- Volume 75, Issue 3, 1994
Volume 75, Issue 3, 1994
- Review Article
-
- Animal
-
-
-
Nucleotide sequence and genetic organization of a 7.3 kb region (map unit 47 to 52.5) of Autographa californica nuclear polyhedrosis virus fragment EcoRI-C
More LessThe nucleotide sequence and genetic organization of a 7297 bp region within the EcoRI-C fragment of Autographa califomica multiple nucleocapsid nuclear polyhedrosis virus (AcMNPV) are presented. Eight putative open reading frames were found and their respective amino acid sequences compared with a number of data libraries. ORF 1227 corresponded with gp41 and its predicted protein sequence was found to be 55 amino acids longer at its C terminus than reported previously. Moreover the main part of the ORF 1227 product, including the additional 55 amino acids, showed a high degree of homology with protein p40 of Helicoverpa zea single nucleocapsid nuclear polyhedrosis virus (HzSNPV). Three other ORFs in the analysed AcMNPV region showed homology with ORFs in the HzSNPV sequence, indicating that the general organization of this region is similar in both viruses. However one ORF found in the AcMNPV sequence was absent from the corresponding HzSNPV sequence.
-
-
-
-
Effect of Sinc genotype, agent isolate and route of infection on the accumulation of protease-resistant PrP in non-central nervous system tissues during the development of murine scrapie
More LessMice congenic for the Sinc gene were infected intracerebrally with two scrapie strains, ME7 and 22A. At various times during the incubation period tissues were monitored for the infection-specific form of PrP (PrPSc). Prpsc was fond in brain, spleen, lymph nodes, pancreas, submaxillary gland and thymus. After intraperitoneal inoculation PrPSc was found in spleen, lymph nodes, pancreas and submaxillary glands prior to its detection in brain. The kinetics of accumulation of PrPSc in these tissues was dependent on the infecting strain of agent, on the mouse Sinc genotype and on the route of infection. This study supports using the presence of PrPSc as an indicator of infectivity in brain and extraneural tissues and defines some of the parameters which influence when and where PrPSc is first found.
-
-
-
Retention of low copy number human papillomavirus DNA in cultured cutaneous and mucosal wart keratinocytes
Cultured wart keratinocytes have previously been described as having a limited proclivity to maintain episomal human papillomavirus (HPV) DNA. To investigate the nature of episome loss, and to determine keratinocyte-specific factors involved in it, we have examined a large series of anogenital and oral wart keratinocyte cultures, tracing episomal copy number with culture passage. We report that a higher proportion of oral wart keratinocytes maintain episomal HPV DNA to first passage (70 % compared with 37 % of anogenital wart cultures) when screened by slot blot hybridization. Furthermore, oral wart keratinocytes maintain episomal HPV copy through a greater number of passages (60 % positive at passage 2 compared with 2 % of anogenital wart cultures) with this technique. When anogenital cultures were examined at first passage for HPV infection by PCR with Southern blot hybridization of the product, a further 34% were found to be HPV-positive. To determine the mechanism of loss of episomal DNA from these cultures we examined the relative HPV copy number in cells which adhered to the culture vessel following passage and in those which did not adhere. Those which remained floating contained episomal HPV at high copy number whereas those which adhered were negative by slot blotting. The adherent cells, however, remained positive by PCR at subsequent passages until senescence. We conclude that a subpopulation of HPV-positive keratinocytes may be maintained in culture through serial passage until senescence.
-
-
-
Quantitative analysis of genomic polymorphism of herpes simplex virus type 1 strains from six countries: studies of molecular evolution and molecular epidemiology of the virus
Using the presence or absence of 63 variable restriction endonuclease (RE) sites selected from 225 sites with six REs, genomic polymorphism of 242 herpes simplex virus type 1 (HSV-1) strains from six countries (Japan, Korea, China, Sweden, U.S.A. and Kenya) was quantitatively analysed. Twenty-five of the 63 sites were found to differ between Korean and Kenyan strains. In contrast, only three and six sites were found to differ between isolates from Sweden and the U.S.A. and between those from Korea and China, respectively, suggesting that they are closely related to each other. In this way, characterization of 63 sites enabled us to categorize 186 distinct HSV-1 genotypes from 242 individuals. Some strains from Japan, Korea and China shared the same genotypes, indicating that they are phylogenetically closely related. Many significant correlation coefficients ( | r | > 0·42; P <0·01) between pairs of sites were found in isolates from the three Asian countries (Japan, Korea and China) as well as in those from Sweden and the U.S.A., suggesting that HSV-1 strains from within the same ethnic groups are evolution- arily closer. The average number of nucleotide substitutions per nucleotide, as defined by nucleotide diversity (π), was estimated for HSV-1 genomes within (πx or πY) and between (π XY) countries. On the basis of 225 sites, nucleotide diversity for Kenyan isolates was 0·0056, almost three times higher than that for Korean isolates, implying that Kenyan HSV-1 genomes are much more diverse than those from Korea. In addition, the diversity between HSV-1 isolates from different countries (πxy) was highest between isolates from the three Asian countries and Kenya (0·0075 to 0·0081) and lowest among those from the three Asian countries (0·0032 to 0·0040). The mutation rate (λ) for HSV-1 was estimated to be 3·5 × 10 −8/site/year. All these findings show that the evolution of HSV-1 may be host-dependent and very slow.
-
-
-
Molecular and biological characterization of simian immunodeficiency virus macaque strain 32H proviral clones containing nef size variants
The proviral genome of the 32H reisolate of simian immunodeficiency of macaques (SIVmac32H) has been cloned and sequenced. Including both long terminal repeats, it is 10277 base pairs in length and contains open reading frames for all known SIV genes (gag, pol, vif vpx, vpr, tat, rev, env and nef). This is the first report of an infectious SIVmac molecular clone which contains no premature termination codons. Three molecular clones of SIVmac32H have been constructed differing in sequence only within their last 1·2 kb. Two of the molecular clones, SIVmac32H(pJ5) and SIVmac32H (pC8), differ in the nef coding region by an in-frame deletion of four amino acids in pC8 and two conservative amino acid changes; other nucleotide changes in the 3′ LTR were not associated with known functionally critical motifs. The third clone, SIVmac32H(pB1), contains the last 1·2 kb of the SIVmac251 clone pBK28. The biological properties of virus produced after electroporation of these clones into C8166 cells has been assessed by infection of rhesus and cynomolgus macaques, time to seroconversion and by induction of cytopathic effects upon co-cultivation of infected rhesus peripheral blood lymphocytes with C8166 cells. The viruses obtained from these clones have identical growth kinetics in vitro but differ in their ability to persist in macaques. Macaques infected with pJ5 derived virus remain viraemic longer than macaques infected with pC8-derived virus. PCR analysis of circulating provirus indicates that the nef gent evolved over time in pJ5 virus-infected macaques, whereas late in infection in pC8 virus-infected macaques the nef gene remained invariant in sequence. These results support the observation that a nef deletion mutant of SIVmac239 lost its pathogenic potential and resulted in low-level viraemia when rhesus macaques were infected. Virus challenge pools for vaccine studies have been prepared for pJ5 using both human and monkey cell substrates and these stocks have been titrated both in vitro and in vivo. Virus has also been prepared from pC8 and titrated in vitro. This virus pool is being assessed as an attenuated live-virus vaccine in macaques. Since only virus originating from the SIVmac239 molecular clone is known to cause AIDSlike symptoms in rhesus macaques consistently, the SIVmac32H molecular clones should tell us more about which viral sequence features are important for the pathogenesis of AIDS.
-
-
-
Cis- and trans-regulation of feline immunodeficiency virus: identification of functional binding sites in the long terminal repeat
More LessNuclear protein binding sites in the long terminal repeat (LTR) of feline immunodeficiency virus (FIV) were identified by the method of DNase I footprinting. Using nuclear protein extracts from a feline T lymphoma cell line, several discrete footprints were generated upstream of the transcriptional initiation site (−50 to −150). The specificity of protein binding was examined by competition with oligonucleotides representing consensus DNA binding sites for known transcription factors. Binding to AP-1 (−124) and ATF (−58) motifs was observed, with cross-competition between these sites. A strong footprint signal was also detected over a tandemly repeated C/EBP motif (−94, −86) and an adjacent weaker footprint was found to be specific for an NF1 motif (−72/ −63). The effect on FIV LTR promoter activity of progressively deleting these nuclear factor binding sites was examined by linking LTR deletion mutants to the chloramphenicol acetyltransferase (CAT) gene. Deletion of the AP-1 site caused a 10- to 25-fold loss of CAT activity whereas deletion past the ATP site reduced activity virtually to background levels. The effects of deleting the C/EBP and NF1 sites were less marked and varied according to cell type. Transactivation of the LTR was assayed using constructs linked to a CAT reporter gene. The full-length FIV LTR was not significantly trans-activated. However, the expression of a deleted LTR construct lacking the AP- 4/AP-l site but retaining C/EBP and ATF sites was partially restored by co-infection with FIV or by cotransfection with an infectious molecular clone of FIV (FIV-PPR). These results show that host transcription factors responsive to cellular activation have a major role in regulating FIV expression, and suggest that virus- coded trans-activators acting through U3 may play a role in some cellular environments.
-
-
-
Identification of a protein kinase involved in the phosphorylation of the C-terminal region of human respiratory syncytial virus P protein
More LessP protein, the structural phosphoprotein of the Long strain of respiratory syncytial (RS) virus, is phosphoryl-ated at serine residues. Some of these residues are candidates for modification by casein kinase II, as they are contained in consensus sequences. A cellular protein kinase, able to phosphorylate the P protein in vitro and apparently associated with purified RS virions, has been partially purified from HEp-2 cells. It shows several characteristics similar to those of casein kinase II. The P protein is modified in vitro by this activity mainly at serine residues located near the C terminus, which are also modified during virus infection. Thus, the P protein is phosphorylated in vivo in two regions, a central region as previously described, and another located in the C-terminal part of the molecule. The protein kinase involved in the phosphorylation of the C-terminal domain is similar to a cellular casein kinase II.
-
-
-
HN proteins of human parainfluenza type 4A virus expressed in cell lines transfected with a cloned cDNA have an ability to induce interferon in mouse spleen cells
Primary monkey kidney cells infected with human parainfluenza type 4A virus (HPIV-4A) were treated with various concentrations of formaldehyde. Formaldehyde (0·275%) treatment completely blocked virus production. However, when mouse spleen cells were cocultured with the fixed virus-infected cells, interferon was produced in the culture fluid. On the other hand, when mouse spleen cells were incubated with the fixed virus-infected cells in the presence of anti-HPIV-4A antiserum or a mixture of anti-HN protein monoclonal antibodies, interferon activity could scarcely be detected in the culture fluid. These findings indicated that the fixed virus-infected cells had an ability to induce interferon in mouse spleen cells and that the HN protein was related to interferon induction. Subsequently, a recombinant plasmid was constructed by inserting the cDNA of the HN gene of HPIV-4A into a pcDL-SRα expression vector. Mouse spleen cells produced interferon when cocultured with COS7 cells transfected with the recombinant plasmid, but did not when cocultured with COS7 cells transfected with the vector alone. Furthermore, we established HeLa cells constitutively expressing HPIV-4A HN (HeL-4aHN cells) or F protein (HeLa-4aF cells). Type I (α/β) interferon was detected in culture fluids of mouse spleen cells with HeLa-4aHN cells, but was not detected in those with HeLa-4aF cells. Therefore, it was concluded that the HN glycoproteins on the cell surface were sufficient for interferon induction to occur.
-
-
-
Intracellular proteolysis of poliovirus eclipse particles is abortive
More LessA series of weak bases and the ionophore monensin were tested for their effect on the intracellular processing of type 1 poliovirus in HeLa cells. At concentrations that did not inhibit plaque formation or viral protein synthesis, the compounds suppressed the proteolytic processing of 135S particles and the formation of 110S particles. In addition, some compounds strongly reduced transit of modified particles to the lysosomes. These results suggest that transit to lysosomes and proteolysis of subviral particles are not essential steps in the infectious pathway. The role of 135S particles as intermediates in infection is discussed.
-
-
-
Capsid destabilization is required for antibody-mediated disruption of poliovirus
More LessThree out of 36 poliovirus type 1-specific monoclonal antibodies which, at 37 °C in a medium of normal ionic strength (μ = 0·16), caused only aggregative neutralization (reversible by immune complex dissociation at pH 2) shifted to cause disruptive, acid-irreversible neutralization when the temperature was raised to 39 °C or the ionic strength was lowered to 1/100 of normal. Under both conditions, the antigenic conversion was stoichiometric, but the efficiency was lower at 39 °C than at low ionic strength. Antigenic conversion and irreversible neutralization under both conditions were inhibited by WIN 51711, a capsid-stabilizing compound. Complete inhibition required filling of most of the virion’s binding pockets by this compound.
-
-
-
Identification of cytotoxic T cell epitopes on the VP3 and VP6 rotavirus proteins
More LessIt has been shown previously that the viral glycoprotein VP7 is a major target of cytotoxic T lymphocytes (CTLs) induced by bovine rotavirus RF in C57BL/6 mice. Here we show that these RF-specific CTLs recognize target cells infected with a recombinant vaccinia virus (rW) expressing the SA11 (simian rotavirus) VP6 but not those infected with rWs that express RF VP1, VP2 or VP3 core proteins. After immunization of mice with insect cells infected with recombinant baculoviruses that express the corresponding RF proteins a strong specific CTL response was generated against target cells infected with VP6 rW and VP3 rW, a weak one against the VP2 rW but none against the VP1 rW. Kb-restricted CTL epitopes were identified on VP6 and VP3 using allele-specific motifs. When peptides corresponding to these epitopes were used to restimulate in vitro the spleen cells from RF-immunized mice, CTLs specific for each peptide and its corresponding rW were induced.
-
-
-
The S RNA genome segments of Batai, Cache Valley, Guaroa, Kairi, Lumbo, Main Drain and Northway bunyaviruses: sequence determination and analysis
More LessBunyaviruses have a genome comprising three segments of negative-sense RNA. The smallest RNA segment, S, encodes the nucleocapsid protein, N, and a nonstructural protein, NSs, in overlapping reading frames. The sequences of the S genome RNA segments of seven bunyaviruses (Batai, Cache Valley, Guaroa, Kairi, Main Drain, Northway and Lumbo) were determined from cloned cDNAs obtained using a one-step reverse transcription-PCR protocol. These sequences were compared to those of six viruses previously published, reinforcing earlier conclusions about relationships of the bunyaviruses. Sequence homologies between N proteins correlated with the subdivision of these viruses into three serogroups, Bunyamwera, California and Simbu.
The encoded N proteins are either 233 or 235 amino acids in length, depending on the serogroup, whereas the NSs proteins are more variable (83 to 109 amino acids). Certain nucleotide sequence motifs are conserved in the S segments of the Bunyamwera and California serogroup viruses, including the spacing of the AUG initiation codons for the N and NSs proteins (except Guaroa virus), and a CA-rich motif in the virion-sense RNA just downstream of the predicted mRNA termination site. A duplicated sequence was observed in the 3′non-coding region of the Lumbo virus S segment, which accounts for the significantly longer S genome segment of this virus.
-
-
-
Identification of naturally occurring monoclonal antibody escape variants of louping ill virus
More LessLouping ill virus isolates from Great Britain, Ireland and Norway were compared antigenically by indirect immunofluorescence, haemagglutination-inhibition and neutralization tests using a panel of five envelope-specific and five non-structural protein NS1-specific monoclonal antibodies raised against louping ill virus. The viruses were grouped according to their reactivities with the antibodies. Group 1, members of which were isolated between 1931 and 1987, consisted of 13 viruses that reacted with all antibodies, whereas group 2, members of which were isolated after 1980, consisted of five viruses that were positive with only eight of the 10 monoclonal antibodies. The two monoclonal antibodies that did not react with the group 2 viruses are known to be neutralizing antibodies and the amino acids that they recognize in the viral envelope protein have been identified. We therefore refer to the group 2 viruses as naturally occurring monoclonal antibody escape variants. When compared with group 1 viruses, the escape variants showed reduced virulence for mice in terms of the time taken to kill and/or the proportion that died, following intraperitoneal inoculation. The nucleotide and deduced amino acid sequences of the envelope gene of one escape variant were compared with those of several group 1 viruses. A single amino acid substitution at residue 308 was detected in the envelope protein of the escape variant which corresponds precisely to the position in experimentally selected attenuated monoclonal antibody escape mutants. The importance and potential implications of these naturally occurring variants in louping ill epizootiology and vaccine-based control are discussed.
-
-
-
Complement-dependent neutralization of influenza virus by a serum mannose-binding lectin
More LessThe nature of the β inhibitor in guinea-pig serum and its mechanism of neutralization of influenza virus have been investigated. This inhibitor was shown to be a mannosebinding lectin serologically related to human serum mannose-binding protein. Ca2+-dependent binding of the guinea-pig lectin to influenza virus or to mannan could be detected with polyclonal or monoclonal antibodies against human mannose-binding protein in an ELISA. Furthermore, the monoclonal antibody inhibited both the haemagglutination-inhibiting and virus-neutralizing activities of the guinea-pig lectin. The lectin was active against influenza viruses of both type A and type B. In haemagglutination inhibition it acts independently of complement, apparently by sterically hindering access to the receptor-binding site on the viral haemagglutinin through binding of the lectin to carbohydrate side-chains in the vicinity of this site. Neutralization by the lectin, however, was shown to require activation of the classical complement pathway. To our knowledge, the neutralization of influenza virus by a serum lectin plus complement represents a previously unrecognized mechanism of complement-dependent viral inactivation that may be important in first-line host defence against a variety of enveloped viruses.
-
-
-
Modification of tritiated γ-amino-n-butyric acid transport in rabies virus-infected primary cortical cultures
More LessThe role of brain neurotransmitter transport processes in rabies virus infection of neurons was examined. The uptake and release of γ-amino-n-butyric acid (GABA) in rabies virus-infected embryonic rat cortical neurons was assayed using tritiated ligands. A 45% reduction of [3H]GABA uptake was observed 3 days post-infection, when a maximum level of infectious particle release occurs. At this time, kinetic analysis revealed significant changes in V max, whereas no changes were found in K m values in comparison with the control values. K+ and veratridine-induced [3H]GABA release was increased in infected cultures (98% and 35%, respectively) as compared with control values. The results obtained from rabies virus-infected cultures provide some preliminary evidence of the involvement of GABA in the pathogenesis of rabies.
-
-
-
The entire nucleotide sequence and classification of a hepatitis C virus isolate of a novel genotype from an Indonesian patient with chronic liver disease
Three hepatitis C virus (HCV) isolates were obtained from patients with chronic liver diseases in Indonesia which were not classifiable into any of the genotypes I/1a, II/1b, III/2a, IV/2b or V/3a reported previously. The entire nucleotide sequence was determined for one HCV isolate (HC-G9); the remaining two isolates were of the same genotype based on a > 95 %; similarity within their partial sequences spanning 2927 nucleotides (nt). The HC-G9 genome consisted of 9440 nt including the 5′ untranslated region of 341 nt, an open reading frame of 9033 nt coding for a polyprotein of 3011 amino acids and the 3′ untranslated region of 66 nt (U stretch of 17 to 47 nt at the extreme 3′ terminus excluded). It differed by 20 to 33 %; in nucleotide sequence from any of 14 HCV genomes of genotypes I/1a to IV/2b whose full-length sequences are known. By the unweighted pair-group method with arithmetic mean, HC-G9 was on a major branch (group 1) of the phylogenetic tree of HCV to which genotypes I/1a and II/1b belong. It is proposed, therefore, that the novel genotype for HC-G9 should be called lc. A method was developed to identify genotype lc by PCR with a primer deduced from the core gene that was specific to it. Since genotype lc was detected in seven (15 %;) of 48 HCV RNA samples from Indonesian patients with chronic liver disease, but not in any of 1097 from other districts of the world, it appears to have evolved and remained in Indonesia. In addition to its epidemiological importance, the association of genotype lc HCV with the severity of liver disease and its response to interferons deserve to be evaluated.
-
-
-
Prediction of a putative fusion peptide in the S protein of hepatitis B virus
Sequence analysis of the S protein of hepatitis B virus (HBV) reveals a stretch of 23 hydrophobic amino acids in the amino-terminal region which shows a high degree of similarity with known fusogenic peptides from other viruses. Additionally, this sequence also appears to be highly conserved within the hepadnavirus family. Taken together, the different criteria used in this work suggest fusogenic activity in the amino-terminal region of the S protein of the envelope of HBV.
-
-
-
Comparative characterization of antigenic epitopes in the immunodominant region of the protein encoded by open reading frame 3 in Burmese and Mexican strains of hepatitis E virus
More LessTo analyse the effect of strain-specific sequence variation on the antigenic properties of the protein encoded by the open reading frame 3 (ORF 3) of hepatitis E virus (HEV), two sets of short overlapping peptides spanning amino acids 91 to 123 of this protein from Burmese and Mexican strains were synthesized and tested with sera obtained from outbreaks of enterically transmitted non-A, non-B hepatitis in three different regions of the world (Mexico, Turkmenistan and Kenya). The data suggest strain-specific variation in the antigenic reactivity of the ORF 3 protein. The C-terminal region of this protein contains several antigenic epitopes located in the most variable positions. Individual sera were found to interact with different groups of epitopes from each set of peptides. The antigenic epitopes of the Mexican strain appear to be less conformation-dependent than those of the Burmese strain. The most immunoreactive epitope of the ORF 3 protein from the Mexican strain was localized at amino acid positions 95 to 101. The ORF 3 protein of the Burmese strain contains an immunodominant epitope at amino acid positions 112 to 117. Some of these short peptides may be useful for the development of a diagnostic assay to discriminate between the Burmese and Mexican strains.
-
-
-
Detection of herpes simplex virus type 1-encoded RNA by polymerase chain reaction: different pattern of viral RNA detection in latently infected murine trigeminal ganglia following in vitro or in vivo reactivation
More LessHerpes simplex virus type 1 (HSV-1) establishes latent infection in the sensory ganglia. To investigate the process of reactivation from latency, we used the RNA polymerase chain reaction (RNA-PCR) to detect the expression of several HSV genes. BALB/c mice were inoculated in the anterior ocular chamber with HSV-1 strain KOS and the trigeminal ganglia were examined at least 8 weeks after inoculation. Latency-associated transcripts (LATs) were found in the latently infected ganglia and remained detectable 120 h after explantation. Besides LATs, we detected transcripts for infected cell protein 0 (ICPO) (Vmw110) 24 h after explantation, but RNAs encoding ICP4 (Vmw175), ICP27, thymidine kinase and VP16 (ICP25; Vmw65) remained undetectable for 120 h after explantation. Following in vivo reactivation of HSV-1 by administration of cyclophosphamide and dexamethasone, all viral transcripts including ICPO RNA became detectable. The RNA-PCR enabled us to detect ICPO RNA much earlier than has been previously reported in studies using the Northern blot technique and has laid a foundation for further study of viral and cellular transcripts during reactivation. Our results suggest that the process of reactivation of HSV-1 from trigeminal ganglia may be divided into at least two steps: (i) initiation of ICPO gene transcription and (ii) detectable transcription of the other genes. The second step may be regulated in part by the host immune system, since cyclophosphamide and dexamethasone administration enabled the detection of several viral transcripts.
-
-
-
Large-scale production and characterization of recombinant human immunodeficiency virus type 1 Nef
Sequences encoding the 27K and 25K nef gene products (Nef 27 and Nef 25) were amplified by PCR from a human immunodeficiency virus type 1 infectious clone and subcloned directly into Escherichia coli, yeast and baculovirus expression vectors. The yeast- and baculovirus-derived Nef had native N termini but the expression levels were low. The expression levels of the E. coli-derived glutathione V-transferase-Nef fusion proteins were very high and a major portion was soluble. Large-scale production of E. coli-derived Nef 27 and Nef 25 was carried out by growing recombinant cells in a fermenter under fed-batch conditions followed by affinity purification on glutathione-Sepharose before and after thrombin cleavage. Large quantities of highly purified recombinant Nef proteins have been produced for functional and structural studies. Under nonreducing conditions both Nef 27 and Nef 25 existed as a mixture of monomers, dimers and small amounts of higher oligomers, but when reduced were monomeric. The highly purified Nef proteins had no G protein activities, however Nef 27 was biologically active. When electroporated into uninfected CD4+ T lymphocytes both E. coli-derived Nef 27 and yeast-derived myristy-lated Nef 27 down-regulated the surface expression of CD4, demonstrating that this method can be used to assess the biological activity of purified recombinant Nef.
-
-
-
Lentivirus cross-reactive determinants present in the capsid protein of equine infectious anaemia virus
More LessIn this study we used immune sera from equine infectious anaemia virus (EIAV)-infected horses which uniquely display broad reactivity with different lentivirus capsid proteins (CA) to characterize the cross-reactive determinants of lentivirus CA proteins. In particular, the role of the major homology region (MHR) of lentivirus CA proteins in this serological cross-reactivity was evaluated using both equine immune serum and murine monoclonal antibodies (MAbs) directed against the MHR segment of different lentiviruses. The results of our studies indicate that about 80 %; of sera from long-term experimentally infected ponies or naturally infected horses react with human immunodeficiency virus type 1 CA in Western immunoblot assays. In addition, the cross-reactive determinants on the EIAV CA were localized within the immunodominant carboxyl terminus of the protein (residues 277 to 367). However, the crossreactive determinants recognized by the equine sera do not appear to correlate with linear peptides from the carboxyl terminus of the EIAV CA, including the MHR. These results suggest cross-reactivity between more distant lentiviruses is associated with non-linear determinants. In contrast, MHR-specific MAbs did react with linear peptides by ELISA and distinguished the primate lentiviruses from EIAV and feline immunodeficiency virus. These data support the concept of a highly conserved structural and antigenic organization among the CA proteins of lentiviruses from different species.
-
-
-
A single amino acid change in the E2 spike protein of a virulent strain of Semliki Forest virus attenuates pathogenicity
More LessThe virulent strain SFV4 of Semliki Forest virus (SFV), produced from the infectious clone pSP6-SFV4, is lethal after intranasal (i.n.) infection of adult mice and for pregnant mice after intraperitoneal (i.p.) infection. In contrast, the A7 strain of SFV is avirulent when given i.n. to adult mice, but induces fetal death in pregnant mice after i.p. infection. The nucleotide and deduced amino acid sequences of part of the core and all of the envelope region of A7-SFV were determined and compared to those of SFV4. A7 differed from SFV4 at 80 nucleotides (nt) in the coding sequence, 15 of which were associated with amino acid differences and seven of which (two in the E2 protein and five in El) were nonconservative. The 3′ non-coding sequence of A7 was longer (415 nt) than that of SFV4 (263 nt) and a divergent sequence of 181 nt was present adjacent to the end of the El coding region. The effects on virulence of two mutations in the E2 gene of SFV4, resulting in the non-conservative amino acid substitutions present in A7, were analysed. One mutation (mut 8729 a/c) resulted in only slight attenuation, whereas the other (mut 8902 a/g) resulted in avirulence for pregnant mice. However, mut 8902 a/g was lethal for the majority of developing fetuses after i.p. infection of the mother.
-
-
-
Antigenic and molecular characterization of host cell-mediated variants of equine H3N8 influenza viruses
Antigenic differences between three of six equine influenza virus (H3N8) MDCK cell- and egg-derived pairs have been demonstrated using monoclonal and polyclonal antibodies. Sequencing of the haemagglutinin (HA) genes revealed amino acid changes in four of the six virus pairs. These data contrast with those for human isolates of influenza virus in that it was predominantly tissue culture-isolated equine virus and not egg-derived virus which displayed heterogeneity. Some of the molecular changes involved are located within the vicinity of the cell receptor-binding site (positions 156, 158 and 222) whereas others are in the vicinity of the HA1-HA2 cleavage site (positions 18 and 32 of HA1 and position 12 of HA2). Our results indicate that the host cell can play a part in selecting antigenic variants of equine influenza virus and suggest that the egg, and not cell culture as is the case for human isolates, is the preferred host for vaccine and antigenic studies.
-
-
-
VP2 sequences of recent European ‘very virulent’ isolates of infectious bursal disease virus are closely related to each other but are distinct from those of ‘classical’ strains
More LessPart of genomic segment A of five recent European ‘very virulent’ (W) isolates of infectious bursal disease virus, encoding the major, conformational, protective epitope, have been reverse-transcribed, amplified and cloned. Sequence analysis of 582 bases of VP2 showed that the four U.K. isolates are closely related to each other, differing by no more than two bases, but are distinct from the ‘classical’ type 1 strains (differing by at least 29 bases, or four amino acids, from any other strain and by 15 bases, or three amino acids, from the consensus sequence of all other strains). The U.K. isolates differ from an earlier W isolate from the Netherlands by no more than five bases or two amino acids. The U.K. isolates differ by only one or two bases, with no amino acid changes, from the recently published sequence of a Japanese W isolate.
-
-
-
Identification of major differences in the nucleocapsid protein genes of a Québec strain and European strains of porcine reproductive and respiratory syndrome virus
More LessThe sequence of the 3′-terminal region of the genome of Québec reference strain IAF-exp91 of porcine reproductive and respiratory syndrome virus (PRRSV) was investigated by analysis of four cDNA clones. The 3′-terminal 530 nucleotides (nt) encompassed a large open reading frame with a coding capacity of 123 amino acids (M r 13649). The predicted protein was extremely basic and hence was considered to correspond to the nucleocapsid (N) protein gene. When compared to the homologous sequences of two reference Netherlands strains (Lelystad and isolate 10) of PRRSV, the IAF-exp91 N protein was found to be five amino acids shorter and displayed a high degree of divergence. Overall, IAF-exp91 strain showed identities of 63 %; and 59%; with both reference European strains at the nucleotide and amino acid level, respectively. Two amino acid stretches, STAPM and SQGAS, present respectively at the N- and C-terminal regions of the N protein of European strains, were missing in the IAF-exp91 N protein sequence. The 3 -terminal non-coding region (151 nt) of the IAF-exp91 strain was 22 nt longer than that of the European strains. The aligned nucleotide sequence of this non-coding region exhibited an overall identity of 59 %; with that of the European strains. The Québec reference strain of PRRSV appeared to be related more closely to equine arteritis virus and lactate dehydrogenase-elevating virus than are the two European strains of the virus. Preliminary data obtained by reverse transcription-PCR experiments, using specific or common oligonucleotide primers, suggested that this approach could be useful for distinguishing between PRRSV strains from different geographic origins.
-
-
-
Intratypic genome variability of the coxsackievirus B1 2A protease region
More LessTo analyse the intratypic genome variability of coxsackievirus B1, 17 coxsackievirus B1 isolates were collected over a period of 10 years. Nucleotide sequences of the 2A coding region of the various coxsackievirus B1 isolates and known sequences of other enteroviruses were compared. The maximum diversity observed within the entire group of coxsackievirus B1 isolates was 25 %;. Comparison of deduced amino acid sequences revealed a maximum diversity of 5%;. Phylogenetic analysis demonstrates a close genetic relationship between these clinical isolates and the other different coxsackievirus B serotypes. Further analysis of nucleotide and amino acid sequences of the 2A region of known enteroviruses demonstrated that the genus enterovirus can be subdivided into a coxsackievirus B-like group, including the coxsackie B viruses, coxsackievirus A9, echovirus 11 and swine vesicular disease virus, and a poliovirus-like group including the polioviruses 1 to 3, and the coxsackieviruses A21 and A24. Enterovirus type 70 and bovine enterovirus represent distinct groups. The 2A coding region of coxsackievirus B1 strains cannot be distinguished from that of other members of the coxsackievirus B-like group. Although conservation of the overall amino acid sequence was almost limited to residues essential for proposed enzymatic activity, the predicted secondary structures were well conserved within the genus enterovirus.
-
- Plant
-
-
-
Complete nucleotide sequence of the geminivirus tomato yellow leaf curl virus, Thailand isolate
More LessThe complete genome of a Thailand isolate of the geminivirus tomato yellow leaf curl virus (TYLCV-Th) has been cloned and the nucleotide sequence determined. The genome consists of two DNAs each slightly greater than 2700 nucleotides in length and designated A-DNA and B-DNA. The A-DNA contains six open reading frames (ORFs) capable of encoding proteins with M rs greater than 10K; two ORFs were located on the B-DNA. The predicted ORFs were found at positions on the genome similar to those identified in other geminiviruses. Based upon computer-assisted sequence comparisons with other geminiviruses, TYLCV-Th maintained the greatest degree of similarity with a tomato pathogen isolated in Australia, tomato leaf curl virus. Furthermore, sequence comparisons with a large number of geminiviruses confirm further divisions of the geminiviruses based upon geographical proximity of the viruses. Agroinoculation of Nicotiana benthamiana with a TYLCV-Th A-DNA clone containing a mutation in the capsid protein ORF demonstrated that the coat protein is not essential for TYLCV-Th infection but enhances the progression of the disease.
-
-
-
-
Infectious transcripts from PCR-amplified broad bean mottle bromovirus cDNA clones and variable nature of leader regions in RNA 3 segment
More LessThe genome of broad bean mottle bromovirus (BBMV) contains three positive-sense ssRNA segments, each capped with m7GpppA. Full-length transcribable cDNA clones for four strains of BBMY were constructed by employing reverse transcriptase-PCR (RT-PCR) and a high fidelity Vent DNA polymerase. The transcribed BBMY RNAs contained a 5′ non-viral G residue and, although delayed, produced symptoms similar to those observed in plants infected with authentic virion RNAs. The transcripts replicated inefficiently in protoplasts. In contrast, transcript-derived progeny BBMV RNAs had the repaired termini, were as infectious as the authentic BBMV RNAs and replicated to high levels in protoplasts. In vitro translation of synthetic RNAs confirmed the previously proposed gene expression strategy for BBMV. Sequencing of virion RNAs from the Bawden strain revealed two forms of BBMV RNA 3 components, the longer form containing 21 5′ extra nucleotides derived by the duplication of two short 5′ leader regions. The relative concentration of the two RNA 3 forms was found to be host-dependent, with the longer form prevailing in broad bean and Nicotiana clevelandii infections and the shorter form in bean infections.
-
Volumes and issues
-
Volume 105 (2024)
-
Volume 104 (2023)
-
Volume 103 (2022)
-
Volume 102 (2021)
-
Volume 101 (2020)
-
Volume 100 (2019)
-
Volume 99 (2018)
-
Volume 98 (2017)
-
Volume 97 (2016)
-
Volume 96 (2015)
-
Volume 95 (2014)
-
Volume 94 (2013)
-
Volume 93 (2012)
-
Volume 92 (2011)
-
Volume 91 (2010)
-
Volume 90 (2009)
-
Volume 89 (2008)
-
Volume 88 (2007)
-
Volume 87 (2006)
-
Volume 86 (2005)
-
Volume 85 (2004)
-
Volume 84 (2003)
-
Volume 83 (2002)
-
Volume 82 (2001)
-
Volume 81 (2000)
-
Volume 80 (1999)
-
Volume 79 (1998)
-
Volume 78 (1997)
-
Volume 77 (1996)
-
Volume 76 (1995)
-
Volume 75 (1994)
-
Volume 74 (1993)
-
Volume 73 (1992)
-
Volume 72 (1991)
-
Volume 71 (1990)
-
Volume 70 (1989)
-
Volume 69 (1988)
-
Volume 68 (1987)
-
Volume 67 (1986)
-
Volume 66 (1985)
-
Volume 65 (1984)
-
Volume 64 (1983)
-
Volume 63 (1982)
-
Volume 62 (1982)
-
Volume 61 (1982)
-
Volume 60 (1982)
-
Volume 59 (1982)
-
Volume 58 (1982)
-
Volume 57 (1981)
-
Volume 56 (1981)
-
Volume 55 (1981)
-
Volume 54 (1981)
-
Volume 53 (1981)
-
Volume 52 (1981)
-
Volume 51 (1980)
-
Volume 50 (1980)
-
Volume 49 (1980)
-
Volume 48 (1980)
-
Volume 47 (1980)
-
Volume 46 (1980)
-
Volume 45 (1979)
-
Volume 44 (1979)
-
Volume 43 (1979)
-
Volume 42 (1979)
-
Volume 41 (1978)
-
Volume 40 (1978)
-
Volume 39 (1978)
-
Volume 38 (1978)
-
Volume 37 (1977)
-
Volume 36 (1977)
-
Volume 35 (1977)
-
Volume 34 (1977)
-
Volume 33 (1976)
-
Volume 32 (1976)
-
Volume 31 (1976)
-
Volume 30 (1976)
-
Volume 29 (1975)
-
Volume 28 (1975)
-
Volume 27 (1975)
-
Volume 26 (1975)
-
Volume 25 (1974)
-
Volume 24 (1974)
-
Volume 23 (1974)
-
Volume 22 (1974)
-
Volume 21 (1973)
-
Volume 20 (1973)
-
Volume 19 (1973)
-
Volume 18 (1973)
-
Volume 17 (1972)
-
Volume 16 (1972)
-
Volume 15 (1972)
-
Volume 14 (1972)
-
Volume 13 (1971)
-
Volume 12 (1971)
-
Volume 11 (1971)
-
Volume 10 (1971)
-
Volume 9 (1970)
-
Volume 8 (1970)
-
Volume 7 (1970)
-
Volume 6 (1970)
-
Volume 5 (1969)
-
Volume 4 (1969)
-
Volume 3 (1968)
-
Volume 2 (1968)
-
Volume 1 (1967)