- Volume 84, Issue 6, 2003
Volume 84, Issue 6, 2003
- Review
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Triple gene block: modular design of a multifunctional machine for plant virus movement
More LessMany plant virus genera encode a ‘triple gene block’ (TGB), a specialized evolutionarily conserved gene module involved in the cell-to-cell and long-distance movement of viruses. The TGB-based transport system exploits the co-ordinated action of three polypeptides to deliver viral genomes to plasmodesmata and to accomplish virus entry into neighbouring cells. Although data obtained on both the TGB and well-studied single protein transport systems clearly demonstrate that plant viruses employ host cell pathways for intra- and intercellular trafficking of genomic nucleic acids and proteins, there is no integral picture of the details of molecular events during TGB-mediated virus movement. Undoubtedly, understanding the molecular basis of the concerted action of TGB-encoded proteins in transporting viral genomes from cell to cell should provide new insights into the general principles of movement protein function. This review describes the structure, phylogeny and expression of TGB proteins, their roles in virus cell-to-cell movement and potential influence on host antiviral defences.
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- Animal
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- RNA viruses
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Amino acid mutations in the replicase protein nsP3 of Semliki Forest virus cumulatively affect neurovirulence
More LessIt has been shown previously that an avirulent Semliki Forest virus (SFV) clone, rA774, engineered to carry the nsP3 gene of the virulent clone SFV4 becomes highly neurovirulent and is lethal for adult BALB/c mice. rA774, like several other alphaviruses, has an opal termination codon close to the 5′ end of nsP3 (aa 469), while SFV4 has an arginine residue at this position. Mutation of the opal codon to an arginine residue increases the virulence of rA774 but does not reconstruct the severe neurovirulence of SFV4. Additionally, nsP3 amino acid sequences differ between these two strains by eight amino acids and by a deletion of seven amino acids in the C-terminal third of rA774 nsP3. This study shows that neurovirulence can be reconstituted gradually by exchanging individual amino acids and is fully retained when combinations of two nsP3 mutations, V11→I and L201→F, V11→I and D249→N, A48→E and G70→A or T435→A and F442→L, are introduced into an rA774 derivative carrying R469. The critical role of the arginine codon for neurovirulence was confirmed further by the acquisition of a fully lethal phenotype following the introduction of R469 into a moderately virulent rA774 recombinant carrying the SFV4 nsP1 and nsP2 genes. In conclusion, virulence determinants in SFV are distributed over a wide region of the nonstructural genes.
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The major envelope protein, GP5, of a European porcine reproductive and respiratory syndrome virus contains a neutralization epitope in its N-terminal ectodomain
A set of neutralizing monoclonal antibodies (mAbs) directed against the GP5 protein of European type porcine reproductive and respiratory syndrome virus (PRRSV) has been produced previously ( Weiland et al., 1999 ). This set reacted with a plaque-purified virus (PPV) subpopulation of Dutch isolate Intervet-10 (I-10), but not with the European prototype PRRSV LV. In order to map the neutralization epitope in the GP5 protein of the PPV strain, the ORF5 nucleotide sequence of PPV was determined. When the amino acid sequence derived from this nucleotide sequence was compared with that of PRRSV LV, four amino acid differences were found. Using site-directed mutagenesis, we showed that a proline residue at position 24 of the GP5 sequence of the PPV strain enabled recognition by the neutralizing mAbs. Pepscan analysis demonstrated that the epitope recognized by the neutralizing mAbs stretched from residues 29 to 35. Surprisingly, the reactivity of the mAbs in the Pepscan system was independent of the presence of a proline in position 24. Moreover, residue 24 is located within the predicted signal peptide, implying that either the signal peptide is not cleaved or is cleaved due to the presence of Pro24 such that the epitope remains intact. Our results demonstrate the presence of a neutralization epitope in the N-terminal ectodomain of the GP5 protein of PRRSV and imply a role for the ectodomain of GP5 in the infection of PRRSV.
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Characterization of the structure and variability of an internal region of hepatitis C virus RNA for M1 RNA guide sequence ribozyme targeting
More LessAccessibility to folded RNA and low potential of variation in the target RNA are crucial requirements for ribozyme therapy against virus infections. In hepatitis C virus (HCV), the sequence of the 5′UTR is conserved but the highly folded RNA structure severely limits the number of accessible sites. To expand investigation of targeting in the HCV genome, we have considered an internal genomic region whose sequence variation has been widely investigated and which has a particularly conserved RNA structure, which makes it accessible to the human RNase P in vitro. We have first mapped the accessibility of the genomic RNA to complementary DNAs within this internal genomic region. We performed a kinetic and thermodynamic study. Accordingly, we have designed and assayed four RNase P M1 RNA guide sequence ribozymes targeted to the selected sites. Considerations of RNA structural accessibility and sequence variation indicate that several target sites should be defined for simultaneous attack.
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Hepatitis C virus IRES efficiency is unaffected by the genomic RNA 3′NTR even in the presence of viral structural or non-structural proteins
More LessHepatitis C virus (HCV) translation is mediated by an IRES structure. Instead of a poly(A) tail, the 3′ end of the genome contains a tripartite 3′NTR composed of a non-conserved region, a polypyrimidine tract and a highly conserved stretch of 98 nt, termed the 3′X region. Using a set of bicistronic recombinant DNA constructs expressing two reporter genes separated by the HCV IRES, it was determined whether the HCV 3′NTR sequence, in the presence or absence of HCV proteins, played a role in the efficiency of HCV IRES-dependent translation ex vivo. Bicistronic expression cassettes were transfected into hepatic and non-hepatic cell lines. These results show that neither the entire 3′NTR nor the 3′X sequence alters IRES-dependent translation efficiency, whatever the cell line tested. A potential effect of the 3′NTR on IRES-dependent translation in the presence of HCV proteins was investigated further. Neither non-structural nor structural HCV proteins had any effect on the efficiency of IRES in this system. In addition, in order to mimic HCV genome organization, monocistronic expression cassettes containing the IRES and a Core–DsRed fusion gene were constructed with or without the 3′NTR. In this context, no effect of the 3′NTR on IRES translation efficiency was observed, even in the presence of HCV proteins. These data demonstrate that HCV translation is not modulated by the viral genomic 3′NTR sequence, even in the presence of HCV structural or non-structural proteins.
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T helper responses to Japanese encephalitis virus infection are dependent on the route of inoculation and the strain of mouse used
More LessT helper cytokine and IgG subtype responses were studied in three strains of mice (C57BL/6J, Swiss albino, BALB/c; n=90 per strain) immunized with live Japanese encephalitis virus (JEV) by intraperitoneal (IP), subcutaneous (SC) and peroral (PO) routes. Lymphocytes obtained from the spleens of immunized and control mice were stimulated in vitro with JEV for 48 h and the supernatants were assayed for the presence of the cytokines IL-4 and IFN-γ. JEV-specific IgG isotypes were also measured in the sera of immunized mice. T helper cytokine responses in mice immunized with JEV were found to be strain- and route-specific in the three species tested. Moreover, they were also dependent on the type of immunogen used (live vs killed virus), as well as the number of doses administered. C57BL/6J and BALB/c mice were more uniform in their T helper responses compared with the outbred Swiss albino mice and induced a good Th1 response (P<0·001). Among the three routes evaluated, the IP and SC routes consistently elicited a Th1 response compared with the PO route (P<0·001), where an initial Th2-type response reverted to a Th1 response after repeated immunization. Live JEV induced a Th1 response while the commercial killed vaccine induced a predominant Th2 profile.
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Molecular epidemiology of dengue virus type 3 in Venezuela
During the past 40 years, dengue haemorrhagic fever and dengue shock syndrome (DHF/DSS) have emerged in humans, with approximately 3 million cases reported and over 58 000 deaths. Dengue virus serotypes 1, 2 and 4 (DENV-1, -2 and -4) have been co-circulating in Venezuela for at least the past 10 years, causing minor or major outbreaks of dengue fever (DF) and DHF/DSS. The first recorded outbreak due to DENV-3 in Venezuela dates to 1964 and the virus then seems to have disappeared. However, DENV-3 re-appeared recently (in July, 2000) in Venezuela after 32 years of absence and produced a prolonged major outbreak, which, by the end of 2001, involved 83 180 cases of dengue, mostly DF (92 %). Previous phylogenetic studies revealed that the DENV-3 circulating during the 1960s Latin American outbreak was a genotype V virus. To gain a better understanding of the nature of the current epidemic, the complete sequence was determined of the envelope (E) gene of 15 Venezuelan DENV-3 viruses isolated during 2000 and 2001 from patients presenting with different disease severity. Sequence data were used in phylogenetic comparisons with global samples of DENV-3. Analysis revealed that the strain circulating in Venezuela is closely related to isolates that were previously present in Panama and Nicaragua in 1994 and since then have spread through Central American countries and Mexico. This study also confirms previous reports showing that the DENV-3 strain currently circulating in the Americas is related to the strain that caused DHF epidemics in Sri Lanka and India in 1989–1991 (genotype III). Finally, no evidence of the re-emergence of the strain that circulated in Venezuela in the late 1960s and 1970s (genotype V) was found.
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Infection competition against grouper nervous necrosis virus by virus-like particles produced in Escherichia coli
More LessDragon grouper (Epinephelus lanceolatus) nervous necrosis virus (DGNNV) comprises 180 copies of capsid protein that encapsulate a bipartite genome of single-stranded (+)-RNAs. This study reports that virus-like particles (VLPs) are formed in Escherichia coli expressing the full-length ORF encoding the DGNNV capsid protein. Two sizes of VLPs are observed. The heavier particles resemble the native piscine nodavirus in size and stain permeability, while the lighter ones are approximately two-thirds of the full size. The recombinant VLPs block attachment of native virus to the surface of cultured fish nerve cells, blocking infection by the native virus.
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Comparisons of the complete genomes of Asian, African and European isolates of a recent foot-and-mouth disease virus type O pandemic strain (PanAsia)
More LessDuring the last 12 years, a strain of foot-and-mouth disease (FMD) virus serotype O, named PanAsia, has spread from India throughout Southern Asia and the Middle East. During 2000, this strain caused outbreaks in the Republic of Korea, Japan, Russia (Primorsky Territory), Mongolia and South Africa (KwaZulu-Natal Province), areas which last experienced FMD outbreaks in 1934, 1908, 1964, 1974 and 1957, respectively. In February 2001, the PanAsia strain spread to the United Kingdom where, in just over 7 months, it caused outbreaks on 2030 farms. From the UK, it quickly spread to the Republic of Ireland, France and the Netherlands. Previous studies that utilized RT-PCR to sequence the VP1-coding region of the RNA genomes of approximately 30 PanAsia isolates demonstrated that the UK virus was most closely related to the virus from South Africa (99·7 % nucleotide identity). To determine if there was an obvious genetic reason for the apparently high level of fitness of this new strain, and to further analyse the relationships between the PanAsia viruses and other FMDVs, complete genomes were amplified using long-range PCR techniques and the PCR products were sequenced, revealing the sequences for the entire genomes of five PanAsia isolates as well as an animal-passaged derivative of one of them. These genomes were compared to two other PanAsia genomes. These analyses revealed that all portions of the genomes of these isolates are highly conserved and provided confirmation of the close relationship between the viruses responsible for the South Africa and UK outbreaks, but failed to identify any genetic characteristic that could account for the unprecedented spread of this strain.
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The implications of virus diversity within the SAT 2 serotype for control of foot-and-mouth disease in sub-Saharan Africa
More LessSAT 2 is the serotype most often associated with outbreaks of foot-and-mouth disease (FMD) in livestock in southern and western Africa and is the only SAT type to have been recorded outside the African continent in the last decade. Its epidemiology is complicated by the presence of African buffalo (Syncerus caffer), which play an important role in virus maintenance and transmission. To assess the level of genetic complexity of this serotype among viruses associated with both domestic livestock and wildlife, complete VP1 gene sequences of 53 viruses from 17 countries and three different host species were analysed. Phylogenetic analysis revealed eleven virus lineages, differing from each other by at least 20 % in pairwise nucleotide comparisons, four of which fall within the southern African region, two in West Africa and the remaining five in central and East Africa. No evidence of recombination between these lineages was detected, and thus we conclude that these are independently evolving virus lineages which occur primarily in discrete geographical localities in accordance with the FMD virus topotype concept. Applied to the whole phylogeny, rates of nucleotide substitution are significantly different between topotypes, but most individual topotypes evolve in accordance with a molecular clock at an average rate of approximately 0·002 substitutions per site per year. This study provides an indication of the intratypic complexity of the SAT 2 serotype at the continental level and emphasizes the value of molecular characterization of diverse FMD field strains for tracing the origin of outbreaks.
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Mapping epitopes in equine rhinitis A virus VP1 recognized by antibodies elicited in response to infection of the natural host
Equine rhinitis A virus (ERAV) is an important respiratory pathogen of horses and is of additional interest because of its close relationship and common classification with foot-and-mouth disease virus (FMDV). As is the case with FMDV, the VP1 capsid protein of ERAV has been shown to be a target of neutralizing antibodies. In FMDV VP1, such antibodies commonly recognize linear epitopes present in the βG–βH loop region. To map linear B cell epitopes in ERAV VP1, overlapping fragments spanning its length were expressed in Escherichia coli as glutathione S-transferase (GST) fusion proteins. These fusion proteins were tested for reactivity with sera from ERAV-infected horses and with polyclonal sera from ERAV-immunized rabbits and mice. Regions at the N- and C-termini as well as the βE–βF and the βG–βH loop regions contained B cell epitopes that elicited antibodies in the natural host. GST fusion proteins of these regions also elicited antibodies following immunization of rabbits and mice, which, in general, strongly recognized native ERAV VP1 but which were non-neutralizing. It is concluded that the N-terminal region of ERAV VP1, in particular, contains strong B cell epitopes.
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Rabies virus matrix protein regulates the balance of virus transcription and replication
More LessRNA synthesis by negative-strand RNA viruses (NSVs) involves transcription of subgenomic mRNAs and replication of ribonucleoprotein complexes. In this study, the envelope matrix (M) protein of rabies virus (RV) was identified as a factor which inhibits transcription and stimulates replication. Transcription, but not replication, of RV minigenomes or of full-length RV was decreased by expression of heterologous M. Since RV assembly involving M and the glycoprotein G renders virus synthetically quiescent, an RV was generated with the M and G genes substituted by placeholders. Surprisingly, RNA synthesis by this recombinant was characterized not only by an increased transcription rate but also by a diminished accumulation of replication products. This phenotype was reversed in a dose-dependent manner by providing M in trans, showing that M is a replication-stimulatory factor. The role of M in determining the balance of replication and transcription was further exploited by generation of a recombinant RV with attenuated M expression, which is highly active in transcription. Regulation of RNA synthesis by matrix proteins may represent a general mechanism of nonsegmented NSVs, which is probably obscured by the silencing activity of M during virus assembly.
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An immunostimulatory oligodeoxynucleotide containing a cytidine-guanosine motif protects senescence-accelerated mice from lethal influenza virus by augmenting the T helper type 1 response
More LessThe SAM-P1 strain of senescence-accelerated model mice shows an impaired T helper type 1 (Th1) immune response upon infection with influenza virus, which results in high susceptibility to the virus. Treatment of spleen cells from SAM-P1 mice with an immunostimulatory oligodeoxynucleotide containing a cytidine-guanosine motif (CpG ODN) in vitro increased the ratio of the titre of IFN-γ to that of IL-4. Administration of CpG ODN to SAM-P1 mice generated satisfactory virus-specific cytotoxic T-lymphocyte responses and natural killer cell activation and the virus-specific immunoglobulin (Ig) isotype switched from IgG1 to IgG2a. Virus growth in the lungs of CpG ODN-treated SAM-P1 mice was cleared quickly and mice survived the lethal influenza virus infection. It could be inferred that a possible mechanism of CpG ODN for normalization of senescence-associated dysregulation of the Th1/Th2 balance involves the upregulated expression of CD154 and CD40 molecules on immune-competent cells. These results suggest that CpG ODN could contribute to the development of a protective strategy against infectious diseases, especially among immunocompromised elderly persons, by stimulating Th1 immune responses.
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Evidence that avian reovirus σA protein is an inhibitor of the double-stranded RNA-dependent protein kinase
More LessThe results of a previous study demonstrated that avian reovirus is highly resistant to the antiviral effects of interferon and suggested that the double-stranded RNA (dsRNA)-binding σA protein might play an important role in that resistance. To gather more evidence on the interferon-inhibitory activity of σA protein, its gene was cloned into the prokaryotic maltose-binding protein (MBP) gene fusion vector pMalC and into the recombinant vaccinia virus WRS2. The two recombinant σA proteins displayed a dsRNA-binding affinity similar to that of σA protein synthesized in avian reovirus-infected cells. Interestingly, MBP–σA, but not MBP, was able to relieve the translation-inhibitory activity of dsRNA in reticulocyte lysates by blocking the activation of endogenous dsRNA-dependent enzymes. In addition, transient expression of σA protein in HeLa cells rescued gene expression of a vaccinia virus mutant lacking the E3L gene, and insertion of the σA-encoding gene into vaccinia virus conferred protection for the virus against interferon in chicken cells. Further studies demonstrated that expression of recombinant σA in mammalian cells interfered with dsRNA-dependent protein kinase (PKR) function. From these results we conclude that σA is capable of reversing the interferon-induced antiviral state by down-regulating PKR activity in a manner similar to other virus-encoded dsRNA-binding proteins.
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Mutational analysis of two zinc-finger motifs in the nucleocapsid protein of simian immunodeficiency virus mac239
More LessTo clarify the physiological function of two zinc-finger (ZF) motifs in the nucleocapsid (NC) protein of simian immunodeficiency virus (SIV), we constructed three mutant viruses with alterations in either or both motifs using a molecular clone of SIVmac (SIVmac239). An immunoblot analysis of the cell lysates transfected with DNA mutated in either the first (ZF1) or second (ZF2) motif showed that the amount of partially processed Gag products (Pr46) was greater than that produced by the wild-type (WT). The genomic RNA contents in the viral particles released from the transfected cells were measured by quantitative RT-PCR. Values for the ZF1 and ZF2 mutants and the double mutant were 26, 20 and 7 % that of the WT, respectively, indicating that the two ZF motifs of SIVmac239 NC protein function almost equivalently with respect to RNA encapsidation and processing of Gag precursors. Despite the presence of some genomic RNA in the mutant viruses, they lost all viral infectivity. To determine the reason for this, we examined (using PCR) to which step viral DNA synthesis proceeded in the mutant viruses. We did not see any block up to the step of minus-strand DNA synthesis. However, plus-strand DNA synthesis after plus-strand transfer did not occur in any of the mutant viruses. These findings indicated that the mutations in the ZF motifs of SIVmac led to a loss of infectivity due partly to impairment of DNA synthesis, in addition to inefficient encapsidation of genomic RNA.
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- DNA viruses
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Comparison of virus production in chicken embryo fibroblasts infected with the WR, IHD-J and MVA strains of vaccinia virus: IHD-J is most efficient in trans-Golgi network wrapping and extracellular enveloped virus release
More LessModified vaccinia virus Ankara (MVA) is an attenuated strain derived from vaccinia virus (VV) Ankara that grows efficiently in primary chicken embryo fibroblasts (CEFs) and baby hamster kidney cells only. MVA produces significantly more of the enveloped forms of VV in infected CEFs compared with VV strain Copenhagen. In the present study, production of the different infectious forms of VV was compared in CEFs infected with MVA or with two well-characterized replication-competent VV strains, WR and IHD-J. In a time-course experiment, the infectivity associated with the extracellular enveloped virus (EEV), the cell-associated enveloped virus (CEV) and intracellular mature and enveloped viruses was determined. Further, the production of the different viral forms was quantified by electron microscopy (EM). The data collectively indicate that IHD-J is most efficient in producing all of the trans-Golgi network-wrapped forms and releases the highest titres of EEVs into the extracellular medium, with WR being least efficient. MVA initially replicated with faster kinetics, resulting in more intracellular virus and CEVs between 8 and 24 h post-infection (p.i.). As assessed by EM, the faster growth kinetics of MVA resulted in 3·5-fold more CEVs at the cell surface at 24 h p.i., compared with both WR and IHD-J. Accordingly, we found that despite the presence of two in-frame deletions in the A36R gene of MVA, this virus was able to make actin tails in CEFs.
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Phenotypic and genotypic characterization of clinical isolates of herpes simplex virus resistant to aciclovir
More LessA panel of 10 clinical isolates of herpes simplex virus (HSV) deficient in the expression of thymidine kinase (TK) and phenotypically resistant to aciclovir was characterized. Sequence analysis revealed a variety of mutations in TK (nucleotide substitutions, insertions and deletions), most of which resulted in truncated TK polypeptides. In line with previous reports, the most common mutation was a single G insertion in the ‘G-string’ motif. One HSV-1 isolate and two HSV-2 isolates appeared to encode full-length polypeptides and, in each case, an amino acid substitution likely to be responsible for the phenotype was identified. Pathogenicity was determined using a zosteriform model of HSV infection in BALB/c mice. The majority of isolates appeared to show impaired growth at the inoculation site compared with wild-type virus. They also showed poor replication in the peripheral nervous system and little evidence of zosteriform spread. One exception was isolate 4, which had a double G insertion in the G-string but, nevertheless, exhibited zosteriform spread. These studies confirmed that TK-deficient viruses display a range of neurovirulence with respect to latency and zosteriform spread. These results are discussed in the light of previous experience with TK-deficient viruses.
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Characterization of a neurovirulent aciclovir-resistant variant of herpes simplex virus
A clinical isolate of herpes simplex virus type 1 that is aciclovir resistant but neurovirulent in mice was described previously. The mutation in this virus is a double G insertion in a run of seven G residues that has been shown previously to be a mutational hotspot. Using a sensitive assay, it has been demonstrated that preparations of this virus are able to induce low but consistent levels of thymidine kinase (TK) activity. However, this activity results from a high frequency mutational event that inserts a further G into the ‘G-string’ motif and thus restores the TK open reading frame. Passage of this virus through the nervous system of mice results in the rapid selection of the TK-positive variant. Thus, this variant is the major component in virus reactivated from latently infected ganglia. Mutation frequency appears to be influenced by the genetic background of the virus.
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The genome of herpesvirus papio 2 is closely related to the genomes of human herpes simplex viruses
More LessInfection of baboons (Papio species) with herpesvirus papio 2 (HVP-2) produces a disease that is clinically similar to herpes simplex virus (HSV-1 and HSV-2) infection of humans. The development of a primate model of simplexvirus infection based on HVP-2 would provide a powerful resource to study virus biology and test vaccine strategies. In order to characterize the molecular biology of HVP-2 and justify further development of this model system we have constructed a physical map of the HVP-2 genome. The results of these studies have identified the presence of 26 reading frames that closely resemble HSV homologues. Furthermore, the HVP-2 genome shares a collinear arrangement with the genome of HSV. These studies further validate the development of the HVP-2 model as a surrogate system to study the biology of HSV infections.
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Five unique open reading frames of infectious laryngotracheitis virus are expressed during infection but are dispensable for virus replication in cell culture
More LessThe chicken alphaherpesvirus infectious laryngotracheitis virus (ILTV) exhibits several unique genetic features including an internal inversion of a conserved part of the unique long genome region. At one end, this inversion is preceded by a cluster of five open reading frames (ORFs) of 335–411 codons, designated ORF A to ORF E, that are not present in any other known herpesvirus genome. In this report we analysed expression of these genes and identified the corresponding viral RNA and protein products. Northern blot analyses showed 3′-coterminal transcripts of ORFs A and B, and monocistronic mRNAs of ORFs C and D. ORF E is part of a 3′-coterminal transcription unit that includes the conserved glycoprotein H and thymidine kinase genes. Monospecific antisera obtained after immunization of rabbits with bacterial fusion proteins allowed detection of the protein products of ORF A (40 kDa), ORF B (34 kDa), ORF C (38 and 30 kDa), ORF D (41 kDa) and ORF E (44 kDa) in ILTV-infected cells. For functional analyses, five virus recombinants possessing deletions within the individual ORFs and concomitant insertions of a reporter gene cassette encoding green fluorescent protein were generated. All virus mutants were replication competent in cell culture, but exhibited reduced virus titres or plaque sizes when compared to wild-type ILTV. These findings indicate that the ILTV-specific ORF A to ORF E genes might be important for virus replication in the natural host organism.
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Genetic characterization of the unique short segment of Phocid herpesvirus type 1 reveals close relationships among alphaherpesviruses of hosts of the order Carnivora
More LessTo further characterize phocid herpesvirus type 1 (PhHV-1) at the molecular level, a cluster of genes comprising the complete unique short (Us) region of PhHV-1 has been cloned and sequenced. Within this region, ORFs were detected that code for the equivalent of the Us 2− protein of herpes simplex virus (HSV), a putative protein kinase, and for the glycoprotein equivalents gG, gD, gI and gE. In addition, two small ORFs downstream of gE, homologous to the Us 8·5 and Us 9 proteins of HSV were identified. Comparative analysis of the ORF encoding the gD equivalent of PhHV-1 identified the corresponding proteins of the alphaherpesviruses canine herpesvirus and, to lesser degree, feline herpesvirus as the closest relatives.
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Rescue of human cytomegalovirus strain AD169 tropism for both leukocytes and human endothelial cells
Endothelial cell-tropism- and leukocyte- (polymorphonuclear- and monocyte-) tropism (leukotropism) are two important biological properties shared by all recent clinical isolates of human cytomegalovirus (HCMV). These properties are lost during extensive propagation of HCMV isolates in human fibroblasts, as shown by reference laboratory-adapted strains AD169 and Towne. Here we show that strain AD169 may reacquire both properties in vitro, endothelial (both venous and arterial) cell-tropism preceding leukotropism (predominantly involving monocytes). Restriction fragment length polymorphism analysis and sequencing performed on the original virus inoculum from human fibroblasts and serial passages on endothelial cells confirmed virus identity. Thus, fundamental biological properties may be lost and reacquired in vitro according to the cell culture system employed. The lack of a 15 kb DNA fragment in the strain AD169 genome does not prevent the rescue of these biological functions, thus indicating that they are likely to be encoded by viral genes located elsewhere.
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Human cytomegalovirus requires cellular deoxycytidylate deaminase for replication in quiescent cells
More LessWe have previously observed that the expression of two thymidylate biosynthesis enzymes, dihydrofolate reductase and thymidylate synthase (TS), is upregulated in quiescent human fibroblasts infected with human cytomegalovirus (HCMV). Here, we have demonstrated that HCMV increases expression of the cellular deoxycytidylate deaminase (dCMP deaminase), which provides the substrate for TS by converting dCMP to dUMP. We observed an increase in dCMP deaminase protein levels, whereas deoxyuridine triphosphatase (dUTPase), another cellular enzyme that may provide dUMP by hydrolysing dUTP, was undetectable. The essential requirement of cellular dCMP deaminase for productive HCMV replication was further emphasized by showing that a precursor of a potent dCMP deaminase inhibitor, zebularine, suppressed virus replication and DNA synthesis. These results suggest that HCMV exploits the host's dCMP deaminase activity to replicate in quiescent cells.
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Updated Epstein–Barr virus (EBV) DNA sequence and analysis of a promoter for the BART (CST, BARF0) RNAs of EBV
Two sequences required for activity of the Epstein–Barr virus BART RNA promoter in transfection assays have been identified by site-directed mutagenesis. One contains a consensus AP-1 site; the other has some similarity to Ets and Stat consensus binding sites. Candidate sequences were suggested by mapping a region of unmethylated DNA in EBV around the BART promoter followed by in vivo footprinting the promoter in the C666-1 nasopharyngeal carcinoma cell line, which expresses BART RNAs. The data are presented in the context of a revised EBV DNA sequence, known as EBV wt, that is proposed as a future standard sequence for EBV.
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The latency-associated nuclear antigen of Kaposi's sarcoma-associated herpesvirus interacts preferentially with the terminal repeats of the genome in vivo and this complex is sufficient for episomal DNA replication
More LessThe genome of Kaposi's sarcoma-associated herpesvirus (KSHV) persists in latently infected cells as a circular episome. The latency-associated nuclear antigen (LANA) has been shown to tether viral DNA fragments to chromosomes and is proposed to maintain the KSHV genome. In order to identify the in vivo-binding sites for LANA on the whole KSHV genome and to analyse the function of this protein–DNA interaction, different in vivo systems have been developed. Chromatin immunoprecipitation experiments using three different cell lines latently infected with KSHV demonstrated that LANA binds preferentially and directly to the terminal repeats (TRs) but not to other regions of the viral chromosome in vivo. In contrast, in vitro LANA–DNA binding was much less specific. To identify autonomously replicating sequences within the KSHV genome, BCBL-1 cells were transfected with cosmids representing the entire genome. Cosmid Z2, consisting of the right end of the unique region and TRs, persisted as an episome in short-term assays. Long term, stable episome replication was observed with constructs derived from Z2 containing TRs only. LANA expression constructs containing a variable number of TRs replicated stably as episomes in uninfected cells. A 424 bp subfragment of the 801 bp TR could mediate episome replication. These studies show that LANA is a trans-acting protein that binds preferentially to TRs in vivo and these two elements are sufficient for episome replication. These results also suggest that the LANA expression plasmids reported here could be utilized as episomal vectors in a manner similar to Epstein–Barr virus-based vectors.
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The latency protein LANA2 from Kaposi's sarcoma-associated herpesvirus inhibits apoptosis induced by dsRNA-activated protein kinase but not RNase L activation
More LessKaposi's sarcoma-associated herpesvirus (KSHV) uses several strategies to counteract the interferon (IFN) system. In this study, the relationship of the protein LANA2 from KSHV to the IFN-activated protein kinase (PKR) and 2-5A system was analysed. It was found that LANA2 could not abrogate apoptosis or RNA degradation mediated by the 2-5A system. However, expression of LANA2 inhibited apoptosis triggered by PKR. LANA2 also counteracted the PKR-mediated inhibition of protein synthesis and partially blocked PKR-induced phosphorylation of eIF-2α. Analysis of PKR-induced activation of caspases 3 and 9 revealed that LANA2 abrogated activation of caspase 3 but not of caspase 9. These findings show that LANA2 is able to interfere with downstream events triggered by PKR. Hence, LANA2 should be considered as a KSHV defence protein against IFN.
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Transcription mapping of human herpesvirus 8 genes encoding viral interferon regulatory factors
More LessThe human herpesvirus 8 (HHV-8) genome contains four tandemly arranged genes encoding viral interferon regulatory factors (vIRF-1 to 4) located between genes 57 and 58. Transcript mapping techniques were employed to determine the sizes, ends and splicing patterns of mRNAs specified by these genes in HHV-8-infected cell lines untreated or chemically induced into the lytic growth cycle. Depending on the cell line used, vIRF-3 transcription was minimally or not induced (i.e. expressed with latent kinetics), whereas the other vIRFs were inducible (i.e. expressed with lytic kinetics). Each gene possessed its own promoter (or promoters) and polyadenylation sites, and all but vIRF-1 were spliced from two exons. vIRF-1 was transcribed in uninduced and induced cells from a single initiation site preceded by a TATA box, with the possible use of an additional TATA box and initiation site in uninduced cells. In induced cells, vIRF-2 was transcribed from a single major initiation site preceded by a TATA box, and vIRF-4 was expressed from two sites each preceded by a TATA box. Transcripts for these genes were insufficiently abundant in uninduced cells to map the 5′-ends. vIRF-3 lacks an obvious TATA box and exhibited heterogeneous 5′-ends in uninduced and induced cells. These data clarify and extend our understanding of the structure and transcription of the HHV-8 vIRF genes.
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Glycoproteins M and N of human herpesvirus 8 form a complex and inhibit cell fusion
More LessGlycoproteins M (gM) and N (gN) are well conserved across the herpesvirus family and their involvement in virus penetration and egress is well described, especially for alphaherpesviruses. Because there was no previous study on the homologues of human herpesvirus 8 glycoproteins M (gM8) and N (gN8), we analysed their biochemical and functional characteristics. We found that: (i) gM8 aggregated following heat treatment; (ii) gM8 was a virion component; (iii) gM8 and gN8 were N-glycosylated; (iv) gM8 formed a specific complex with gN8; and (v) gN8 was required for functional processing of gM8. Co-expression of gM8 and gN8 inhibited cell fusion induced either by a combination of herpes simplex virus type 1 glycoproteins or by Molony murine leukaemia virus envelope protein. These results indicate that, in addition to the similar biochemical properties, the fusion inhibition reported previously only for alphaherpesviruses is a function conserved in the gammaherpesvirus subfamily.
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Human papillomavirus type 6 virus-like particles present overlapping yet distinct conformational epitopes
The epitope for a human papillomavirus (HPV) type 6 conformation-dependent, neutralizing monoclonal antibody (mAb) was partially mapped using HPV L1 recombinant virus-like particles (VLPs). The mAb H6.J54 is cross-reactive with the closely related HPV types 6 and 11. By making HPV-6-like amino acid substitutions in the cottontail rabbit papillomavirus (CRPV) major capsid protein L1, we were able to transfer H6.J54 binding activity into a CRPV/HPV-6 hybrid L1 protein. Full binding activity was achieved with only nine amino acid changes and identified a region centred on the HPV-6 residues 49–54. This region has previously been shown to be a critical part of HPV-6 type-specific epitopes. Fine mapping of the region by scanning a series of alanine substitution mutations showed that in HPV-6 VLPs this type-common epitope overlaps HPV-6 type-specific epitopes.
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Seroepidemiology of the human polyomaviruses
More LessTo assess the stability of polyomavirus antibodies in serial samples over time and the incidence and age-specific prevalence of polyomavirus infections, we established enzyme immunoassays (EIAs) using purified yeast-expressed virus-like particles (VLPs) containing the VP1 major capsid proteins of JC virus (JCV) and the AS and SB strains of BK virus (BKV). A random subsample of 150 Finnish women who had serum samples taken during the first trimester of pregnancy and had a second pregnancy during a 5 year follow-up period was selected, grouped by age of first pregnancy. The polyomavirus antibody levels were similar in samples taken during the first and second pregnancies (correlation coefficient 0·93 for BKV SB and 0·94 for JCV). Analysis of serum samples from 290 Swedish children aged 1–13 years, grouped by age in 2 year intervals, demonstrated that BKV seropositivity increased rapidly with increasing age of the children, reaching 98 % seroprevalence at 7–9 years of age, followed by a minor decrease. JCV seroprevalence increased only slowly with increasing age and reaching 72 % positivity among mothers >25 years of age. The age-specific seroprevalence of the human polyomaviruses measured using this VLP-based EIA was similar to previous serosurveys by other methods. The stability of the antibodies over time indicates that polyomavirus seropositivity is a valid marker of cumulative virus exposure, and polyoma VLP-based EIAs may therefore be useful for epidemiological studies of these viruses.
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Genetic variability of porcine parvovirus isolates revealed by analysis of partial sequences of the structural coding gene VP2
The 3′-terminal 853 nt (and the putative 283 aa) sequence of the VP2-encoding gene from 29 field strains of porcine parvovirus (PPV) were determined and compared both to each other and with other published sequences. Sequences were examined using maximum-parsimony and statistical analyses for nucleotide diversity and sequence variability. Among the nucleotide sequences of the PPV field strains, 26 polymorphic sites were encountered; 22 polymorphic sites were detected in the putative amino acid sequence. Mapping polymorphic sites of protein data onto the three-dimensional (3D) structure of PPV VP2 revealed that almost all substitutions were located on the external surface of the viral capsid. Mapping amino acid substitutions to the alignment between PPV VP2 sequences and the 3D structure of canine parvovirus (CPV) capsid, many PPV substitutions were observed to map to regions of recognized antigenicity and/or to contain phenotypically important residues for CPV and other parvoviruses. In spite of the high sequence similarity, genetic analysis has shown the existence of at least two virus lineages among the samples. In conclusion, these results highlight the need for close surveillance on PPV genetic drift, with an assessment of its potential ability to modify the antigenic make-up of the virus.
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Transcriptional analysis of the white spot syndrome virus major virion protein genes
More LessWhite spot syndrome virus (WSSV) is a member of a new virus family (Nimaviridae) infecting crustaceans. The regulation of transcription of WSSV genes is largely unknown. Transcription of the major WSSV structural virion protein genes, vp28, vp26, vp24, vp19 and vp15, was studied to search for common promoter motifs for coordinate expression. The temporal expression of these genes and both 5′ and 3′ ends of the mRNA were determined, using infected crayfish gill tissue as a RNA source. RT-PCR showed that all five genes are expressed late in infection compared to the early ribonucleotide reductase large subunit gene. 5′ RACE studies revealed a consensus late transcription initiation motif for only two of the five major virion protein genes. This motif was only found in one other upstream region of the putative translational start site of a gene with unknown function (ORF 158). No other conserved sequence motifs could be detected in the sequences surrounding the transcriptional start sites of the five major virion protein genes. All 5′ ends were located about 25 nt downstream of an A/T rich sequence, including the consensus TATA-box sequence for vp15. The absence of a consensus motif is distinct from gene regulation of other large dsDNA viruses and suggests a unique regulation of WSSV transcription, in line with its unique taxonomic position.
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Use of Spring beauty latent virus to identify compatible interactions between bromovirus components required for virus infection
More LessSpring beauty latent virus (SBLV) is a member of the genus Bromovirus, and is closely related to Brome mosaic virus (BMV) and Cowpea chlorotic mottle virus (CCMV). Compatible interactions between viral components are required for successful infection of plants by BMV and CCMV. To further our understanding of interactions between bromovirus components, we used SBLV to produce reassortants among the three bromoviruses. We found that SBLV RNA 2 functioned with heterologous bromovirus RNA 1 in infections of whole plants and protoplasts of Nicotiana benthamiana, although SBLV RNA 1 did not function with heterologous bromovirus RNA 2. A DNA-based transient assay for 1a and 2a proteins, which are encoded by RNAs 1 and 2, respectively further suggested that SBLV 2a protein may function in combination with heterologous bromovirus 1a protein. Moreover, analysis of the ability of reassortants to spread locally revealed that an RNA 2-mediated interaction between viral components may be required for efficient cell-to-cell movement of bromoviruses.
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Infectivity of recombinant strawberry vein banding virus DNA
More LessInfectivity of the cloned DNA genome of strawberry vein banding virus (SVBV) was demonstrated by particle bombardment of 4-week-old strawberry (Fragaria vesca L. var. UC-5) plants with gold particles coated with the putative full-length 7·9 kb viral DNA. Vein banding symptoms developed on 15 % of inoculated plants 6–7 weeks post-inoculation. An approximate 1·25-mer of the viral DNA was cloned into the binary vector pCGN1547. Particle bombardment of this construct into strawberry plants gave an infection rate of 75 %. The construct was used for transformation of Agrobacterium tumefaciens, and infiltration of these cells into healthy strawberry leaves resulted in development of vein banding symptoms in 100 % of inoculated plants. Gel electrophoresis, Southern blot hybridization with an SVBV probe and sequence analyses of PCR-amplified DNA fragments were used to confirm SVBV infection in symptomatic plants.
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Volumes and issues
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Volume 105 (2024)
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Volume 104 (2023)
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Volume 102 (2021)
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Volume 101 (2020)
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Volume 7 (1970)
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Volume 6 (1970)
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Volume 5 (1969)
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Volume 4 (1969)
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Volume 3 (1968)
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Volume 2 (1968)
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Volume 1 (1967)