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PubMed1980

Partial amino-terminal sequences of the polyoma nonhistone proteins VP1, VP2, and VP3 synthesized in vitro.

The three polyoma virus capsid proteins VP1, VP2, and VP3 were synthesized in vitro in the presence of several radiolabeled amino acids and, after purification on sodium dodecyl sulfate-polyacrylamide gels, were subjected to sequential Edman degradation. The partial amino-terminal amino acid sequences obtained were compared with the sequence of amino acids predicted from the polyoma virus DNA sequencing (Arrand et al., J. Virol. 33:606--618, 1980). Together, these results showed that the 5' ends of the VP1, VP2, and VP3 coding sequences are located 1,217, 289, and 634 nucleotides, respectively, from the junction of HpaII restriction fragments 3 and 5.

Amino Acid Sequence↗

PubMed2000

Molecular evolution of a type 1 wild-vaccine poliovirus recombinant during widespread circulation in China.

Type 1 wild-vaccine recombinant polioviruses were isolated from poliomyelitis patients in China from 1991 to 1993. We compared the sequences of 34 recombinant isolates over the 1,353-nucleotide (nt) genomic interval (nt 2480 to 3832) encoding the major capsid protein, VP1, and the protease, 2A. All recombinants had a 367-nt block of sequence (nt 3271 to 3637) derived from the Sabin 1 oral poliovirus vaccine strain spanning the 3'-terminal sequences of VP1 (115 nt) and the 5' half of 2A (252 nt). The remaining VP1 sequences were closely (up to 99.5%) related to those of a major genotype of wild type 1 poliovirus endemic to China up to 1994. In contrast, the non-vaccine-derived sequences at the 3' half of 2A were more distantly related (<90% nucleotide sequence match) to those of other contemporary wild polioviruses from China. The vaccine-derived sequences of the earliest (April 1991) isolates completely matched those of Sabin 1. Later isolates diverged from the early isolates primarily by accumulation of synonymous base substitutions (at a rate of approximately 3.7 x 10(-2) substitutions per synonymous site per year) over the entire VP1-2A interval. Distinct evolutionary lineages were found in different Chinese provinces. From the combined epidemiologic and evolutionary analyses, we propose that the recombinant virus arose during mixed infection of a single individual in northern China in early 1991 and that its progeny spread by multiple independent chains of transmission into some of the most populous areas of China within a year of the initiating infection.

Base Sequence↗

PubMed2002

Functional mimicry of a discontinuous antigenic site by a designed synthetic peptide.

Functional reproduction of the discontinuous antigenic site D of foot-and-mouth disease virus (FMDV) has been achieved by means of synthetic peptide constructions that integrate each of the three protein loops that define the antigenic site into a single molecule. The site D mimics were designed on the basis of the X-ray structure of FMDV type C-S8c1 with the aid of molecular dynamics, so that the five residues assumed to be involved in antigenic recognition are located on the same face of the molecule, exposed to solvent and defining a set of native-like distances and angles. The designed site D mimics are disulfide-linked heterodimers that consist of a larger unit containing VP2(71-84), followed by a polyproline module and by VP3(52-62), and a smaller unit corresponding to VP1(188-194) (VP=viral protein). Guinea pig antisera to the peptides recognized the viral particle and competed with site D-specific monoclonal antibodies, while inoculation with a simple (not covalently joined to one another) admixture of the three VP1-VP3 sequences yielded no detectable virus-specific serum conversion. Similar results have been reproduced in two bovines. Antisera to the peptides also moderately neutralize FMDV in cell cultures and partially protect guinea pigs against challenge with the virus. These results demonstrate functional mimicry of the discontinuous site D by the peptides, which are therefore obvious candidates for a multicomponent, peptide-based vaccine against FMDV.

Amino Acid Sequence↗

PubMed2003

Recombinant murine L20B cell line supports multiplication of group A coxsackieviruses.

Sensitive, reliable, and rapid methods of virus culture are essential for wild poliovirus isolation and identification from stool specimens collected from cases of acute flaccid paralysis. Recently, recombinant murine cell lines expressing human poliovirus receptor (CD155) on the cell surface have become available. These cells are sensitive selectively to poliovirus because the poliovirus receptor is not used by other enteroviruses. In early field studies non-polio enteroviruses were not isolated from stool samples of cases of acute flaccid paralysis using L20B cells. For the past 3 years, L20B cells were used extensively in our laboratory for virus culture. The objective of the present study was to identify non-polio enteroviruses causing cytopathic changes in L20B cells. Stool specimens collected from 1,153 cases of acute flaccid paralysis and 2,670 apparently healthy children were tested for enteroviruses using RD and L20B cell lines. A small number of viruses other than poliovirus causing cytopathic effect in L20B cells were isolated. Such isolates detected in other polio network laboratories in India were also included. The virus isolates were typed using partial VP1 nucleotide sequence analysis and virus neutralization tests. Of the 111 viruses studied, 8 were non-enteroviruses. Among the 103 non-polio enteroviruses, 73 were identified as group A coxsackieviruses. Of the 30 isolates that could not be characterized, 1 remained unidentified even by sequence analysis and 29 did not reach high titers in L20B as well as RD cells. In conclusion, coxsackie A viruses multiply in L20B cells causing cytopathic effect. Coxsackievirus A8 and coxsackievirus A10 were predominant among the eight coxsackie A virus types so far identified.

Animals↗

PubMedDate not supplied

Synthetic peptides as functional mimics of a viral discontinuous antigenic site.

Functional reproduction of discontinuous antigenic site D of foot-and-mouth disease virus (FMDV) has been achieved by means of synthetic peptide constructions that integrate into a single molecule each of the three protein loops that define the antigenic site. The site D mimics are designed on the basis of the X-ray structure of FMDV type C-S8c1 with the aid of molecular dynamics, so that the five residues assumed to be involved in antigenic recognition are located on the same face of the molecule, exposed to solvent and defining a set of native-like distances and angles. The designed site D mimics are disulphide-linked heterodimers that consist of a larger unit containing VP2(71-84), followed by a polyproline module and by VP3(52-62), and a smaller unit corresponding to VP1(188-194). Guinea pig antisera to the peptides recognize the viral particle and compete with site D-specific monoclonal antibodies, while inoculation with a simple (non-covalently bound) admixture of the three VP1-VP3 sequences yields no detectable virus-specific serum conversion. Similar results have been reproduced in two cattle. Antisera to the peptides are also moderately neutralizing of FMDV in cell culture and partially protective of guinea pigs against challenge with the virus. These results demonstrate functional mimicry of the discontinuous site D by the peptides, which are therefore obvious candidates for a multicomponent peptide-based vaccine against FMDV.

Amino Acid Sequence↗

PubMed1992

Does the VP1 gene of foot-and-mouth disease virus behave as a molecular clock?

We have carried out a phylogenetic study of the evolution of the VP1 gene sequence from different serological types and subtypes of foot-and-mouth disease virus (FMDV). The maximum-likelihood method developed by Hasegawa and co-workers (Hasegawa et al. 1985) for the estimation of evolutionary parameters and branching dates has been used to decide between alternative models of evolution: constant versus variable rates. The results obtained indicate that a constant rate model, i.e., a molecular clock, seems to be the most plausible one. However, additional information suggests the possibility that the appearance of serotype CS has been accompanied by an episode of rapid evolution (Villaverde et al. 1991). We discuss the possibility that this evolution of RNA viruses was due to episodic positive Darwinian selection, which would have helped the new variant to escape the immunogenic pressure from the hosts.

Aphthovirus↗

PubMed2005

Molecular epidemiology of enterovirus 71 over two decades in an Australian urban community.

Enterovirus 71 (EV71), first isolated in 1969, has been responsible for numerous outbreaks of hand, foot and mouth disease (HFMD) with a small proportion of cases associated with neurological disease. Since 1997 there has been a significant increase in both the prevalence and virulence of EV71 in the Asia-Pacific region. We have examined the genetic diversity of EV71 in a large Australian city (Sydney N.S.W.) over a nineteen-year period. We determined the VP1 gene sequence of forty-eight EV71 strains isolated between 1983 and 2001. Analysis by molecular phylogeny revealed the presence of four subgenogroups B2, B4, C1 and C2. The results indicate that the major lineage circulating in Sydney N.S.W. was subgenogroup C1 with a recent switch in dominance to B4 in 2000 and 2001.

Australia↗

PubMed1992

Possible approaches to develop vaccines against hepatitis A.

More than a decade ago, successful replication of hepatitis A virus (HAV) in cell culture opened the way to the development of live attenuated and inactivated vaccine candidates. Serial passages of HAV in cell culture led to attenuation as demonstrated by experiments in non-human primates. Several live vaccine candidates obtained through serial passages have been evaluated in volunteers. Significant improvements in the yield of viral antigen from infected cell cultures stimulated the development of killed vaccine candidates. These formalin-inactivated vaccines contain the viral capsid antigens assembled into viral particles. The immunogenic potential of the vaccine candidates depends strongly on the preservation of the configuration of the capsid proteins. Synthetic peptides covering immunogenic sequences of VP1 as well as soluble capsid proteins expressed as fusion proteins in Escherichia coli were therefore only weakly immunogenic when injected at high concentrations in rabbits. On the other hand, tamarin monkeys immunized with a live recombinant vaccinia expressing P1 were protected against virulent challenge. There are, however, considerable drawbacks related to the use of live vaccinia as a carrier virus. Chimeric polio-HAV VP1 viruses have been constructed. These hybrid viruses were not able to induce an immune response, probably because of configurational constraints of poliovirus on the inserted HAV epitopes. More recently, encouraging data on empty virus particles expressed in baculovirus and vaccinia virus systems have been reported.

Animals↗

PubMed2005

Molecular epidemiology of hepatitis A in Noord-Brabant, The Netherlands.

BACKGROUND: Previous studies on the molecular epidemiology of hepatitis A virus (HAV) in Amsterdam, The Netherlands, show that subgenotype 1A is mainly seen among homosexual men practising anonymous oral-anal sex in saunas and darkrooms, while subgenotype 1B is usually detected among children originating from Morocco, and subgenotype 3A is mostly found among travellers to Pakistan. OBJECTIVE: We studied the genotype distribution in a more rural area of The Netherlands, Noord-Brabant, and compared it with Amsterdam. STUDY DESIGN: We collected blood and feces samples from 34 HAV IgM(+) individuals who were reported from August 2001-March 2003 at the Municipal Health Service (MHS) Heart for Brabant (Brabant). We also collected feces samples from nine household contacts of whom the HAV IgM status was not known. HAV RNA was isolated and subsequently amplified by reverse transcriptase polymerase chain reaction (RT-PCR) at the VP1-P2a and the VP3-VP1 region, sequenced and analysed. RESULTS AND CONCLUSIONS: In most cases, relations between risk groups and HAV subgenotypes in Noord-Brabant were similar to those in Amsterdam. Next to genotypes 1 and 3 we also detected a genotype 2/7 strain in a Noord-Brabant case. Also, in contrast to the Amsterdam study, sporadic transmission occurred among various risk groups. Children involved in a school-related outbreak were infected with strains identical to one that was previously isolated from a man who has sex with men (MSM). Also, Dutch patients having no epidemiological link with Turkish or Moroccan children harboured strains imported from high-endemic countries. Furthermore, we report a special case in which HAV may be causally involved in meningitis. The results of this study show that the molecular epidemiology of HAV in The Netherlands can be more complicated than previously anticipated and that HAV phylogenetic studies can provide important information for the design of appropriate public health measures.

Adolescent↗

PubMed2005

The Lim Benyesh-Melnick antiserum pools for serotyping human enterovirus cell culture isolates--still useful, but may fail to identify current strains of echovirus 18.

Neutralisation tests are often used as a second line method of serotyping human enterovirus cell culture isolates after failure of indirect immunofluorescence. We reviewed the results of 85 consecutive neutralisation tests performed routinely on presumptive enterovirus isolates using the Lim Benyesh-Melnick antiserum pools after failure of immunofluorescence on most of them, in order to assess the value of these antiserum pools. Confirmation of serotype was determined by partial VP1 genome sequencing. Forty isolates were serotyped successfully and 45 isolates were untypable by the LBM pools, although 16 of the latter would not be expected to be typable by this means. Of eighteen echovirus 18 isolates tested, only two were typable by neutralisation. Of 15 coxsackievirus A16 isolates only nine were typable by neutralisation. Neutralisation with the LBM pools remains a useful second line method of serotyping human enteroviruses despite difficulties with at least two serotypes.

Animals↗

PubMed2000

Natural transmission of foot-and-mouth disease virus between African buffalo (Syncerus caffer) and impala (Aepyceros melampus) in the Kruger National Park, South Africa.

VP1 gene sequences of SAT-2 type foot-and-mouth disease (FMD) viruses recovered from impala and African buffalo in the Kruger National Park (KNP) were used to determine intra- and interspecies relationships of viruses circulating in these wildlife populations. On this basis five distinct lineages of SAT-2 virus were identified in routine sampling of oesophageopharyngeal epithelium from buffalo between 1988 and 1996. Different lineages were associated with discrete geographic sampling localities. Over the period 1985-95, four unrelated epizootics occurred in impala in defined localities within the KNP. Evidence for natural transmission of FMD between buffalo and impala is presented for the most recent 1995 outbreak, with data linking the 1985 and 1988/9 impala epizootics to viruses associated with specific buffalo herds.

Animals↗

PubMed2003

Frequency, viral loads, and serotype identification of enterovirus infections in Scottish blood donors.

BACKGROUND: Enteroviruses are non-enveloped, frequently pathogenic RNA viruses infecting humans. Infection is potentially transmissible through blood or blood component transfusion from donor in the viremic phase before seroconversion for antibody. To investigate the threat to blood safety from enteroviruses, a large-scale survey of frequency and levels of viremia in blood donors was conducted. STUDY DESIGNS AND METHODS: Blood donations from Scotland over a period of 22 calendar months were screened for enterovirus RNA sequences by PCR. Positive samples were quantified, and serotypes were identified by nucleotide sequencing of VP1. RESULTS: From a total of 3658 pools of 95 donations tested, 73 samples that were enterovirus-positive were identified (corrected annual frequency 0.024% or 1 in 4000). The highest rates of viremia were in late summer months (e.g., 0.055%, 1 in 1800 in July) and lowest from January to May (0.009 and 0.012%). Viral loads ranged from 500 (the lower cutoff of the assay) to greater than 100,000 amplifiable enterovirus template copies per mL. Coxsackievirus A16, echoviruses 11 and 30, and enterovirus 71 were most often identified. CONCLUSIONS: The detection of enterovirus-positive blood units indicates the potential for enteroviral transmission by blood components. Although the infrastructure established for PCR-based screening for HCV RNA would allow parallel screening for enteroviruses, any decision concerning donor testing would require further information on the outcome of transfusion-acquired enterovirus infections.

Blood↗

PubMed1985

Expression of human choriogonadotropin in monkey cells using a single simian virus 40 vector.

We have inserted the cDNAs coding for both polypeptide subunits, alpha and beta, of human choriogonadotropin (hCG) into a single simian virus 40 expression vector in such a way that they replace the viral VP2 and VP1 coding sequences, respectively. Monkey cells infected with this virus and the appropriate helper virus produce dimeric hCG. hCG produced in this system was shown to chromatograph identically to standard hCG preparations on gel filtration and to be biologically active in the mouse uterine weight assay.

Animals↗

PubMed2004

Epizootic occurrence of haemorrhagic nephritis enteritis virus infection of geese.

Recent outbreaks of haemorrhagic nephritis enteritis in geese flocks of 3 to 10 weeks in age in Hungary were investigated. Mortality varied between 4% and 67%. Affected birds generally died suddenly. Occasional clinical signs included tremors of the head and neck, subcutaneous haemorrhages and excretion of faeces containing partly digested blood. At necropsy the most frequent findings were a turgid wall and reddish mucosa of the intestines and reddish discolouration of the swollen kidneys, but oedema and haemorrhages of the subcutaneous connective tissue, hydropericardium and ascites were also seen. In subacute cases, visceral gout was frequently observed. Histological examination revealed zonal necrosis of the tubular epithelial cells with haemorrhages in the kidney. Other histological findings were serous hepatitis with fatty infiltration, necrotizing haemorrhagic enteritis and haemorrhages in the different organs including the brain. Experimental geese infected parenterally with crude liver and spleen homogenates prepared from diseased birds died after 8 to 20 days without premonitory signs, and had typical gross and histological lesions. Attempts to isolate cytopathic virus on different tissue cultures failed. The presence of polyomavirus was proven by polymerase chain reaction. Five isolates were further investigated by analysing their complete VP1 gene sequence. All tested strains were very closely related to each other on the basis of the nucleotide sequence, and they were identical at the deduced amino acid level.

Aging↗

PubMed2005

Human parechovirus infections in Dutch children and the association between serotype and disease severity.

BACKGROUND: Human parechoviruses (HPeVs) are members of the family Picornaviridae and are classified into 3 known serotypes: HPeV1, HPeV2, and the recently identified HPeV3. HPeV1 and HPeV2 infections are most commonly associated with mild respiratory or gastrointestinal symptoms and occasionally with severe disease conditions, such as flaccid paralysis and encephalitis. HPeV3 infection has been associated with transient paralysis and neonatal infection and has until now only been reported in Japan and Canada. METHODS: Culture isolates considered to be enterovirus on the basis of cell culture but that were found to be enterovirus negative by 5' untranslated region reverse-transcriptase polymerase chain reaction (5'UTR RT-PCR) during the period December 2000 through January 2005 were selected. Isolates were tested by HPeV 5'UTR RT-PCR and were genotyped by sequencing the VP1 region. Phylogenetic analysis was performed, and the association with clinical symptoms was established. RESULTS: Thirty-seven (12%) of the 303 isolates that tested positive for enterovirus by cell culture were in fact HPeV. The majority of the HPeV-positive isolates (n = 27) could be identified as HPeV1. The remaining 10 isolates, which were grown from samples obtained in 2001, 2002, and 2004, could be typed as the recently identified HPeV3. HPeV was exclusively detected in children aged < 3 years. Children infected with HPeV3 were significantly younger than children infected with HPeV1, and sepsis-like illness and central nervous system involvement were more frequently reported in children infected with HPeV3. CONCLUSIONS: We report HPeV infections in young children during the period of 2000-2005 and show an association between HPeV3 infection and sepsis-like illness and central nervous system involvement in neonates.

Central Nervous System Infections↗

PubMed2003

The implications of virus diversity within the SAT 2 serotype for control of foot-and-mouth disease in sub-Saharan Africa.

SAT 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.

Africa South of the Sahara↗

PubMed2002

Comparison of molecular and conventional methods for typing of enteroviral isolates.

Twenty-eight enteroviral isolates obtained from various clinical specimens were typed by Lim-Benyesh-Melnick (LBM) pool-based neutralization, PCR-restriction fragment length polymorphism (RFLP), and partial sequencing of the VP1 region of the enteroviral genome. Sequencing was found to be a good alternative to LBM typing, while PCR-RFLP was inadequate for identification of enteroviral isolates.

Bacterial Typing Techniques↗

PubMed1995

Encapsidation of poliovirus replicons encoding the complete human immunodeficiency virus type 1 gag gene by using a complementation system which provides the P1 capsid protein in trans.

Poliovirus genomes which contain small regions of the human immunodeficiency virus type 1 (HIV-1) gag, pol, and env genes substituted in frame for the P1 capsid region replicate and express HIV-1 proteins as fusion proteins with the P1 capsid precursor protein upon transfection into cells (W. S. Choi, R. Pal-Ghosh, and C. D. Morrow, J. Virol. 65:2875-2883, 1991). Since these genomes, referred to as replicons, do not express capsid proteins, a complementation system was developed to encapsidate the genomes by providing P1 capsid proteins in trans from a recombinant vaccinia virus, VV-P1. Virus stocks of encapsidated replicons were generated after serial passage of the replicon genomes into cells previously infected with VV-P1 (D. C. Porter, D. C. Ansardi, W. S. Choi, and C. D. Morrow, J. Virol. 67:3712-3719, 1993). Using this system, we have further defined the role of the P1 region in viral protein expression and RNA encapsidation. In the present study, we constructed poliovirus replicons which contain the complete 1,492-bp gag gene of HIV-1 substituted for the entire P1 region of poliovirus. To investigate whether the VP4 coding region was required for the replication and encapsidation of poliovirus RNA, a second replicon in which the complete gag gene was substituted for the VP2, VP3, and VP1 capsid sequences was constructed. Transfection of replicon RNA with and without the VP4 coding region into cells resulted in similar levels of expression of the HIV-1 Gag protein and poliovirus 3CD protein, as indicated by immunoprecipitation using specific antibodies. Northern (RNA) blot analysis of RNA from transfected cells demonstrated comparable levels of RNA replication for each replicon. Transfection of the replicon genomes into cells infected with VV-P1 resulted in the encapsidation of the genomes; serial passage in the presence of VV-P1 resulted in the generation of virus stocks of encapsidated replicons. Analysis of the levels of protein expression and encapsidated replicon RNA from virus stocks after 21 serial passages of the replicon genomes with VV-P1 indicated that the replicon which contained the VP4 coding region was present at a higher level than the replicon which contained a complete substitution of the P1 capsid sequences. These differences in encapsidation, though, were not detected after only two serial passages of the replicons with VV-P1 or upon coinfection and serial passage with type 1 Sabin poliovirus.(ABSTRACT TRUNCATED AT 400 WORDS)

Amino Acid Sequence↗