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PubMedDate not supplied

Cloning and expression of the VP2 gene of an infectious bursal disease virus.

A serotype I infectious bursal disease virus (IBDV) strain HZ96 was isolated in Hangzhou, China, in 1996 and attenuated by adaptation to chicken embryo fibroblast cells. The VP2 gene of strain HZ96 was amplified by reverse transcription-polymerase chain reaction, cloned, and sequenced. Compared with the VP2 sequences of other IBDV strains, HZ96 is most related to two attenuated strains, CU-1 and PBG98, and two attenuated Chinese strains, Harbin and CJ801bkf. HZ96 shares nucleotide sequence homology 98.9% with CU-1 and PBG98, 98.5% with Harbin, and 98.6% with CJ801bkf. Most of the sequence variations observed between HZ96 and other strains are located in the middle variable region from nucleotides 637 to 996. Similar to other attenuated IBDVs, HZ96 has unique substitutions at residues 279 (Asp to Asn) and 284 (Ala to Thr), suggesting that these two substitutions may be directly related to adaptation of the virus to cell culture and attenuation of its virulence. As part of our effort to develop a submit vaccine for IBDV, the VP2 gene of HZ96 was cloned into a heat-inducible expression vector and expressed in Escherichia coli system. A protein band with expected molecular weight of 52 kD was detected by direct protein staining and western blotting.

Amino Acid Sequence↗

PubMed1990

Mutagenesis of the 3' nontranslated region of Sindbis virus RNA.

A cDNA clone from which infectious RNA can be transcribed was used to construct 42 site-specific mutations in the 3' nontranslated region of the Sindbis virus genome. The majority of these mutations were made in the 3'-terminal 19-nucleotide conserved sequence element and consisted of single nucleotide substitutions or of small (1 to 8) nucleotide deletions. An attempt was made to recover mutant viruses after transfection of SP6-transcribed RNA into chicken cells. In most cases, viable virus was recovered, but almost all mutants grew more poorly than wild-type virus when tested under a number of culture conditions. In the case of mutations having only a moderate effect, the virus grew as well as the wild type but was slightly delayed in growth. Mutations having a more severe effect led to lower virus yields. In many cases, virus growth was more severely impaired in mosquito cells than in chicken cells, but the opposite phenotype was also seen, in which the mutant grew as well as or better than the wild type in mosquito cells but more poorly in chicken cells. One substitution mutant, 3NT7C, was temperature sensitive for growth in chicken cells and severely crippled for growth in mosquito cells. Insertion mutations were also constructed which displaced the 19-nucleotide element by a few nucleotides relative to the poly(A) tail. These mutations had little effect on virus growth. Deletion of large regions (31 to 293 nucleotides long) of the 3' nontranslated region outside of the 19-nucleotide element resulted in viruses which were more severely crippled in mosquito cells than in chicken cells. From these results, the following principles emerge. (i) The entire 3' nontranslated region is important for efficient virus replication, although there is considerable plasticity in this region in that most nucleotide substitutions or deletions made resulted in viable virus and, in some cases, in virus that grew quite efficiently. Replication competence was particularly sensitive to changes involving the C at position 1, the A at position 7, and a stretch of 9 U residues punctuated by a G at position 14. (ii) The panel of mutants examined collectively deleted the entire 3' nontranslated region. Only mutants in which 8 nucleotides in the 3' terminal 19 nucleotides had been deleted or in which the 3' terminal C was deleted were nonviable. Although the 3' terminal C was essential for replication, it could be displaced by at least 7 nucleotides from its 3' terminal position adjacent to the poly(A) tract.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗

PubMed2001

Recovery of infectious human parainfluenza type 2 virus from cDNA clones and properties of the defective virus without V-specific cysteine-rich domain.

A full-length cDNA clone was constructed from the genome of the human parainfluenza type 2 virus (hPIV2). First, Vero cells were infected with recombinant vaccinia virus expressing T7 RNA polymerase, and then the plasmid encoding the antigenome sequence was transfected into Vero cells together with polymerase unit plasmids, NP, P, and L, which were under control of the T7 polymerase promoter. Subsequently, the transfected cells were cocultured with fresh Vero cells. Rescue of recombinant hPIV2 (rPIV2) from cDNA clone was demonstrated by finding the introduced genetic tag. As an application of reverse genetics, we introduced one nucleotide change (UCU to ACU) to immediate downstream of the RNA-editing site of the V gene in the full-length hPIV2 cDNA and were able to obtain infectious viruses [rPIV2V(-)] from the cDNA. The rPIV2V(-) possessed a defective V protein that did not have the unique cysteine-rich domain in its carboxyl terminus (the V-protein-specific domain). The rPIV2V(-) showed no growth in CV-1 and FL cells. Replication of the rPIV2V(-) in these cells, however, was partially recovered by adding anti-interferon (IFN)-beta antibody into the culture medium, showing that the rPIV2V(-) is highly sensitive against IFN and that no growth of rPIV2V(-) in CV-1 and FL cells is mainly due to its hypersensitivity to endogenously produced IFN. These findings indicate that the V-protein-specific domain of hPIV2 is related to IFN resistance. On the other hand, the rPIV2V(-) efficiently replicated in Vero cells, which are known as a IFN-non-producers. However, the virus yields of rPIV2V(-) in Vero cells were 10- to100-fold lower than those of control rPIV2, although syntheses of the viral-specific proteins and their mRNAs in rPIV2V(-)-infected Vero cells were augmented up to 48 p.i. in comparison with those of rPIV2. Furthermore, the rPIV2V(-) virions showed anomalous in size as compared with rPIV2 virions. These results suggest that the V protein plays an important role in the hPIV2 assembly, maturation, and morphogenesis.

Animals↗

PubMed1987

Expression of bacterial chloramphenicol acetyltransferase gene in tobacco plants mediated by TMV-RNA.

We have constructed three tobacco mosaic virus (TMV) cDNA derivatives by modification of the full-length cDNA clone from which infectious TMV-RNA can be transcribed in vitro. A coatless TMV construct lacks most of the coat protein gene and chimeric TMV constructs retain the bacterial chloramphenicol acetyltransferase (CAT) gene in place of the coat protein gene. When in vitro transcripts from these cDNA derivatives were inoculated on the local lesion tobacco plants, TMV-specific lesions were produced. In the case of the TMV-CAT chimeras, however, the lesions were small compared to those of wild-type TMV and those produced by transcript derived from the coatless construct. Northern blot analysis of RNA extracted from the inoculated leaves of the systemic host plants revealed replication of the derivative genomic RNAs and production of their own subgenomic RNAs corresponding to the coat protein mRNA. The TMV-CAT chimeras produced biologically active CAT in the inoculated leaves of the systemic host. CAT activity increased at least until 2 weeks post-inoculation and was approximately 0.1 units/mg of tissue at 10 days post-inoculation. Thus, TMV-RNA may be utilized as a new plant expression vector.

Blotting, Northern↗

PubMed1993

3'-terminal nucleotide sequences important for the accumulation of cowpea mosaic virus M-RNA.

The location of nucleotide sequences important in determining the extent of cowpea mosaic virus M-RNA accumulation in cowpea protoplasts has been analyzed by deletion mutagenesis of full-length cDNA clones from which infectious transcripts can be produced in vitro. The results suggest that cis-acting sequences which direct replication of M-RNA by B-RNA-encoded products are located within the 5'-terminal 524 nucleotides and the 3'-terminal 151 nucleotides. RNA secondary structure predictions for the 3'-terminal 151 nucleotides of both genomic RNAs (Eggen et al. (1989) Virology 173, 456-464) indicate that the terminal nucleotides form a stable secondary structure composed of a Y-shaped stem-loop and a simple A-U-rich stem-loop. The latter structure has been implicated in B-RNA replication. We have examined the role of the Y-shaped structure in M-RNA accumulation by site-directed mutagenesis of putative base-pairing combinations in the two minor stems. The results suggest that efficient replication is dependent on the formation of both of these minor stem structures.

Base Sequence↗

PubMed1994

Evidence that the alpha a gene of barley stripe mosaic virus encodes determinants of pathogenicity to oat (Avena sativa).

Complementary DNA clones from which infectious RNA can be transcribed were used to map the genetic determinants for oat (Avena sativa L.) pathogenicity of barley stripe mosaic virus (BSMV). Pseudorecombinant analysis of BSMV strains ND18 (nonpathogenic to oat) and CV42 (pathogenic to oat) indicated that the ability to systemically infect oat mapped to RNA alpha. A homologous recombinant of ND18 (18 alpha TKTKIN), possessing nucleotides 2218 to 2454 from CV42 RNA alpha, induced symptoms on oat similar to those generated by wild-type CV42. Six amino acids encoded by the alpha a gene differ between ND18 (PQSQTK) and CV42 (TKTKIN) in this region. Fine structure recombinants that encoded subsets of the six amino acid changes either were slow in their infection phenotype (18 alpha TKTK and 18 alpha TQSQIN) relative to recombinant 18 alpha TKTKIN or were not infectious (18 alpha KTKIN and 18 alpha KTK) to inoculated oat plants. Neither coat protein antigen nor viral RNA was detected in inoculated plants that did not display symptoms. All recombinants infected isolated oat protoplasts, however, as determined by Northern and Western blots of extracts from inoculated oat protoplasts. The accumulation of viral genomic RNAs in protoplasts among the recombinants tested was similar, except for that of recombinant 18 alpha TKTK which was reduced by 30%. The data support an earlier suggestion (Y. Zheng, and M. C. Edwards, 1990, J. Gen. Virol. 71, 1865-1868) that resistance of oat to BSMV strain ND18 is due to restricted virus movement in planta and provide evidence that the alpha a protein itself may be involved in the movement of BSMV in the infected plant.

Amino Acid Sequence↗

PubMed1994

Satellite tobacco mosaic virus sequence variants with only five nucleotide differences can interfere with each other in a cross protection-like phenomenon in plants.

The type strain of satellite tobacco mosaic virus (STMV) contains two major variants, designated type 5 (T5) and type 6 (T6), which can be easily distinguished by RNase protection analyses. Clones containing cDNA of representative T5 and T6 STMV genomes have only five single-base differences in the entire 1059-nucleotide genome, and RNA transcribed from each clone is highly infectious when inoculated onto tobacco plants. The different RNase protection assay patterns can be used as genetic markers to identify individual STMV variants and to follow the interactions of variants and their progeny during coinfections in plants. The study described here investigated the effects of coinoculation and various delayed inoculations of T5 and T6 variants on the composition of the progeny STMV populations in systemically infected tobacco tissues. When T5 and T6 STMV RNAs were coinoculated or inoculated with 1-hr delays, the progeny from individual plants most often contained a mixture of T5 and T6 genomes. However, when there was a 24-hr delay between inoculations, the balance of T5 and T6 components in the progeny populations shifted toward predominance of the first variant inoculated. With delays of 3 or 7 days only the first variant was evident in the progeny populations, indicating that established replication of one STMV variant interferes with replication of another in a manner similar to the cross protection phenomenon.

Helper Viruses↗

PubMed1998

Sequence element required for efficient -1 ribosomal frameshifting in red clover necrotic mosaic dianthovirus.

The RNA-1 of the bipartite red clover necrotic mosaic dianthovirus (RCNMV) genome encodes the 88-kDa polymerase. The polymerase is translated from both 5' proximal and internal open reading frames by a -1 ribosomal frameshifting event. A shifty heptanucleotide conforming to the simultaneous slippage model is identified, and a downstream stem-loop structure and atypical pseudoknot are predicted. A beta-glucuronidase reporter assay identified a 118-nucleotide element containing both the shifty heptanucleotide and the predicted secondary structures that were required for efficient -1 ribosomal frameshift expression in vivo. A series of site-directed and compensatory mutations affecting the base-paired regions of the predicted secondary structure were introduced into a RCNMV RNA-1 cDNA clone from which infectious transcripts were derived. Mutations that destroyed the predicted pseudoknot had no effect on frameshifting efficiency in vitro or infectivity of the virus, whereas mutations destabilizing the stem-loop structure abolished both ribosomal frameshifting in vitro and biological activity. These results demonstrate the essential role of a predicted secondary structure that does not involve a pseudoknot in the expression of the RCNMV polymerase by ribosomal frameshifting.

Amino Acid Sequence↗

PubMed2001

Recombinant equine arteritis virus as an expression vector.

Equine arteritis virus (EAV) is the prototypic member of the family Arteriviridae, which together with the Corona- and Toroviridae constitutes the order Nidovirales. A common trait of these positive-stranded RNA viruses is the 3'-coterminal nested set of six to eight leader-containing subgenomic mRNAs which are generated by a discontinuous transcription mechanism and from which the viral open reading frames downstream of the polymerase gene are expressed. In this study, we investigated whether the unique gene expression strategy of the Nidovirales could be utilized to convert them into viral expression vectors by introduction of an additional transcription unit into the EAV genome directing the synthesis of an extra subgenomic mRNA. To this end, an expression cassette consisting of the gene for a green fluorescent protein (GFP) flanked at its 3' end by EAV-specific transcription-regulating sequences was constructed. This genetic module was inserted into the recently obtained mutant infectious EAV cDNA clone pBRNX1.38-5/6 (A. A. F. de Vries, et al., 2000, Virology 270, 84-97) between the genes for the M and the G(L) proteins. Confocal fluorescence microscopy of BHK-21 cells electroporated with capped RNA transcripts derived from the resulting plasmid (pBRNX1.38-5/6-GFP) demonstrated that the GFP gene was expressed in the transfected cells, while the gradual spread of the infection through the cell monolayer showed that the recombinant virus was replication competent. The development of the cytopathic effect was, however, much slower than in cells that had received equivalent amounts of pBRNX1.38-5/6 RNA, indicating that the vector virus had a clear growth disadvantage compared to its direct precursor. Immunoprecipitation analyses of proteins from metabolically labeled BHK-21 cells infected with supernatant of the transfected cultures confirmed that the recombinant virus vector was viable and expressed viral genes as well as the GFP gene. Reverse transcription-PCR of the viral mRNAs extracted from cells infected with the vector virus revealed that it directed the synthesis of nine instead of eight different EAV RNAs. These findings were corroborated by hybridization analyses. Mapping of the leader-to-body junctions of the ninth mRNA indicated that the 3' part of the GFP gene contains cryptic transcription signals which gave rise to at least five different RNA species ranging in size from 1277 to 1439 nt [without oligo(A) tract]. Furthermore, translation of the unintended mRNA resulted in the production of an extended version of the EAV M protein. Serial passage of the recombinant virus vector led to its gradual replacement by viral mutants carrying deletions in the GFP gene. The reduction in viral fitness associated with the insertion of the expression cassette into the EAV genome apparently caused genetic instability of the recombinant virus.

Amino Acid Sequence↗

PubMed2003

Transformation and oncogenesis by jaagsiekte sheep retrovirus.

Jaagsiekte sheep retrovirus (JSRV) is an exogenous retrovirus of sheep that induces a contagious lung cancer, ovine pulmonary adenocarcinoma (OPA). JSRV is a potent carcinogen in the experimental setting, inducing end-stage tumors at around 6 weeks of age when newborn lambs are inoculated intratracheally. Despite this rapid oncogenesis, inspection of the JSRV genome sequence does not reveal any obvious viral oncogenes. In this review, recent advances in studies of JSRV oncogenic transformation are described. Molecular cloning of an infectious and oncogenic JSRV provirus was instrumental in the studies. DNA transfection of JSRV proviral DNA into mouse NIH3T3 cells results in morphological transformation, indicating that the JSRV genome carries an oncogene. Further experiments identified the JSRV envelope protein as the transforming gene, and a PI3 kinase docking site in the cytoplasmic tail of the transmembrane (TM) protein was shown to be necessary for transformation. Avian DF-1 cells infected with an avian retroviral vector (RCAS) expressing the JSRV envelope protein also undergo tumorigenic transformation. Possible mechanisms of transformation are discussed, and a cooperating role for insertional activation of proto-oncogenes in tumorigenesis is also considered. The transforming potential of the JSRV envelope protein may be necessary for JSRV infection and replication in vivo.

3T3 Cells↗

PubMed1995

A single amino acid substitution in the capsid protein VP1 of coxsackievirus B3 (CVB3) alters plaque phenotype in Vero cells but not cardiovirulence in a mouse model.

We previously described a large plaque attenuant (p14V-1) derived from a cardiovirulent Coxsackievirus B3 (CVB3) and showed that there were no major determinants of either attenuation or plaque phenotype in the 5' nontranslated region (5'NTR). Part of the region encoding the last 124 amino acids of VP3 and the first 106 amino acids of VP1 of the attenuant was then sequenced and compared to the wild-type. Three nucleotide changes were found in the VP1 coding region: a silent single base change at nucleotide position 2467 (C to U) and a double-base change at position 2690-1 (AA to GT), which leads to a change from lysine to serine at amino acid position 80. This mutation maps to the begining of B-C loop of the three-dimensional structure of VP1 of CVB3, where a distinct surface projection is formed. Two infectious chimeric cDNA clones were constructed, based on a cardiovirulent cDNA construct. In one construct, the 5'NTR and the VP3-VP1 region were from p14V-1 and in the other, only the VP3-VP1 region was from this attenuant. Both chimeric viruses produced large plaques on Vero cell monolayers, similar to p14V-1 but larger than the prototypic cardiovirulent virus. In vivo experiments showed that both chimeric viruses induced myocarditis in a murine model, similar to wild-type virus. We conclude that mutation serine-80 in capsid protein VP1 of p14V-1 is a determinant of the large plaque phenotype but is not responsible for attenuation.

Amino Acid Sequence↗

PubMed2006

The intraleader AUG nucleotide sequence context is important for equine arteritis virus replication.

The 5(-terminal leader sequence of the equine arteritis virus (EAV) genome contains an open reading frame (ORF) with an AUG codon in a suboptimal context for initiation of protein synthesis. To investigate the significance of this intraleader ORF (ILO), an expression plasmid was generated carrying a DNA copy of the subgenomic mRNA7 behind a T7 promoter. Capped RNA transcribed from this construct was shown to direct, in an in vitro translation system, the synthesis of leader peptide as well as N protein. Site-directed mutations aimed to either optimize or weaken the sequence context of the ILO start codon affected leader peptide synthesis as predicted; no peptide was detected when the initiation codon was incapacitated. Translation of the downstream N gene was inversely affected by leader peptide production, consistent with a ribosomal leaky scanning mechanism. To investigate the role of the leader peptide in the EAV replication life cycle we generated, using an infectious EAV cDNA clone, two mutant viruses in one of which the ILO start codon was in an optimal Kozak context for translation initiation while in the other the codon was again incapacitated. Surprisingly, both mutant viruses were equally viable and exhibited similar phenotypes in BHK-21 cells. However, their replication kinetics and viral yields were reduced relative to that of the wild-type parental virus, as were their plaque sizes. Importantly, the mutations introduced into the viruses appeared to be rapidly and precisely repaired upon passaging. Already after one viral passage a significant fraction of the viruses had regained the wild-type sequence as well as its phenotype. The results demonstrate that EAV replication is not dependent on the synthesis of the intraleader peptide. Rather, the leader peptide does not seem to have any function in the EAV life cycle. As we discuss, the available data indicate that the ILO 5( nucleotide sequence per se, not its functioning in translation initiation, is of critical importance for EAV replication.

Animals↗

PubMed1995

Mutational analysis of the 3'-terminal extra-cistronic region of poliovirus RNA: secondary structure is not the only requirement for minus strand RNA replication.

A series of mutations were introduced in the 3'-terminal untranslated region (3'-UTR) of full-length infectious poliovirus cDNA clones, and following transfection of COS-1 cells the ability of these constructs to generate viable viral particles and/or to support viral RNA synthesis was assayed. Substitution of the 3'-UTR of poliovirus RNA with the equivalent sequences of HAV RNA abrogated viral RNA replication, whereas the introduction of extended 'foreign' sequences between the open reading frame and the 3'-UTR was well tolerated. Point mutation that either destabilized the stem-and-loop structure or altered the sequence of the loop in domain 'Y' (nomenclature as per Pilipenko et al., [Nuclei Acids Res. 20 (1992) 1739-1745]) abolished both the infectivity and viral RNA synthesis. These were not restored by compensatory mutation that reconstructed the native secondary structure of this domain, suggesting that the secondary/tertiary folding of the 3'-UTR is not the only determinant for template recognition at initiation of RNA synthesis, but rather that a specific primary sequence is indeed required.

Animals↗

PubMed1988

[Experimental studies on maedi-visna].

In order to study pathogenicity of sheep lentiviruses, to obtain monospecific sera and to perfect ELISA, 3 experiments with different strains were carried out for 4 yr. In expt 1, one clone only of a French maedi-visna strain (564-79) elicits a clear seroconversion in inoculated sheep. In expt 2, K1514 is more immunogenic than K796 and PPV: intratracheal route seems more efficient than intracerebral route. Sheep infected by ts mutants (expt 3) are early positive as wild strain K796. Nevertheless, the level of positivity is less important than for the parental strain, suggesting that the defect of the ts mutants is not limiting their replication in vivo. An important result is the lack of clinical signs and anatomical and histopathological lesions, in spite of frequent isolations of virus from buffy coat cells. These results suggest that: different enhancing factors have to be taken in account in the apparition of clinical signs; all the clones are not infectious; viral infection might be effective with several types of virions.

Animals↗

PubMed1990

Mutation of conserved N-glycosylation sites around the CD4-binding site of human immunodeficiency virus type 1 GP120 affects viral infectivity.

Infection by the human immunodeficiency virus type 1 (HIV-1) is initiated through interaction of its exterior envelope glycoprotein gp120 with the CD4 receptor on target cells. To address the possible role of N-glycosylation of HIV-1 gp120 in binding CD4, we mutated different conserved N-glycosylation site Asn-residues in the vicinity of the putative CD4 binding site, as single mutations or in combinations. Authentic and mutant gp120 proteins were produced using the baculovirus expression system. All mutant proteins were produced and secreted at similar levels and could be immunoprecipitated with an HIV(+)-serum. Furthermore, all glycosylation mutants retained the full capacity to bind CD4 except for a triple mutant which showed reduced binding. Different gp120 mutant genes were then introduced in an infectious proviral DNA clone. Upon transfection of MT-2 cells, the authentic HIV-1 clone induced maximal virus production after 6 days. In the case of the triple glycosylation mutant, comparable virus production was first reached after a delay of about 12 days. Moreover, in contrast to native HIV, the mutant virus induced no typical multinucleated giant cells. These results suggest that the attached carbohydrates around the CD4-binding site of gp120, may contribute to the generation of this protein domain required for high affinity receptor interaction.

Acquired Immunodeficiency Syndrome↗

PubMed1991

Comparison of regulatory sequences and enhancer activities of SV40 variants isolated from patients with neurological diseases.

The genomic regulatory regions of human isolates of SV40 were compared by nucleotide sequencing and by biological assays of their transcriptional enhancers. SVAST is a defective genome and SVMEN an infectious genome molecularly cloned from human astrocytoma and meningioma, respectively (Krieg and Scherer, 1984). SVPML is an isolate from a case of progressive multifocal leukoencephalopathy (Weiner et al., 1972). SVAST contains a duplication of the regulatory region. One copy is virtually identical to that of SV40; the other bears a deletion of the minimal enhancer and is associated with an insertion of early/late sequences derived from the terminus of replication. The latter copy, together with its insertion, is inserted at nucleotide 4966 (SV40 number). SVMEN has an almost exact deletion of one of the 72-base-pair repeats of the natural SV40 enhancer. The regulatory sequences of SVPML were reported earlier (Martin, 1989). The enhancer activity in CV-1 cells of the lytic virus, SVPML, is 36% that of SV40. In contrast, the SVAST and SVMEN enhancers are more effective, being equal to or 64% that of SV40, respectively. The possibility that SVPML and SVAST/SVMEN originated from different variants of SV40 is discussed.

DNA, Viral↗

PubMed2004

Oral administration of a mite allergen expressed by zucchini yellow mosaic virus in cucurbit species downregulates allergen-induced airway inflammation and IgE synthesis.

BACKGROUND: Sublingual-swallow immunotherapy in house dust mite-related asthma has a good safety profile and improves respiratory function and bronchial hyperreactivity. Zucchini yellow mosaic virus (ZYMV) is envisaged as a promising viral vector for expressing large quantity of foreign proteins in cucurbit species. OBJECTIVE: We sought to investigate whether oral feeding of dust mite allergen expressed by ZYMV in a cucurbit species can suppress allergen-induced inflammation and IgE synthesis. METHODS: An infectious plant virus clone, p35SZYMV2-26, that contains the full-length cDNA to the genomic RNA of a Taiwan isolate of ZYMV, driven by the cauliflower mosaic virus 35S promoter, was engineered as an in vivo viral vector to express Dermatophagoides pteronyssinus group 5 allergen (Der p 5) in cucurbit species. Female BALB/c mice were intraperitoneally sensitized with Escherichia coli bacteria-expressed Der p 5 and orally treated with the virus-expressed Der p 5 (vDer p 5) extracted from the recombinant virus-infected squash plants. Der p 5-specific immunoglobulins were measured by ELISA, and bronchoalveolar lavage assays were used to measure airway inflammation. RESULTS: Infectivity assays and immunoblotting revealed that large quantities of free-form vDer p 5 are produced in the recombinant virus-infected squash plants. The recombinant virus carried and expressed the Der p 5 allergen in squash plants for at least 1 year after numerous passages. In animal tests, squash extract containing vDer p 5 inhibited Der p 5-specific IgE synthesis and airway inflammation. CONCLUSION: Our results suggest that oral feeding with allergen produced by the plant viral vector provides a novel approach for the therapy of allergic asthma.

Administration, Oral↗

PubMed2006

Natural polymorphisms in the protease gene modulate the replicative capacity of non-B HIV-1 variants in the absence of drug pressure.

BACKGROUND: Genetic variation in the HIV-1 pol gene, which encodes the main targets for anti-HIV drugs, may favors different susceptibility and resistance pathways to antiretroviral agents. Several amino acid substitutions occur frequently in some non-B viruses at positions associated with drug resistance in clade B viruses. The clinical relevance of those polymorphisms is unclear. OBJECTIVE: To evaluate the effect of two natural protease (PR) polymorphisms, K20I and M36I, which are frequently found in non-B subtypes, on the virus replicative capacity in the presence and absence of protease inhibitors (PI). STUDY DESIGN: Infectious HIV-1 clones carrying K20I, M36I or K20I/M36I were designed. Their replication kinetics were analyzed by viral competition in the absence of PI. Susceptibility to six different PI was phenotypically assessed in clones and in recombinant viruses carrying non-B proteases from 16 drug-naive individuals. RESULTS: In the absence of drug, the M36I clone replicated more rapidly than wt (wild type) or the double mutant K20I/M36I. Natural polymorphisms 20I and/or 36I improved the virus replicative capacity under drug pressure, reducing the susceptibility to saquinavir and indinavir, with IC(50) values 2-3.5-fold higher than wt. All but one drug-naive individual carrying non-B viruses were fully susceptibility to all tested PI, suggesting that additional substitutions within the PR might compensate the reduced PI susceptibility caused by K20I and/or M36I. CONCLUSION: Natural PR polymorphisms in non-B HIV-1 variants can influence in vitro the virus replication capacity in the presence and/or absence or certain PI. Hypothetically, the improved viral replication of mutant 36I might favor a more rapid spreading of non-B subtypes of HIV-1.

Coculture Techniques↗