction of Wnt-1 or Wnt-3 transcripts in the respective tumors, indicating that MMTV integration resulted in activated expression of these genes. Semiquantitative RT-PCR analyses showed that overexpression of Wnt-1 or Wnt-3 was a common occurrence in MMTV-infected WAP-TGFalpha tumors, and some noninfected WAP-TGFalpha tumors also showed evidence of elevated Wnt-3 transcripts. Collectively, these results reveal cooperative induction of mammary gland tumorigenesis by simultaneous deregulation of EGF-like (TGFalpha) and Wnt growth factors.

Animals↗

PubMed2002

Complexities in ETS-domain transcription factor function and regulation: lessons from the TCF (ternary complex factor) subfamily. The Colworth Medal Lecture.

The ETS-domain transcription factor family can be divided into a series of subfamilies. Elk-1 represents the founding member of the ternary complex factor (TCF) subfamily. By focusing on the TCF subfamily, we can demonstrate the complexities that exist in the function and regulation of ETS-domain transcription factors. This article focuses on Elk-1 in detail and summarizes the functions of other TCFs. The key themes covered include the domain structure of the TCFs, the mechanisms of complex formation with serum response factor, regulation of TCFs by mitogen-activated protein kinase cascades, and transcriptional regulatory properties of the TCFs. Finally, the emerging role of the TCFs in vivo is discussed. A picture is developing indicating that, while these proteins exhibit significant sequence and functional conservation, key differences in their structure and regulation are being identified which may relate to unique functions of these proteins in vivo.

Amino Acid Sequence↗

PubMed2001

Technical Advance. Tobacco rattle virus as a vector for analysis of gene function by silencing.

Virus vectors carrying host-derived sequence inserts induce silencing of the corresponding genes in infected plants. This virus-induced gene silencing (VIGS) is a manifestation of an RNA-mediated defence mechanism that is related to post-transcriptional gene silencing (PTGS) in transgenic plants. Here we describe an infectious cDNA clone of tobacco rattle virus (TRV) that has been modified to facilitate insertion of non-viral sequence and subsequent infection to plants. We show that this vector mediates VIGS of endogenous genes in the absence of virus-induced symptoms. Unlike other RNA virus vectors that have been used previously for VIGS, the TRV construct is able to target host RNAs in the growing points of plants. These features indicate that the TRV vector will have wide application for gene discovery in plants.

Base Sequence↗

PubMed2000

Development of clover yellow vein virus as an efficient, stable gene-expression system for legume species.

A highly infectious cDNA clone of clover yellow vein virus (pClYVV) was tested as a viral vector, especially for legume species. The genes for green fluorescent protein (GFP) and soybean glutamine synthetase (GS) were inserted between the genes for P1 and HC-Pro on pClYVV to create three recombinant plasmids: pClYVV-GFP, pClYVV-GFP-GS, and pClYVV-GFP:GS. In the former two constructs all the junctions between the inserted proteins contained the sequences of protease cleavage recognition sites, whereas the third construct expressed a fusion of GFP and GS. Western blot analyses showed that GFP and GS appeared to have been precisely excised from the viral polyprotein with the viral proteases (P1 and NIa). Under UV irradiation, green fluorescence was detected in infected broad bean, kidney bean, and soybean plants. The stability of the constructs in the symptomatic tissues was confirmed by RT-PCR and Western blot analyses. The plants expressing GS together with GFP became tolerant to the herbicide glufosinate, and flowered early. As the GS gene, one of the nodulin genes for nitrogen fixation, is expressed in legume species, this system will be useful for examining the function of genes important to legume plants.

Blotting, Western↗

PubMed1999

Characterization of human T-cell leukemia virus type I integrase expressed in Escherichia coli.

The C-terminal part of the pol gene of the human T-cell leukemia virus type I (HTLV-I) is predicted to encode the integrase (IN) of the virus; however, this protein has not yet been detected in virions or infected cells. We expressed the putative IN from an infectious molecular clone of HTLV-I in Escherichia coli. Comparison with protein resulting from coexpression of HTLV-I protease (PR) and Pol in insect cells indicated that the bacterially expressed protein is identical with or very similar to IN released from a PR-Pol precursor by proteolytic cleavage. HTLV-I IN was purified from E. coli under native conditions. The protein behaved like a dimer in size-exclusion chromatography. It carried out activities characteristic of retroviral IN with high efficiency, displaying a strong preference for U5-derived vs. U3-derived sequences in the processing and strand-transfer reactions. In the disintegration reaction, HTLV-I IN not only accepted the double-stranded branched substrate corresponding to the product of a strand-transfer reaction, but was also able to carry out a phosphoryl transfer on a branched molecule with a single-stranded or a single adenosine overhang.

Aspartic Acid Endopeptidases↗

PubMedDate not supplied

PRRSV, the virus.

Porcine reproductive and respiratory syndrome virus (PRRSV) is a positive-strand RNA virus that belongs to the Arteriviridae family. PRRSV grows in primary alveolar macrophages and in monkey kidney cell lines. The genomic RNA is approximately 15 kb. The genome encodes the RNA replicase (ORF1a and ORF1b), the glycoproteins GP2 to GP5, the integral membrane protein M, and the nucleocapsid protein N (ORFs 2 to 7). A comparison of nucleotide sequences of different strains indicates that European and North American strains represent two distinct antigenic types. Various PRRSV-specific monoclonal antibodies and recombinant structural proteins have been produced. Well-defined PRRSV mutants can be generated with the recently developed infectious cDNA clone of PRRSV.

Animals↗

PubMedDate not supplied

Immortalized goat milk epithelial cell lines replicate CAEV at high level.

Primary milk epithelial cells were isolated from CAEV-uninfected goats and three cell lines designated TIGMEC-1, TIGMEC-2 and TIGMEC-3 were established. The three cell lines retained their morphological characteristics of epithelial cells and expressed specific epithelial cytokeratin marker as well as the immortalizing SV40 large T antigen. The kinetics of growth of TIGMEC1, TIGMEC2 and TIGMEC3 cell lines showed a doubling time of 24-48 hours while the parental cell lines became senescent after the passage 6 in cell culture. Like the parental primary cells, the three cell lines were found to be highly sensitive to CAEV-pBSCA, an infectious molecular clone of CAEV-CO strain, and to a French isolate CAEV-3112. TIGMEC cell lines infected with CAEV-pBSCA became chronically infected producing high virus titers in absence of cytopathic effects. These cell lines may be useful for study of the possible physiological alterations in mammary epithelial cells infected with small ruminant lentiviruses and their consequences on milk quality. On an other hand, these cell lines can be used to study their role in virus transmission and pathogenesis.

Animals↗

PubMed2002

Mutations that permit efficient replication of hepatitis C virus RNA in Huh-7 cells prevent productive replication in chimpanzees.

The development of a subgenomic replicon derived from the hepatitis C virus (HCV) strain Con1 enabled the study of viral RNA replication in Huh-7 cells. The level of replication of replicons, as well as full-length Con1 genomes, increased significantly by a combination of two adaptive mutations in NS3 (E1202G and T1280I) and a single mutation in NS5A (S2197P). However, these cell culture-adaptive mutations influenced in vivo infectivity. After intrahepatic transfection of chimpanzees, the wild-type Con1 genome was infectious and produced viral titers similar to those produced by other infectious HCV clones. Repeated independent transfections with RNA transcripts of a Con1 genome containing the three adaptive mutations failed to achieve active HCV infection. Furthermore, although a chimpanzee transfected with RNA transcripts of a Con1 genome with only the NS5A mutation became infected, this mutation was detected only in virus genomes recovered from serum at day 4; viruses recovered at day 7 had a reversion back to the original Con1 sequence. Our study demonstrates that mutations that are adaptive for replication of HCV in cell culture may be highly attenuating in vivo.

Adaptation, Physiological↗

PubMed2003

Coding potential of laboratory and clinical strains of human cytomegalovirus.

Six strains of human cytomegalovirus have been sequenced, including two laboratory strains (AD169 and Towne) that have been extensively passaged in fibroblasts and four clinical isolates that have been passaged to a limited extent in the laboratory (Toledo, FIX, PH, and TR). All of the sequenced viral genomes have been cloned as infectious bacterial artificial chromosomes. A total of 252 ORFs with the potential to encode proteins have been identified that are conserved in all four clinical isolates of the virus. Multiple sequence alignments revealed substantial variation in the amino acid sequences encoded by many of the conserved ORFs.

Chromosomes, Artificial, Bacterial↗

PubMed2000

Hepatitis C virus lacking the hypervariable region 1 of the second envelope protein is infectious and causes acute resolving or persistent infection in chimpanzees.

Persistent infection with hepatitis C virus (HCV) is among the leading causes of chronic liver disease. Previous studies suggested that genetic variation in hypervariable region 1 (HVR1) of the second envelope protein, possibly in response to host immune pressure, influences the outcome of HCV infection. In the present study, a chimpanzee transfected intrahepatically with RNA transcripts of an infectious HCV clone (pCV-H77C) from which HVR1 was deleted became infected; the DeltaHVR1 virus was subsequently transmitted to a second chimpanzee. Infection with DeltaHVR1 virus resulted in persistent infection in the former chimpanzee and in acute resolving infection in the latter chimpanzee. Both chimpanzees developed hepatitis. The DeltaHVR1 virus initially replicated to low titers, but virus titer increased significantly after mutations appeared in the viral genome. Thus, wild-type HCV without HVR1 was apparently attenuated, suggesting a functional role of HVR1. However, our data indicate that HVR1 is not essential for the viability of HCV, the resolution of infection, or the progression to chronicity.

Animals↗

PubMed1986

Infectious RNA derived by transcription from cloned cDNA copies of the genomic RNA of an insect virus.

RNA transcripts of cloned cDNA of the genomic RNAs of BBV (black beetle virus) are infectious to cultured cells of Drosophila melanogaster. Individual transcripts had approximately 10% of the infectivity of the corresponding authentic virion RNA. Progeny virus resulting from transcript infection was phenotypically indistinguishable from the progenitor virus used to generate the original cDNA forms as judged by sucrose density gradient sedimentation, specific infectivity, plaque morphology, and serology. Although the transcript RNAs used to produce this virus had 20 nonviral bases headed by a capping group at their 5' termini, these 20 bases were absent in the progeny viral RNAs. The cDNA forms, and therefore the resulting transcript RNAs, should be readily modifiable by the techniques of recombinant DNA technology both for viral studies and for the insertion of foreign genes into the viral genome and thus into the host cytoplasm.

Animals↗

PubMed1987

Mutational analysis of the human immunodeficiency virus: the orf-B region down-regulates virus replication.

Mutations were made by recombinant DNA techniques in an infectious molecular clone of the human immunodeficiency virus San Francisco isolate 2 (HIVSF2) [formerly the prototype isolate of the acquired immunodeficiency syndrome-associated retrovirus (ARV-2)]. The effect of these changes on the replicative and cytopathologic properties of the virus was studied by transfecting modified virus clones into cultured human cells. Mutations in the gag, pol, env, and tat regions precluded virus replication and cytopathology in lymphoid cells. A mutation in orf-A dramatically reduced but did not abolish virus replication. Mutant viruses with deletions in the orf-B region were highly cytopathic and replicated to approximately 5-fold higher levels than wild-type virus. They also produced approximately 5-fold more viral DNA in infected lymphoid cells than did wild-type virus. Thus, the orf-B region may function to down-regulate virus replication. This mutational analysis of the HIVSF2 genome is a means of assessing genes regulating viral replication and cytopathology.

Cloning, Molecular↗

PubMed1988

Evidence for the direct involvement of the rhinovirus canyon in receptor binding.

Evidence is presented that indicates a deep crevice located on the surface of human rhinovirus type 14 is involved in virion attachment to cellular receptors. By using mutagenesis of an infectious cDNA clone, 11 mutants were created by single amino acid substitutions or insertions at positions 103, 155, 220, 223, and 273 of the structural protein VP1. Seven of the recovered mutants had a small plaque phenotype and exhibited binding affinities significantly lower than wild-type virus. One mutant, in which glycine replaced proline at amino acid position 155, showed a greatly enhanced binding affinity. Single-cycle growth kinetics suggested that 5 of the mutants had delayed growth cycles due to intracellular deficiencies apart from receptor binding.

HeLa Cells↗