Showing papers in "Archives of Virology in 2001"
TL;DR: The resultant primer set is suitable for all influenza A viruses to generate full-length cDNAs, to subtype viruses, to sequence their DNA, and to construct expression plasmids for reverse genetics systems.
Abstract: To systematically identify and analyze the 15 HA and 9 NA subtypes of influenza A virus, we need reliable, simple methods that not only characterize partial sequences but analyze the entire influenza A genome. We designed primers based on the fact that the 15 and 21 terminal segment specific nucleotides of the genomic viral RNA are conserved between all influenza A viruses and unique for each segment. The primers designed for each segment contain influenza virus specific nucleotides at their 3'-end and non-influenza virus nucleotides at the 5'-end. With this set of primers, we were able to amplify all eight segments of N1, N2, N4, N5, and N8 subtypes. For N3, N6, N7, and N9 subtypes, the segment specific sequences of the neuraminidase genes are different. Therefore, we optimized the primer design to allow the amplification of those neuraminidase genes as well. The resultant primer set is suitable for all influenza A viruses to generate full-length cDNAs, to subtype viruses, to sequence their DNA, and to construct expression plasmids for reverse genetics systems.
1,924 citations
TL;DR: Seventy-eight bovine viral diarrhoea viruses recently collected in Austria, France, Hungary, Italy, Slovakia, Spain and UK were genetically typed in the 5′-untranslated and autoprotease regions of the pestivirus genome, suggesting significant antigenic similarities within the BVDV-1 genotype.
Abstract: Seventy-eight bovine viral diarrhoea viruses (BVDV) recently collected in Austria, France, Hungary, Italy, Slovakia, Spain and UK were genetically typed in the 5'-untranslated (5'UTR) and autoprotease (Npro) regions of the pestivirus genome. Seventy-six of the isolates were BVDV-1 and two French isolates were of the BVDV-2 genotype. Phylogenetic analysis of the 5'UTR (245 nt), including additional BVDV-1 sequences from USA, Canada, Germany, New Zealand, Mozambique and Sweden, taken from GenBank and from our previous works, indicated that these viruses were clustered not only into the two generally accepted groups (BVDV-1a-"NADL like" and BVDV-1b-"Osloss like"), but altogether into 11 phylogenetic groups. Similar clustering was observed with Npro region sequences (385 nt) and the highest bootstrap values (over 95%) were obtained by phylogeny combining 5'UTR and Npro sequences. Some associations between the genetic grouping and the origin of the isolates were apparent, probably reflecting historical trade contacts. Considering the variability of isolates it is recommended that diagnostic PCR primers should be re-examined to ensure coverage of all BVDV-1 groups. The genogroups were less clearly differentiated by monoclonal antibody typing, suggesting significant antigenic similarities within the BVDV-1 genotype.
352 citations
TL;DR: The distribution of phages in different bacterial phylogenetic divisions is shown and siphoviridae or phages with long, noncontractile tails comprise 61% of tailed phages.
Abstract: Over 5100 bacterial viruses have been examined in the electron microscope since 1959. About 4950 phages (96%) are tailed and only 186 phages (3.6%), are cubic, filamentous, or pleomorphic. Phages belong to 13 virus families and occur in over 140 bacterial genera. Phages are listed by morphotypes and host genera. Siphoviridae or phages with long, noncontractile tails comprise 61% of tailed phages. The distribution of phages in different bacterial phylogenetic divisions is shown.
324 citations
TL;DR: It is concluded that the Italian HPAI viruses arose from low pathogenicity strains, and that a deletion in the NA stalk followed by the acquisition of additional glycosylation near the receptor binding site of HA1 may be an adaptation of H7 viruses to a new host species i.e. domestic poultry.
Abstract: Outbreaks of avian influenza due to an H7N1 virus of low pathogenicity occurred in domestic poultry in northern Italy from March 1999 until December 1999 when a highly pathogenic avian influenza (HPAI) virus emerged. Nucleotide sequences were determined for the HA1 and the stalk region of the neuraminidase (NA) for viruses from the outbreaks. The HPAI viruses have an unusual multibasic haemagglutinin (HA) cleavage site motif, PEIPKGSRVRRGLF. Phylogenetic analysis showed that the HPAI viruses arose from low pathogenicity viruses and that they are most closely related to a wild bird isolate, A/teal/Taiwan/98. Additional glycosylation sites were present at amino acid position 149 of the HA for two separate lineages, and at position 123 for all HPAI and some low pathogenicity viruses. Other viruses had no additional glycosylation sites. All viruses examined from the Italian outbreaks had a 22 amino acid deletion in the NA stalk that is not present in the N1 genes of the wild bird viruses examined. We conclude that the Italian HPAI viruses arose from low pathogenicity strains, and that a deletion in the NA stalk followed by the acquisition of additional glycosylation near the receptor binding site of HA1 may be an adaptation of H7 viruses to a new host species i.e. domestic poultry.
291 citations
TL;DR: This review summarizes the existing knowledge on this important group of viruses and their vector in this region and concludes that the proliferation and rapid dissemination of whitefly-transmitted viruses of important food and industrial crops in Latin America have been the consequence of drastic changes in traditional cropping systems.
Abstract: The proliferation and rapid dissemination of whitefly-transmitted viruses of important food and industrial crops in Latin America, have been the consequence of drastic changes in traditional cropping systems. Some of the expanding non-traditional cash and export crops, such as soybean and several vegetables, have served as suitable reproductive hosts for the whitefly Bemisia tabaci. This insect pest has been shown to transmit at least 20 different geminiviruses that affect different commercial and basic food crops in Latin America. This review summarizes the existing knowledge on this important group of viruses and their vector in this region.
288 citations
TL;DR: A universal primer, designed from the consensus sequences that code for the conserved sequence GNNSGQP in the NIb region of members of the family Potyviridae, was used to amplify by RT-PCR the 3′-terminal genome regions from infected plant samples representing 21 different viruses in the family.
Abstract: A universal primer (Sprimer: 5′-GGX AAY AAY AGY GGX CAZ CC-3′, X = A, G, C or T; Y = T or C; Z = A or G), designed from the consensus sequences that code for the conserved sequence GNNSGQP in the NIb region of members of the family Potyviridae, was used to amplify by RT-PCR the 3′-terminal genome regions from infected plant samples representing 21 different viruses in the family. Sequencing of some of the fragments (c. 1.7 kb) showed that the type strain (ATTC PV-107) of Oat necrotic mottle virus is not a distinct species in the genus Rymovirus, but is synonymous with Brome streak mosaic virus (genus Tritimovirus) and that Celery mosaic virus is a distinct member of the genus Potyvirus not closely related to any other sequenced species. Potyviruses infecting crops in China were also investigated, showing that viruses on cowpea and maize in Hangzhou, Zhejiang province were respectively Bean common mosaic virus and Sugarcane mosaic virus and that one on garlic in Nanjing, Jiangsu province was Onion yellow dwarf virus. Fragments were also sequenced from Chinese isolates of Lettuce mosaic virus and Soybean mosaic virus (from Hangzhou), Turnip mosaic virus (2 different isolates from Zhejiang province) and RNA1 of Wheat yellow mosaic virus (from Rongcheng, Shandong province).
275 citations
TL;DR: Close analysis of this iteron-related domain (IRD) revealed consistent correlations between specific Rep residues and defined nucleotides of its cognate iteron, thus providing important insights about the molecular code which dictates the Rep preference for specific DNA sequences.
Abstract: Geminiviruses encode a replication initiator protein, Rep, which binds in a sequence-specific fashion to iterated DNA motifs (iterons) functioning as essential elements for virus-specific replication. By using the iterons of more than one hundred geminiviruses as heuristic devices, we have identified a Rep subdomain 8 to 10 residues in length, whose primary structure varies among viruses harboring different iterons, but which is similar among viruses with identical iterons, regardless of their differences in host range, insect vector, geographical origin or genome structure. Close analysis of this iteron-related domain (IRD) revealed consistent correlations between specific Rep residues and defined nucleotides of its cognate iteron, thus providing important insights about the molecular code which dictates the Rep preference for specific DNA sequences. A model of potential Rep-iteron contacts is proposed. The identified IRD is adjacent to a conserved motif characteristic of a superfamily of rolling-circle (RC) replication proteins, and secondary structure predictions suggest that those Rep subdomains form together the core of a novel DNA-binding domain possessing a β-sheet as recognition subdomain, which is apparently conserved in the replication proteins of nanoviruses, circoviruses, microviruses, and a variety of ssDNA plasmids of eubacteria, archaebacteria and red algae. The evolutionary implications of these findings are discussed.
213 citations
TL;DR: This is the first demonstration of RT-PCR detection on a panel of H7 strains using only one primer set and the H7 origin and the pathogenic potential defined by the presence or absence of basic amino acids at the cleavage site can be determined by sequencing of the PCR product.
Abstract: Avian influenza virus infections are a major cause of morbidity and rapid identification of the virus has important clinical, economical and epidemiological implications. We have developed a one-tube Reverse Transcriptase Polymerase Chain Reaction (RT-PCR) for the rapid diagnosis of avian influenza A. A panel of reference influenza strains from various hosts including avian species, human, swine and horse were evaluated in a one tube RT-PCR using primers designed for the amplification of a 218 bp fragment of the NP gene. The PCR products were detected by PCR-ELISA by use of an internal catching probe confirming the NP influenza A origin. The PCR-ELISA was about 100 times more sensitive than detection of PCR products by agarose gel electrophoresis. RT-PCR and detection by PCR-ELISA is comparable in sensitivity to virus propagation in eggs. We also designed primers for the detection of the influenza. A subtypes H5 and H7 shown to have pathogenic potential in poultry. The H5 primers cover the cleavage site of the HA gene and specifically amplify influenza A subtype H5. The H7 primers also cover the HA cleavage site and detected all H7 reference strains investigated. In addition, the H7 primers also amplified very weak and/or additional bands on an agarose gel from other subtypes. However, the H7 origin and the pathogenic potential defined by the presence or absence of basic amino acids at the cleavage site can be determined by sequencing of the PCR product. As far as we know this is the first demonstration of RT-PCR detection on a panel of H7 strains using only one primer set.
159 citations
TL;DR: An informative molecular marker is described that permits provisionalBegomovirus identification and classification using a begomoviral sequence that is smaller than the presently accepted, but less accessible CP sequence.
Abstract: Polymerase chain reaction (PCR) was applied to detect and establish provisional identity of begomoviruses through amplification of a ∼ 575 bp fragment of the begomoviral coat protein gene (CP), referred to as the ‘core’ region of the CP gene (core CP). The core CP fragment contains conserved and unique regions, and was hypothesized to constitute a sequence useful for begomovirus classification. Virus relationships were predicted by distance and parsimony analyses using the A component (bipartite viruses) or full genome (monopartite viruses), CP gene, core CP, or the 200 5′-nucleotides (nt) of the CP. Reconstructed trees and sequence divergence estimates yielded very similar conclusions for all sequence sets, while the CP 5′-200 nt was the best strain discriminator. Alignment of the core CP region for 52 field isolates with reference begomovirus sequences permitted provisional virus identification based on tree position and extent of sequence divergence. Geographic origin of field isolates was predictable based on phylogenetic separation of field isolates examined here. A ‘closest match’ or genus-level identification could be obtained for previously undescribed begomoviruses using the BLAST program to search a reference core CP database located at our website and/or in GenBank. Here, we describe an informative molecular marker that permits provisional begomovirus identification and classification using a begomoviral sequence that is smaller than the presently accepted, but less accessible CP sequence.
145 citations
TL;DR: The results suggested that the evolution of equine-2 influenza virus resembled the multiple evolution pathways of influenza B virus, indicating antigenic distinctions among these viruses.
Abstract: We reported previously that equine-2 influenza A virus (H3N8) had evolved into two genetically and antigenically distinct “Eurasian” and “American” lineages. Phylogenetic analysis, using the HA1 gene of more recent American isolates, indicated a further divergence of these viruses into three evolution lineages: A South American lineage, a Kentucky lineage, and a Florida lineage. These multiple evolution pathways were not due to geographic barriers, as viruses from different lineages co-circulated. For the Kentucky lineage, the evolution rate was estimated to be 0.89 amino acid substitutions per year, which agreed with the previously estimated rate of 0.8. For the South American lineage, the evolution rate was estimated to be only 0.27 amino acid substitutions per year. This low evolution rate was probably due to a unique alternating Ser138 to Ala138 substitutions at antigenic site A. For the Kentucky lineage, there was a preference for sequential nonsynonymous substitutions at antigenic site B, which was also a “hot spot” for amino acid substitutions. Convalescent sera had minimal cross-reactivity to viruses of different lineages, indicating antigenic distinctions among these viruses. In contrast to human H3N2 viruses, our results suggested that the evolution of equine-2 influenza virus resembled the multiple evolution pathways of influenza B virus.
141 citations
TL;DR: The production, preliminary characterisation and applications of monoclonal antibodies (mabs) against six porcine circovirus 2 isolates are described and one of the seven mabs tested demonstrated neutralising activity against PCV2 isolates from Canada and France.
Abstract: The production, preliminary characterisation and applications of monoclonal antibodies (mabs) against six porcine circovirus 2 isolates are described. A total of 14 stable hybridomas were produced, of which 7 were characterised. All of the mabs characterised were of IgG isotype. All the mabs tested reacted by IIF with acetone-fixed cell cultures infected with PCV2 isolates from Canada, France, Spain, Denmark, USA and UK. No cross-reactivity with a porcine circovirus 1 field isolate was demonstrated using the panel of mabs tested. In addition, one of the seven mabs tested demonstrated neutralising activity against PCV2 isolates from Canada and France. The use of selected PCV2-specific mabs for the development of virus detection methodologies is described.
TL;DR: Results confirm field and experimental data documenting reinfection of the respiratory and enteric tracts of cattle, suggesting that, in closed herds, respiratory or enteric tract reinfections may constitute a source of BCV transmissible to cows (WD) or neonatal or feedlot calves.
Abstract: A 1-step RT-PCR assay, targeting a 730 bp fragment of the nucleocapsid (N) gene of bovine coronavirus (BCV), and a nested PCR assay, targeting a 407 bp fragment of the N gene, were developed to detect BCV in nasal swab and fecal samples of calves experimentally exposed to BCV. Both 1-step RT-PCR and nested PCR recognized cell culture passaged isolates of 10 bovine respiratory coronavirus (BRCV), 5 calf diarrhea (CD) and 8 winter dysentery (WD) strains of BCV, but not transmissible gastroenteritis coronavirus or bovine rotavirus. The sensitivity of the 1-step RT-PCR and nested PCR was compared to that of an antigen-capture ELISA. The lowest detection limit of the 1-step RT-PCR and nested PCR as determined by using tenfold serial dilutions of the BRCV 255 and 440 strains in BCV negative nasal swab suspensions from preexposure gnotobiotic calves was 2 x 10(4) and 2 x 10(2) TCID50/0.1 ml for each strain, respectively. The lowest detection limit of the antigen-capture ELISA as determined by using the same serially diluted samples was 1 x 10(6) TCID50/0.1 ml for each strain. Therefore, the 1-step RT-PCR and nested PCR assays were 50 and 5000 times, respectively more sensitive than the antigen-capture ELISA to detect BRCV in nasal swab suspensions. To investigate in vivo cross-protection between the BRCV and CD or WD strains of BCV and to detect nasal and fecal shedding of BCV using the 1-step RT-PCR, nested PCR and antigen-capture ELISA, 6 colostrum-deprived and two gnotobiotic calves were inoculated with a BRCV, a CD or a WD strain of BCV and then challenged 3-4 weeks later with either BRCV, CD or WD strains of BCV. All calves developed diarrhea after inoculation and BCV antigen (ELISA) or RNA (RT-PCR) was detected in the diarrheic fecal samples or the corresponding nasal swab samples. In addition, low amounts of BCV were also detected only by nested PCR in the fecal and nasal swab samples before and after diarrhea. No respiratory clinical signs were observed during the entire experimental period, but elevated rectal temperatures were detected during diarrhea in the BCV-inoculated calves. All calves recovered from infection with the BRCV, CD, or WD strains of BCV were protected from BCV-associated diarrhea after challenge exposure with either a heterologous or homologous strain of BCV. However, all calves challenged with heterologous BCV strains showed subclinical BCV infection evident by detection of nasal and fecal shedding of BCV RNA detected only by nested PCR. Such results confirm field and experimental data documenting reinfection of the respiratory and enteric tracts of cattle, suggesting that, in closed herds, respiratory or enteric tract reinfections may constitute a source of BCV transmissible to cows (WD) or neonatal or feedlot calves. In addition, the present 1-step RT-PCR and nested PCR assays were highly sensitive to detect BCV in nasal swab and fecal specimens. Therefore, these assays should be useful to diagnose BCV infections in calves and adult cows.
TL;DR: The nucleotide sequences of segments S1 to S6 of a Chinese isolate of Rice black-streaked dwarf virus were determined, providing the first complete sequence of a plant pathogenic member of the genus Fijivirus.
Abstract: The nucleotide sequences of segments S1 to S6 of a Chinese isolate of Rice black-streaked dwarf virus (RBSDV) were determined. This provides the first complete sequence of a plant pathogenic member of the genus Fijivirus. The complete ten-segment genome has 29141 nucleotides, making it the largest reovirus genome so far reported. Each of the segments S1–S6 is predicted to encode a single major protein. Protein comparisons indicated that S1 encoded an RNA dependent RNA polymerase, with similarities to that encoded by S1 of Nilaparvata lugens reovirus (NLRV). S2 and S3 appeared to be homologous to S3 and S4 respectively of both Fiji disease virus (FDV) and NLRV. The protein encoded on S4 showed some similarity to that of NLRV S2. The proteins encoded on S5 and S6, though similar in size to those of NLRV S5 and S6, had no detectable homologies to them or to any other known protein.
TL;DR: Virus replication was partially restored by the addition of 2-aminopurine (2-AP), an inhibitor of dsRNA inducible protein kinase (PKR) and restored the viral RNA content per cell to near normal levels, suggesting that inhibition of viral RNA synthesis was through PKR.
Abstract: Porcine reproductive and respiratory syndrome virus (PRRSV) belongs to a group of RNA viruses that establish persistent infections. A proposed strategy for evading immunity during persistent PRRSV infection is by preventing the induction of IFN activity in pigs and/or by blocking the activation of antiviral proteins in permissive cells. IFN-γ mRNA expression was observed in the lymph nodes and lungs of pigs infected with wild-type PRRSV strain SDSU-23983. Pretreatment of MARC-145 cells with IFN-γ inhibited wild-type (SDSU-23983 P6) and culture-adapted (SDSU-23983 P136) PRRS viruses in a dose-dependent manner and at relatively low concentrations. The effect of IFN-γ on virus replication included reductions in the number of infected cells, virus yield, and RNA content in single cells. Virus replication was partially restored by the addition of 2-aminopurine (2-AP), an inhibitor of dsRNA inducible protein kinase (PKR). The addition of 2-AP also restored the viral RNA content per cell to near normal levels, suggesting that inhibition of viral RNA synthesis was through PKR. The principal difference between P6 and P136 isolates was the recovery of P136 replication with lower concentrations of 2-AP. Immunostaining with anti-PKR antibody showed a redistribution of PKR from the cytoplasm into nucleoli of infected cells.
TL;DR: Comparisons of pathogenesis of acute and latent infections with closely related bovine herpesvirus types 1 and 5 in their natural host indicate that, after primary infection, BHv-1 and BHV-5 displayed similar biological features and consequently need to be considered together for the control of BhV-1 infection.
Abstract: This study was conducted to compare the pathogenesis of acute and latent infections with closely related bovine herpesvirus types 1 (BHV-1) and 5 (BHV-5) in their natural host. Two groups of eight calves were inoculated intranasally with BHV-1 or BHV-5. Although BHV-1 and BHV-5 similarly replicate in the nasal mucosa after inoculation, both viruses differ markedly in their ability to cause disease, BHV-5 being responsible of some fatal encephalitis while BHV-1 inducing rhinotracheitis. Virus isolation and immunohistochemistry demonstrated that BHV-5 replicates extensively in neurons of the central nervous system (CNS) and in respiratory cells of lungs, tracheal and nasal mucosae. Invasion of the CNS likely occurs through the trigeminal and olfactory pathways. Both groups developed cross-neutralising antibodies during this experiment suggesting partial clinical cross-protection afforded by the two infections. Three months after primary infection, experimental reactivation showed that BHV-5 was able to establish latency in the trigeminal ganglia but also the CNS of surviving calves. Moreover, laboratory findings suggested that BHV-5 could also persist in the tracheal and nasal mucosae. These results indicate that, after primary infection, BHV-1 and BHV-5 displayed similar biological features and consequently need to be considered together for the control of BHV-1 infection.
TL;DR: Data suggest that the expression of episomal BSV observed during the in vitro procedure is correlated with the presence of an integrated form, similar to that observed during micropropagation of banana streak virus.
Abstract: Banana streak virus (BSV) is causing increasing concern in almost every producing area of banana and plantain (Musa spp.) worldwide. This situation appeared partially linked to some breeding lines and micropropagated hybrids. A complete BSV sequence integrated into the genome of a triploid plantain has been recently characterised and it has been hypothesised that it could give rise to infectious virus via recombination. In this study, we evaluated the effect of a routine micropropagation procedure on the expression of BSV in the FHIA 21 tetraploid hybrid. The widespread presence of integrated sequences and the absence of episomal BSV in thirty FHIA 21 “mother plants” selected for micropropagation were first confirmed by specific PCR and IC-PCR tests. The proliferation stage of the procedure, characterised by an intensive production of neoformed buds, appeared determinant in BSV expression whereas the rooting and acclimatisation stages had little or no effect. The duration in culture and the way of subdividing the clumps of proliferation influenced greatly the percentage of episomal BSV infections, reaching 58% of infected micropropagated lines after six in vitro subcultures. These data suggest that the expression of episomal BSV observed during the in vitro procedure is correlated with the presence of an integrated form.
TL;DR: The percentage of conserved amino acid sites across the 50 SAT-1 viruses compared in this study was 50%.
Abstract: Genetic relationships of 50 SAT-1 type foot-and-mouth disease viruses were determined by phylogenetic analysis of an homologous 417 nucleotide region encoding the C-terminal half of the VP1 gene and part of the 2A segment. Viruses obtained from persistently-infected African buffalo populations were selected in order to assess the regional genetic variation within the host species and compared with ten viruses recovered from recent and historical cases of clinical infection. Phylogenetic reconstructions identified three independently evolving buffalo virus lineages within southern Africa, that correspond with the following discrete geographic localities: (1) South Africa and southern Zimbabwe, (2) Namibia, Botswana and western Zimbabwe, and (3) Zambia, Malawi and northern Zimbabwe. This strict geographic grouping of viruses derived from buffalo was shown to be useful for determining the origin of recent SAT-1 epizootics in livestock.
TL;DR: The isolation and characterisation of porcine circovirus 2 (PCV2) from cases of sow abortion and Porcine dermatitis and nephropathy syndrome suggests that the clinical scope of PCV2 infections requires continuous re-evaluation.
Abstract: We report the isolation and characterisation of porcine circovirus 2 (PCV2) from cases of sow abortion and porcine dermatitis and nephropathy syndrome. The results suggest that the clinical scope of PCV2 infections requires continuous re-evaluation.
TL;DR: Results and high sequence identity among all HAstV antigenic types in the transition region and RNA structural predictions supported a potential recombination site at the ORF1b/ORF2 junction, the first evidence that recombination occurs among human astroviruses.
Abstract: We report a naturally occurring human astrovirus (HAstV) strain detected in two different geographic locations. We identified two isolates of this strain in a diarrhea outbreak at a child care center in Houston, Texas; and two isolates in diarrhea stool samples from two children in Mexico City. All four isolates were detected in stool samples by enzyme immunoassay (EIA). One of the Mexican isolates was typed by EIA and all four isolates were HAstV-5 by typing RT-PCR. The four isolates were >97% nucleotide-identical in two different genomic regions: ORF1a (246nt), and the 3′ end of the genome (471nt). One isolate from each geographic location was further sequenced in the transition region from ORF1b to ORF2 (1255nt) and this region of the two isolates showed ≥ 99% nt identity. Phylogenetic analyses of sequences of eight HAstV antigenic types and the novel strain in the transition region demonstrated the new strain being closely related to HAstV-3 in ORF1b, but closest to HAstV-5 in ORF2. These results and high sequence identity among all HAstV antigenic types in the transition region and RNA structural predictions supported a potential recombination site at the ORF1b/ORF2 junction. This is the first evidence that recombination occurs among human astroviruses.
TL;DR: The MEC associated with diarrhea in mink are morphologically similar to but are genetically distinct from the cultivable MCV and likely represent a new member of the SLV genus.
Abstract: Enteric caliciviruses are emerging pathogens responsible for diarrhea or gastroenteritis in their respective hosts. In this report, mink enteric caliciviruses (MEC) were detected in feces from diarrheic mink by both immune electron microscopy (IEM) and RT-PCR using a broadly reactive primer pair (p289/290) targeting the highly conserved RNA polymerase regions of the enteric caliciviruses, Norwalk-like viruses (NLVs) and Sapporo-like viruses (SLVs). The MEC possess classical caliciviral morphology with typical cup-shaped depressions on the viral surface. Sequence analyses based on nucleotide and predicted amino acid (aa) sequences of the RT-PCR products indicated that MEC is most closely related genetically to SLVs of humans and animals. The MEC shared the highest aa identities (64–71%) in the RNA polymerase region with both human SLVs and the porcine enteric calicivirus (PEC) Cowden strain SLV, indicating that MEC may belong to an individual genogroup or subgroup in the SLV genus. The MEC shared only limited aa identities in the RNA polymerase region with vesiviruses (40–51%) and NLVs (29–33%). The RNA polymerase regions of the cultivable, non-enteric mink caliciviruses (MCV) were also amplified by RT-PCR using the primer pair Pol1/Pol3 based on sequences of vesiviruses, and the primer pair p289/290. Sequence analysis indicated that these MCV shared higher aa identities in the RNA polymerase region with vesiviruses (58–81%) than with SLVs (43–51%) including the MEC, lagoviruses (35–37%) and NLVs (27–35%), suggesting that they are most closely related genetically to vesiviruses. The MEC associated with diarrhea in mink are morphologically similar to but are genetically distinct from the cultivable MCV and likely represent a new member of the SLV genus.
TL;DR: It is demonstrated that SLVs and NLVs could be classified uniformly on the basis of the entire capsid sequences and that the 11 SLV strains could be genetically classified into 3 major genetic groups, genogroups I, II and III, comprised of 5 genetic subgroups.
Abstract: “Sapporo-like viruses” (SLVs) and “Norwalk-like viruses” (NLVs) are an important cause of acute gastroenteritis in humans. While NLVs have been genetically classified into three major genetic groups consisting of 17 genetic subgroups, a classification of SLVs into comparable genetic groups remains to be determined. In an attempt to classify both SLVs and NLVs uniformly, the sequences of 2 SLV strains newly detected from French infants were analysed together with the published sequences of 9 SLV and 19 NLV strains. Distance and phylogenetic analyses were conducted on the sequences of the capsid gene, RNA polymerase gene, 3’ open reading frame (3’ORF), ORF overlapping the capsid gene, and 3’ untranslated region (3’UTR). The histogram showing frequency distribution of pairwise distances and the topology of the phylogenetic tree demonstrated that SLVs and NLVs could be classified uniformly on the basis of the entire capsid sequences and that the 11 SLV strains could be genetically classified into 3 major genetic groups, genogroups I, II and III, comprised of 5 genetic subgroups. The differentiation of the 11 SLV strains into these genetic groups was also maintained in the 4 remaining genome regions, while the sequences at the junction between the RNA polymerase and capsid genes were shown to be genogroup-specific.
TL;DR: It is revealed that the constructed chimeric protein may have utility as a serological diagnostic reagent and for further immunological studies that may provide new insights on mechanisms of protective immunity to ASFV.
Abstract: A chimera of the two immunodominant African swine fever (ASF) virus proteins p54 and p30 was constructed by insertion of the gene CP204L into a Not I restriction site of E183L gene. The resulting chimeric protein p54/30, expressed by a recombinant baculovirus in insect cells and in Trichoplusia ni larvae, retained antigenic determinants present in both proteins and reacted in Western blot with a collection of sera from inapparent ASF virus carrier pigs. Remarkably, pigs immunized with the chimeric protein developed neutralizing antibodies and survived the challenge with a virulent African swine fever virus, presenting a reduction of about two logs in maximum viremia titers with respect to control pigs. In conclusion, this study revealed that the constructed chimeric protein may have utility as a serological diagnostic reagent and for further immunological studies that may provide new insights on mechanisms of protective immunity to ASFV.
TL;DR: The results indicate that the genetic variability of echovirus 30 is significantly lower than that of other previously characterized enteroviruses.
Abstract: The genetic relationships between 131 echovirus type 30 (E-30) field isolates were studied using phylogenetic analysis of three genomic intervals: VP4/VP2 (420 nt), the entire VP1 and VP1/2A (150 nt). The strains had been isolated between 1975–1998, in different European countries, and in Israel and Japan. The maximum genetic variation was 15.7% in the VP4/VP2 region, 21.3% across the VP1/2A junction and 16.7% in the VP1-gene. The clustering patterns were very similar in all three regions. Two distinct genotypes were observed among the European strains, one of which was prevailing, spanning most of the investigated period. The same genotype was previously described to be the most prevalent circulating lineage of E-30 in Northern America. Interestingly, the two other genotypes comprising the prototype strain Bastianni and the oldest European isolates circulating before 1976, respectively, had apparently disappeared. Furthermore, the oldest lineages of the prevailing genotype had likewise disappeared and the recently isolated strains in the prevailing genotype were genetically quite homogenous, even when isolated in geographic regions far apart. These results indicate that the genetic variability of echovirus 30 is significantly lower than that of other previously characterized enteroviruses. Furthermore, one single, major genotype showed epidemic spread across two continents. Interestingly, despite the low nucleotide variability, maximum amino acid sequence variability in VP1 was surprisingly high, 8.0%, suggesting possible antigenical differences.
TL;DR: The virus which causes this maize disease in China is rice black-streaked dwarf virus, which contains a single open reading frame which potentially encoded a protein with 558 amino acids.
Abstract: Three virus isolates from maize with rough dwarf in different provinces in China were analyzed at the molecular level. When compared to an isolate from diseased rice plants in Hubei Province, all four isolates had identical genomic RNA electrophoretic profiles, which were composed of ten double-stranded (ds) RNAs. Full-length cDNAs of segment 10 (S10) from each of the four isolates were cloned by RT-PCR and the complete sequences were determined. Analysis of the sequences revealed that each consisted of 1801 nucleotides and contained a single open reading frame (ORF) which potentially encoded a protein with 558 amino acids. Further, the sequences showed more than 97.0% and 98.0% identity atnucleotide and amino acid levels, respectively. In addition, theiridentities to rice black-streaked dwarf virus S10 were significantlyhigher than those to maize rough dwarf virus S10. Based on theseresults, it is suggested that the virus which causes this maize diseasein China is rice black-streaked dwarf virus.
TL;DR: A 3599 nucleotide portion of the genomic RNA of a UK isolate of Pepino mosaic virus, isolated from tomato, has been sequenced and shows that all the tomato isolates share over 99% identity, but only between 96–97% identity with the Peruvian pepino isolate.
Abstract: A 3599 nucleotide portion of the genomic RNA of a UK isolate of Pepino mosaic virus (PepMV), isolated from tomato, has been sequenced (Accession No. AF340024). The region sequenced includes the 3'-end of the RNA polymerase, the triple gene block (TGB), the coat protein (CP) and 3' untranslated region (UTR). In addition, the CP sequences of another 15 PepMV isolates, including 14 European tomato isolates and a Peruvian pepino isolate, have been determined and compared. This analysis shows that all the tomato isolates share over 99% identity, but only between 96-97% identity with the Peruvian pepino isolate.
TL;DR: Degenerate primers were used to detect and amplify cDNA of viruses of the genera Carlavirus, Allexivirus and Potyvirus from garlic plants with mosaic symptoms growing in Zhejiang province, China, which is the first report of allexiviruses from China.
Abstract: Degenerate primers were used to detect and amplify cDNA of viruses of the genera Carlavirus, Allexivirus and Potyvirus from garlic plants with mosaic symptoms growing in Zhejiang province, China. Plants contained a complex mixture of viruses and strains. Three distinct stains of Garlic latent virus were detected; the most frequent one was completely sequenced and partial sequences were obtained for the other two. The complete sequence (8363 nt) was 76.4% identical to a Korean isolate. Two allexiviruses were detected and completely sequenced. One (8319 nt) was identified as Garlic virus X and comparisons showed that a published Korean isolate (which had 90.2% identical nucleotides) had an N-terminal deletion in the serine-rich ORF4. The other isolate (8451 nt), tentatively named Garlic virus E, appeared to be a new member of the genus. Phylogenetic analyses of the different viral proteins and distinctive conserved sequence motifs within the genus are discussed. This is the first report of allexiviruses from China. Using potyvirus primers, three distinct isolates of Onion yellow dwarf virus and one of Leek yellow stripe virus were detected and the 3′-terminal sequences of their genomes were determined. In a coat protein phylogenetic analysis, the new isolates were most closely related to other published isolates from Japan and China.
TL;DR: The characterization of immunorelevant linear B-cell epitopes of the Open Reading Frame 2-encoded protein (Orf2) fromPCV2 by an enzyme-linked immunosorbent assay (ELISA) using experimental antisera collected from pigs inoculated with a PCV2 isolate suggests that epitope B-133 is a serological marker of PCV1 infection that could be used for the detection of PCv2 antibody response.
Abstract: Post-weaning multisystemic wasting syndrome (PMWS) is a recently identified disease of pigs linked to the emergence of a new porcine circovirus (PCV2). We report here the characterization of immunorelevant linear B-cell epitopes of the Open Reading Frame 2-encoded protein (Orf2) from PCV2 by an enzyme-linked immunosorbent assay (ELISA) using experimental antisera collected from pigs inoculated with a PCV2 isolate. Two epitopes spanning residues 69 to 83 and 117 to 131 were specific to PCV2. Antibodies to the 117 to 131 epitope (B-133) were detected in 22% and 100% of specific pathogen-free (SPF) pig sera 6 and 11 weeks post inoculation, respectively. Cross-sectional studies performed with field sera collected from PMWS-affected herds showed B-133 antibodies in 5% of 8 to 10 week-old pigs, 38% of 13–14 week-old pigs, 62% of 16 to 19 week-old pigs, 56% of 20 to 25 week-old pigs and 45% of 26 to 31 week-old pigs. All these data suggest that epitope B-133 is a serological marker of PCV2 infection that could be used for the detection of PCV2 antibody response.
TL;DR: There seems to be a worldwide trend with strains ofgenotype B appearing earlier than strains of genotype C which took over later in the dominance, which is similar to previous outbreaks of enterovirus infection in Taiwan.
Abstract: Taiwan suffered a severe and widespread outbreak of enterovirus infection in 1998. More than 400 children were hospitalized, with seventy-eight fatalities due to central nerve system (CNS) involvement and cardiopulmonary collapse. Enterovirus 71 (EV71) was incriminated as the causative agent for the fatal cases. To understand the viral molecular epidemiology in this outbreak, fragments of 207-bp length of the VP4 region in 23 Taiwanese EV 71 isolates were sequenced. Pair-wise comparison revealed a 17.5–24.4% difference between the isolates and the prototype BrCr. However, all the changes in the VP4 region of the isolated strains were synonymous substitutions. Phylogenetic analysis was performed on these 23 isolates and 21 others deposited in GenBank. In this study, forty-four EV71 isolates from the world were separated into three distinct genotypes: A, B and C. The EV71 prototype strain, BrCr/70, is the only strain of genotype A. Group B included strains from the United States, Japan and Taiwan. Most strains in genotype B were isolated prior to 1990. Group C consisted of strains from Japan and Taiwan. Most strains of genotype C were isolated after 1990, they were further divided into 3 clusters: i.e. C-1, C-2 and C-3. In Taiwan, two genotypes, B and C-3, were co-circulating during the outbreak in 1998, although a minor group of genotype B may have appeared in Taiwan before 1986. The majority of the isolates clustered in genotype C-3. Genotype C showed a higher evolutionary rate than genotype B (3.9 × 10−3vs. 1.4 × 1010−3) in the VP4 region. There seems to be a worldwide trend with strains of genotype B appearing earlier than strains of genotype C which took over later in the dominance.
TL;DR: It is demonstrated that the embryonic fibroblastic cells from a feral-origin strain (SPR) expressed 74 kDa Mx2 protein, which prevented the accumulation of viral transcripts and proteins of hantaviruses when the Mx1 gene was constitutively expressed in transfected Vero cells.
Abstract: The antiviral potential of Mx2 protein remains unknown, because the Mx2 gene in commonly used strains of laboratory mice is nonfunctional. Our previous study showed that functional Mx2 protein in some feral-origin strains was induced upon interferon treatment, was localized in the cytoplasm, and inhibited vesicular stomatitis virus replication. In the present study, we have demonstrated that the embryonic fibroblastic cells from a feral-origin strain (SPR) expressed 74 kDa Mx2 protein, which prevented the accumulation of viral transcripts and proteins of hantaviruses when the Mx2 gene was constitutively expressed in transfected Vero cells. Furthermore, the cells showed significantly lower titers of the virus than control cells. In contrast, influenza virus replication was not affected by the expression of Mx2 protein in the Vero cells.
TL;DR: A novel tospovirus serologically distinct from all established tospo- virus species was found in Thailand in Physalis minima L, and N protein sequence comparisons revealed closest relationship to the species Watermelon bud necrosis virus, Watermelon silver mottle virus and Peanut bud necrot virus and a distant relationship to Peanut yellow spot virus.
Abstract: A novel tospovirus serologically distinct from all established tospo- virus species was found in Thailand in Physalis minima L. The S RNA of this virus was cloned by a new RT-PCR approach revealing a nucleotide sequence of 3257 nucleotides. The ambisense RNA segment encoded a nonstructural protein (NSs) of 469 amino acids, with a predicted Mr of 53.2 kDa, and a nucleoprotein (N) of 279 amino acids and a Mr of 31.0 kDa, so far the largest N protein known for any tospovirus species. N protein sequence comparisons revealed closest relationship to the species Watermelon bud necrosis virus (58% identity), Watermelon silver mottle virus and Peanut bud necrosis virus (57%) and a distant relationship to Peanut yellow spot virus (23%) and Peanut chlorotic fanspot virus (22%).