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Volume 82,
Issue 1,
2001
Volume 82, Issue 1, 2001
- Animal: RNA Viruses
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Molecular strategy for ‘serotyping’ of human enteroviruses
More LessTo explore further the phylogenetic relationships between human enteroviruses and to develop new diagnostic approaches, we designed a pair of generic primers in order to study a 1452 bp genomic fragment (relative to the poliovirus Mahoney genome), including the 3′ end of the VP1-coding region, the 2A- and 2B-coding regions, and the 5′ moiety of the 2C-coding region. Fifty-nine of the 64 prototype strains and 45 field isolates of various origins, involving 21 serotypes and 6 strains untypeable by standard immunological techniques, were successfully amplified with these primers. By determining the nucleotide sequence of the genomic fragment encoding the C-terminal third of the VP1 capsid protein we developed a molecular typing method based on RT–PCR and sequencing. If field isolate sequences were compared to human enterovirus VP1 sequences available in databases, nucleotide identity score was, in each case, highest with the homotypic prototype (74.8 to 89.4%). Phylogenetic trees were generated from alignments of partial VP1 sequences with several phylogeny algorithms. In all cases, the new classification of enteroviruses into five identified species was confirmed and strains of the same serotype were always monophyletic. Analysis of the results confirmed that the 3′ third of the VP1-coding sequence contains serotype-specific information and can be used as the basis of an effective and rapid molecular typing method. Furthermore, the amplification of such a long genomic fragment, including non-structural regions, is straightforward and could be used to investigate genome variability and to identify recombination breakpoints or specific attributes of pathogenicity.
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Deletion or substitution of the aphthovirus 3′ NCR abrogates infectivity and virus replication
More LessThe 3′ noncoding region (NCR) of the genomic picornaviral RNA is believed to contain major cis-acting signals required for negative-strand RNA synthesis. The 3′ NCR of foot-and-mouth disease virus (FMDV) was studied in the context of a full-length infectious clone in which the genetic element was deleted or exchanged for the equivalent region of a distantly related swine picornavirus, swine vesicular disease virus (SVDV). Deletion of the 3′ NCR, while maintaining the intact poly(A) tail as well as its replacement for the SVDV counterpart, abrogated virus replication in susceptible cells as determined by infectivity and Northern blot assays. Nevertheless, the presence of the SVDV sequence allowed the synthesis of low amounts of chimeric viral RNA at extended times post-transfection as compared to RNAs harbouring the 3′ NCR deletion. The failure to recover viable viruses or revertants after several passages on susceptible cells suggests that the presence of specific sequences contained within the FMDV 3′ NCR is essential to complete a full replication cycle and that FMDV and SVDV 3′ NCRs are not functionally interchangeable.
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Genetic variability among group A and B respiratory syncytial viruses in Mozambique: identification of a new cluster of group B isolates
Respiratory syncytial virus (RSV) is the major cause of acute lower respiratory tract infection in children and vulnerable adults, but little is known regarding RSV infection in Africa. In this report, a recent RSV outbreak in Mozambique was studied and results showed that 275 of 3192 (8·6%) nasopharyngeal aspirates tested were RSV-positive by ELISA. RSV presents two antigenic groups (A and B) with a high genetic and antigenic variability between and within them. Analysis by a new RFLP assay of RT–PCR amplified N protein gene products showed a higher prevalence of group B RSV than that of group A (85% versus 15%). However, genetic variability of the G protein gene was higher among group A RSV strains. The frequency and pattern of glycosylation sites were also quite different between both groups. In addition, two different phylogenetic clusters of Mozambican viruses were found within each group, but only sequences from cluster B-I were relatively distinct from previously described isolates. The implications of such differences in the antigenic and immunogenic characteristics of each group are discussed.
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Binding of the La autoantigen to the hepatitis C virus 3′ untranslated region protects the RNA from rapid degradation in vitro
More LessWe have analysed hepatitis C virus (HCV) RNAs in an in vitro RNA degradation assay. We found that the 3′ end of positive polarity HCV RNA is sensitive to cytosolic RNases whereas the 3′ end of negative polarity HCV RNA is relatively stable. Interaction of the HCV 3′ untranslated region with the cellular La protein prevented premature degradation of the HCV RNA. One may speculate that HCV RNAs interact with La protein in infected cells to prevent premature degradation of the viral RNAs.
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Zinc-binding properties of Junín virus nucleocapsid protein
The arenavirus nucleocapsid protein (N) is a highly basic 63 kDa protein with a dual function during the virus life-cycle. First, it is involved in essential steps of genome replication, promoting the synthesis of the full-length antigenomic copy of S RNA, and second it associates with the genomic RNA to form the nucleocapsid. We have expressed the N protein of Junín virus in E. coli and shown that it binds zinc in vitro. This property is in agreement with the presence in the carboxy-terminal region of the N protein of the CX2HX23CX4C sequence, which resembles a classical zinc-finger motif. The specificity for zinc binding was demonstrated by competition with other divalent metal ions. The ability of the predicted motif to bind zinc was established by analysis of a series of N mutants, including truncated variants and amino acid substitutions. In addition, alternative zinc-binding sites were found.
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T-helper and humoral responses to Puumala hantavirus nucleocapsid protein: identification of T-helper epitopes in a mouse model
More LessPuumala hantavirus (PUUV) is a rodent-borne agent causing nephropathia epidemica in humans, a milder form of haemorrhagic fever with renal syndrome occurring in Fennoscandia, central Europe and western Russia. In this study we characterized the immunogenicity of an E. coli-expressed nucleocapsid (N) protein of PUUV (strain Kazan-E6) in inbred mice (BALB/c, CBA and C57/BL6). The recombinant N (rN) protein raised PUUV-specific antibodies in all three tested murine haplotypes, and all IgG subclasses were detected. Epitope mapping using peptides spanning the N protein revealed that the B-cell recognition sites were mainly located at the amino-terminal part of the protein. Proliferative T-helper (Th) lymphocyte responses were detected in all haplotypes after a single immunization with rN. Several Th-recognition sites, spanning amino acids 6–27, 96–117, 211–232 and 256–277, were identified using overlapping peptides. Peptides representing the identified sites could also prime Th-lymphocytes to proliferate in response to recall with rN protein, thereby confirming the authenticity of the identified sites. The rN-primed Th-lymphocytes produced predominantly interleukin (IL)-2 and gamma interferon, together with lower levels of IL-4 and IL-6, indicating a mixed Th1/Th2 response.
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Characterization of chimeric enzymes between caprine arthritis–encephalitis virus, maedi–visna virus and human immunodeficiency virus type 1 integrases expressed in Escherichia coli
More LessIn order to investigate the functions of the three putative lentiviral integrase (IN) protein domains on viral DNA specificity and target site selection, enzymatically active chimeric enzymes were constructed using the three wild-type IN proteins of caprine arthritis–encephalitis virus (CAEV), maedi–visna virus (MVV) and human immunodeficiency virus type 1 (HIV-1). The chimeric enzymes were expressed in Escherichia coli, purified by affinity chromatography and analysed in vitro for IN-specific endonuclease and integration activities on various DNA substrates. Of the 21 purified chimeric IN proteins constructed, 20 showed distinct site-specific cleavage activity with at least one substrate and six were able to catalyse an efficient integration reaction. Analysis of the chimeric IN proteins revealed that the central domain together with the C terminus determines the activity and substrate specificity of the enzyme. The N terminus appears to have no considerable influence. Furthermore, an efficient integration activity of CAEV wild-type IN was successfully demonstrated after detailed characterization of the reaction conditions that support optimal enzyme activities of CAEV IN. Also, under the same in vitro assay conditions, MVV and HIV-1 IN proteins exhibited endonuclease and integration activities, an indispensable prerequisite of domain-swapping experiments. Thus, the following report presents a detailed characterization of the activities of CAEV IN in vitro as well as the analysis of functional chimeric lentiviral IN proteins.
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Variation of African horsesickness virus nonstructural protein NS3 in southern Africa
More LessNS3 protein sequences of recent African horsesickness virus (AHSV) field isolates, reference strains and current vaccine strains in southern Africa were determined and compared. The variation of AHSV NS3 was found to be as much as 36·3% across serotypes and 27·6% within serotypes. NS3 proteins of vaccine and field isolates of a specific serotype were found to differ between 2·3% and 9·7%. NS3 of field isolates within a serotype differed up to 11·1%. Our data indicate that AHSV NS3 is the second most variable AHSV protein, the most variable being the major outer capsid protein, VP2. The inferred phylogeny of AHSV NS3 corresponded well with the described NS3 phylogenetic clusters. The only exception was AHSV-8 NS3, which clustered into different groups than previously described. No obvious sequence markers could be correlated with virulence. Our results suggest that NS3 sequence variation data could be used to distinguish between field isolates and live attenuated vaccine strains of the same serotype.
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VP5 and the N terminus of VP2 are not responsible for the different pathotype of serotype I and II infectious bursal disease virus
More LessTwo serotypes have been identified in infectious bursal disease virus (IBDV), a member of the family Birnaviridae. A reverse genetics system was used for generation of chimeras in genome segment A of the two serotypes, in which the complete viral VP5 gene and 3′ noncoding region (NCR), or parts thereof, were exchanged. The engineered viruses were characterized in vitro and in vivo in comparison to serotype I and II IBDV. Our results show that IBDV chimeras exhibit a different phenotype in cell culture compared to the wild-type viruses. In in vitro-cultivated bursal-derived cells, chimeric viruses infected B lymphocytes, as does serotype I IBDV. Surprisingly, serotype II virus was also able to infect in vitro-cultivated bursal cells, but these were neither B lymphocytes nor macrophages. After infection of susceptible chickens all chimeras replicated in the bursa of Fabricius (BF), and three chimeric viruses caused mild depletion of bursal cells. In contrast, after infection of chickens with a chimeric IBDV containing exchanged VP5 as well as 3′-NCR, no depletion was detectable. The serotype II strain did not replicate in the BF nor did it cause depletion of bursal cells. Thus, the origin of VP5 does not explain the different pathotype of IBDV serotype I and II.
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- Animal: DNA Viruses
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Hepatitis B virus X protein protects against anti-Fas-mediated apoptosis in human liver cells by inducing NF-κB
More LessThe hepatitis B virus-encoded X antigen (HBxAg) may contribute to the development of liver cancer, in part, by stimulating the growth and survival of infected cells in the face of ongoing immune responses. Given that the Fas ligand/receptor system contributes to the pathogenesis of chronic hepatitis B, experiments were designed to test the hypothesis that HBxAg mediates resistance of liver cells to anti-Fas killing. Accordingly, when HBxAg was introduced into HepG2 cells, it rendered these cells partially resistant to killing by anti-Fas. In HepG2 cells replicating virus, protection against anti-Fas killing was also observed, but to a lesser extent. Survival correlated with the activation of nuclear factor kappa B (NF-κB) by HBxAg. Sensitivity to anti-Fas was observed in control cells, and was re-established in HepG2X cells stably transfected with the dominant negative inhibitor of NF-κB, IκBα. HBxAg activation of NF-κB was also associated with decreased levels of endogenous IκBα mRNA. Hence, HBxAg stimulation of NF-κB promotes the survival of liver cells against Fas killing. This may contribute to the persistence of infected hepatocytes during chronic infection.
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Construction and characterization of recombinant porcine adenovirus serotype 5 expressing the transmissible gastroenteritis virus spike gene
Tamás Tuboly and Éva NagyFive recombinant porcine adenoviruses of serotype 5 (PAdV-5) carrying the full-length or the 5′ 2·2 kb half of the transmissible gastroenteritis virus (TGEV) spike (S) gene were generated by homologous recombination in E. coli strain BJ5183 cells and subsequent transfection of swine testicle cells. The foreign genes were inserted into the E3 region of PAdV-5. One recombinant virus had no deletion in the E3 region, whereas a 1·2 kb fragment was removed from the E3 region in the remainder of the recombinant viruses. One stable construct with a 4·4 kb insertion had a genome size of 109·6% of the wild-type genome, the largest reported for any recombinant adenovirus. Only those viruses that carried the S gene in the left to right orientation expressed the S gene. Three recombinant viruses were tested by oral immunization of pigs and both antibody response and virus shedding were monitored. None of the pigs showed clinical signs and the virus was recovered from rectal swabs until 6–7 days post-infection. Viruses expressing the S gene induced TGEV- and PAdV-5-specific virus-neutralizing antibodies. Moreover, TGEV-specific secretory IgA was detected in the small intestine and in the lungs of the immunized animals.
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The VP1-unique region of parvovirus B19: amino acid variability and antigenic stability
More LessThe unique region of structural protein VP1 of parvovirus B19 (erythrovirus B19) is important for eliciting neutralizing antibodies that are responsible for eliminating the virus from the peripheral blood and for inducing lifelong immunity. Neutralizing human MAbs bind a conformationally defined epitope spanning VP1 residues 30–42. The DNA sequence encoding the VP1-unique region was determined in parvovirus B19 isolated from peripheral blood and amniotic fluid of nine acutely infected pregnant women, five arthritis patients and two chronically infected children. The amino acid sequences of the VP1-unique region exhibited higher variability in comparison with other B19-specific proteins. To analyse the influence of amino acid variations on antibody binding and protein conformation, two variants of the VP1-unique region were selected and expressed in E. coli as intein-fusion proteins. The selected variants displayed a number of amino acid exchanges in the VP1-unique region and had mutations in the determined epitope and adjacent regions. After purification via affinity chromatography, the dissociation constants K D of VP1-specific human MAbs interacting with the variant antigens and a viral prototype of the VP1-unique region were determined with a quartz crystal microbalance biosensor. A value of 5·4×10−8 M was determined for the prototype isolate pJB; the affinity constants for the variant VP1-unique regions were similar. Comparable values were obtained for interaction of antibodies with non-infectious VP1/VP2 capsids produced by recombinant baculovirus and with B19 virions from amniotic fluid. It is concluded that the conformation of the epitope is unaffected by mutations or the environment of the VP1-unique region in virus capsids.
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Oestrogen and progesterone increase the levels of apoptosis induced by the human papillomavirus type 16 E2 and E7 proteins
More LessHuman papillomavirus (HPV) type 16 infects the genital tract and is generally acknowledged to be a causative agent of cervical cancer. HPV infection alone is not sufficient to induce cervical cancer and other factors such as steroid hormones are thought to play a role in the establishment and/or progression of this disease. The HPV-16 E2 protein is required for virus replication and modulates viral gene expression whereas the HPV-16 E7 protein is required for cell transformation. We and others have shown that both the E2 and E7 proteins can induce apoptotic cell death in HPV-transformed and non-HPV transformed cell lines. Here we show that the steroid hormones oestrogen and progesterone can both increase the levels of E2- and E7-induced apoptosis. The oestrogen metabolite 16α-hydroxyoestrone also increases E2- and E7-induced cell death and the dietary component indole-3-carbinol, which reduces the formation of 16α-hydroxyoestrone from oestrogen, blocks the effects of oestrogen. Thus the metabolism of oestrogen to 16α-hydroxyoestrone appears to be required for the effects of this hormone on E2- and E7-induced cell death. We also show that the oestrogen receptor antagonist 3-hydroxytamoxifen blocks the effects of oestrogen on E2- and E7-induced cell death, whereas the anti-progesterone RU486 blocks the effects of both progesterone and oestrogen. We discuss these results in terms of the origin and progression of cervical cancer.
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Role of the cytoplasmic tails of pseudorabies virus glycoproteins B, E and M in intracellular localization and virion incorporation
More LessThe cytoplasmic domains of several herpesviral glycoproteins encompass potential intracellular sorting signals. To analyse the function of the cytoplasmic domains of different pseudorabies virus (PrV) glycoproteins, hybrid proteins were constructed consisting of the extracellular and transmembrane domains of envelope glycoprotein D (gD) fused to the cytoplasmic tails of gB, gE or gM (designated gDB, gDE and gDM), all of which contain putative endocytosis motifs. gD is a type I membrane protein required for binding to and entry into target cells. Localization of hybrid proteins compared to full-length gB, gE and gM as well as carboxy-terminally truncated variants of gD was studied by confocal laser scanning microscopy. The function of gD hybrids was assayed by trans-complementation of a gD-negative PrV mutant. The carboxy-terminal domains of gB and gM directed a predominantly intracellular localization of gDB and gDM, while full-length gD and a tail-less gD mutant (gDc) were preferentially expressed on the cell surface. In contrast gDE, and a gDB lacking the putative gB endocytosis signal (gDBΔ29), were predominantly located in the plasma membrane. Despite the different intracellular localization, all tested proteins were able to complement infectivity of a PrV gD− mutant. Cells which stably express full-length gD and plasma-membrane-associated gD hybrids exhibit a significant resistance to PrV infection, while cells expressing predominantly intracellularly located forms do not. This suggests that the assumed sequestration of receptors by gD, which is supposed to be responsible for the interference phenomenon, occurs at the cell surface.
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Recognition of another member of the malignant catarrhal fever virus group: an endemic gammaherpesvirus in domestic goats
More LessA novel gammaherpesvirus in goats that is herein tentatively designated as caprine herpesvirus-2 was identified based on the sequence of a fragment from the herpesvirus DNA polymerase gene. Sequence alignment analysis revealed that the virus sequence isolated from goats was 67% identical to the homologous sequence from alcelaphine herpesvirus-1, 71% identical to ovine herpesvirus-2 and 73% identical to a recently recognized herpesvirus causing malignant catarrhal fever in white-tailed deer. Combined serological and PCR-survey data demonstrated that this virus is endemic in goats and its transmission pattern may be similar to that of ovine herpesvirus-2 in sheep.
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Detection of avian oncogenic Marek’s disease herpesvirus DNA in human sera
More LessThe avian herpesvirus Marek’s disease virus (MDV) has a worldwide distribution and is responsible for T-lymphoma in chickens. The question as to whether MDV poses a public health hazard to humans was first raised when the virus was isolated in 1967. However, no irrefutable results have been obtained in immunological and virological studies. We used a nested-PCR to detect MDV DNA in human serum samples. A total of 202 serum samples from individuals exposed and not exposed to poultry was tested by nested-PCR for a target sequence located in the MDV gD gene. The assay system was specific and sensitive, making it possible to detect a single copy of the target sequence. Forty-one (20%) of the 202 serum samples tested positive for MDV DNA. The prevalence of MDV DNA was not significantly different in the group exposed to poultry and the group not exposed to poultry. There was also no difference due to age or sex. Alignment of the 41 gD sequences amplified from human sera with eight gD sequences amplified from MDV-infected chicken sera showed a maximum nucleotide divergence of 1·65%. However, four ‘hot-spot’ mutation sites were identified, defining four groups. Interestingly, two groups contained only human MDV-gD sequences. The status of the MDV genome detected in human blood is discussed.
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- Insect
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The sequence of the Helicoverpa armigera single nucleocapsid nucleopolyhedrovirus genome
The nucleotide sequence of the Helicoverpa armigera single-nucleocapsid nucleopolyhedrovirus (HaSNPV) DNA genome was determined and analysed. The circular genome encompasses 131403 bp, has a G+C content of 39·1 mol% and contains five homologous regions with a unique pattern of repeats. Computer-assisted analysis revealed 135 putative ORFs of 150 nt or larger; 100 ORFs have homologues in Autographa californica multicapsid NPV (AcMNPV) and a further 15 ORFs have homologues in other baculoviruses such as Lymantria dispar MNPV (LdMNPV), Spodoptera exigua MNPV (SeMNPV) and Xestia c-nigrum granulovirus (XcGV). Twenty ORFs are unique to HaSNPV without homologues in GenBank. Among the six previously sequenced baculoviruses, AcMNPV, Bombyx mori NPV (BmNPV), Orgyia pseudotsugata MNPV (OpMNPV), SeMNPV, LdMNPV and XcGV, 65 ORFs are conserved and hence are considered as core baculovirus genes. The mean overall amino acid identity of HaSNPV ORFs was the highest with SeMNPV and LdMNPV homologues. Other than three ‘baculovirus repeat ORFs’ (bro) and two ‘inhibitor of apoptosis’ (iap) genes, no duplicated ORFs were found. A putative ORF showing similarity to poly(ADP-ribose) glycohydrolases (parg) was newly identified. The HaSNPV genome lacks a homologue of the major budded virus (BV) glycoprotein gene, gp64, of AcMNPV, BmNPV and OpMNPV. Instead, a homologue of SeMNPV ORF8, encoding the major BV envelope protein, has been identified. GeneParityPlot analysis suggests that HaSNPV, SeMNPV and LdMNPV (group II) have structural genomic features in common and are distinct from the group I NPVs and from the granuloviruses. Cluster alignment between group I and group II baculoviruses suggests that they have a common ancestor.
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Analysis of the capsid processing strategy of Thosea asigna virus using baculovirus expression of virus-like particles
More LessThosea asigna virus (TaV), a putative member of the genus Betatetravirus of the family Tetraviridae, is predicted to have a novel capsid expression strategy compared with other characterized tetraviruses. The capsid precursor protein is cleaved twice to generate three proteins. Two of the proteins, L (58·3 kDa) and S (6·8 kDa), are incorporated into the TaV virion. The third, non-structural protein, produced from the N terminus of the precursor protein, is up to 17 kDa in size and is of unknown function. The TaV capsid precursor protein sequence without the 17 kDa N-terminal region was modelled against the solved structure from Nudaurelia ω virus (NωV) using SwissModel. The TaV model was very similar to the solved structure determined for subunit A of NωV and had features that are conserved between tetraviruses and nodaviruses, including the positioning of the cleavage site between the L and S capsid proteins. The production of virus-like particles (VLPs) using the baculovirus expression system was used to analyse the capsid processing strategy employed by TaV. VLPs were formed in both the presence and absence of the 17 kDa N-terminal region of the capsid precursor. VLPs were not formed when the L and S regions were expressed from separate promoters, indicating that cleavage between the L and S capsid proteins was an essential part of TaV capsid assembly. Expression of the TaV 17 kDa protein in bacteria did not produce intracellular tubules similar to those formed by bacterial expression of the p17 protein from Helicoverpa armigera stunt virus.
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- Plant
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Nucleotide sequence, genome organization and phylogenetic analysis of pineapple mealybug wilt-associated virus-2
More LessThe genome of pineapple mealybug wilt-associated closterovirus-2 (PMWaV-2) was cloned from double-stranded RNA isolated from diseased pineapple and its sequence determined. The 3′-terminal 14861 nt of the single-stranded RNA genome contains ten open reading frames (ORFs) which, from 5′ to 3′, potentially encode a >204 kDa polyprotein containing papain-like protease, methyltransferase and helicase domains (ORF1a), a 65 kDa RNA-dependent RNA polymerase (ORF1b), a 5 kDa hydrophobic protein (ORF2), a 59 kDa heat shock protein 70 homologue (ORF3), a 46 kDa protein (ORF4), a 34 kDa coat protein (ORF5), a 56 kDa diverged coat protein (ORF6), a 20 kDa protein (ORF7), a 22 kDa protein (ORF8) and a 6 kDa protein (ORF9). A 132 nt untranslated region was present at the 3′ terminus of the genome. This genome organization is typical of the monopartite closteroviruses, including the putative +1 ribosomal frameshift allowing expression of ORF1b. Phylogenetic analysis revealed that within the family Closteroviridae the mealybug-transmitted PMWaV-2 is more closely related to other mealybug-transmitted members than to those which are transmitted by aphids or whiteflies. Within this group, PMWaV-2 shares the greatest sequence identity with grapevine leafroll-associated virus-3, another mealybug-transmitted closterovirus.
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Rapid identification of a tobacco mosaic virus epitope by using a coat protein gene-fragment–pVIII fusion library
More LessThis study describes the identification of the epitope recognized by the tobacco mosaic virus (TMV) coat protein (CP)-specific monoclonal antibody 29 (MAb29) by displaying a CP gene-fragment library on pVIII of filamentous phage M13. More than 80% of the clones isolated after one round of panning bound specifically to MAb29. DNA sequencing of ten randomly chosen MAb29-specific clones and subsequent sequence comparison revealed a common seven amino acid epitope (ELIRGTG) representing amino acids 131–137 of the TMV CP. The reactivity of MAb29 in competition ELISA towards glutathione S-transferase fused to this epitope was stronger than that towards full-length wild-type TMV CP, confirming the epitope sequence determined by gene-fragment phage display. This demonstrated that gene-fragment libraries displayed on the phage surface as fusion proteins with the filamentous bacteriophage gene VIII are useful tools for rapid identification of linear epitopes recognized by MAbs.
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