Temperature inactivation of Feline calicivirus vaccine strain FCV F-9 in comparison with human noroviruses using an RNA exposure assay and reverse transcribed quantitative real-time polymerase chain reaction-A novel method for predicting virus infectivity.

A one-step reverse transcription quantitative real-time polymerase chain reaction (RT-QPCR) method in combination with RNase treatment and low copy number samples was developed in order to examine the effect of temperature on the ability of virus capsids to protect their RNA content. The method was applied to a non-cultivable virus (GII.4 norovirus) and Feline calicivirus vaccine strain F-9 (FCV) which is often used as a norovirus surrogate. Results demonstrated that FCV RNA is exposed maximally after 2min at 63.3 degrees C and this correlated with a greater than 4.5log reduction in infectivity as assessed by plaque assay. In contrast human GII.4 norovirus RNA present in diluted clinical specimens was not exposed maximally until 76.6 degrees C, at least 13.3 degrees C greater than that for FCV. These data suggest that norovirus possesses greater thermostability than this commonly used surrogate. Further, these studies indicate that current food processing regimes for pasteurisation are insufficient to achieve inactivation of GII.4 NoVs. The method provides a novel molecular method for predicting virus infectivity.

Prevalence of antibodies to astrovirus types 1 and 3 in children and adolescents in Norfolk, Virginia.

OBJECTIVE: To determine the prevalence of antibody to human astrovirus types 1 (HAstV-1) and 3 (HAstV-3) in children. METHODS: Sera from children hospitalized in Norfolk, VA, for noninfectious conditions were collected for a 1-month period every 6 months from 1993 to 1996 and tested by enzyme immunoassay for antibody to HAstV-1 and HAstV-3 with the use of baculovirus-expressed recombinant capsid proteins as antigens. RESULTS: The seroprevalence of 393 infants and children to HAstV-1 decreased from 67% in infants <3 months of age to 7% by 6 to 8 months of age, consistent with loss of transplacental antibodies. Children acquired HAstV-1 antibody with a peak prevalence of 94% at 6 to 9 years of age (P < 0.001). Antibodies to HAstV-3 exhibited a lower prevalence, with 26% positive at <3 months, 0% at 6 to 11 months and 42% by 6 to 9 years of age. HAstV-1 seroprevalence in children O to 2 months of age decreased from 89% in November, 1993, to 40% in November, 1996 (P = 0.009). CONCLUSIONS: Astrovirus type-specific antibody prevalence can be measured by baculovirus-expressed capsid antigens in an enzyme immunoassay. Children developed antibody to HAstV-1 (94%) and to HAstV-3 (42%) by 6 to 9 years of age indicating frequent exposure to these enteric viruses in infancy and early childhood.

The molecular biology of astroviruses.

Astroviruses (genus Astrovirus) are assigned to a newly established virus family, the Astroviridae. The molecular biology of these agents reveals many features unique amongst the non-enveloped animal viruses and resembles that of members of certain plant virus families. In particular, their possession of a serine protease and use of ribosomal frameshifting to express the RNA polymerase are similar to the luteoviruses. Many aspects of the astrovirus replication strategy are still unclear, but replication may involve a nuclear step and non-structural proteins may influence host cell range.

Identification and sequence determination of the capsid protein gene of human astrovirus serotype 1.

We present the sequence of an open reading frame (ORF) at the 3' end of human astrovirus serotype 1. Primer extension experiments showed that the RNA expressing this gene is shorter than the complete ORF, and could form a protein of M(r) 85,540. The protein was expressed by recombinant baculovirus and was recognized by anti-virion serum, indicating a structural role. Sequence comparison indicates that astrovirus serotypes 1 and 2 differ markedly in the C-terminal half of the protein but are well conserved towards the N-terminus.

Detection of Norwalk virus in the UK by the polymerase chain reaction.

We have developed a polymerase chain reaction for the detection of Norwalk virus using the published sequence of the virus RNA dependent RNA polymerase gene and have used this to clone and sequence this region of a virus from a UK outbreak. We have applied this method to a panel of UK Norwalk-like viruses using both Tet-z and Taq DNA polymerases and found that amplification produces a multiplicity of bands from stool samples. However, in combination with Southern blotting, Taq polymerase amplification detected virus in 13 of a panel of 30 clinical samples known to contain these viruses and also detected astroviruses in a mixed infection. Amplification using Tet-z DNA polymerase was less efficient (6/30) and detected predominantly viruses typed as UK type 2 by solid phase immune electron microscopy.

The 3' terminal sequence of a human astrovirus.

We have determined the sequence for 1,000 bases from the 3' terminus of a human astrovirus serotype 1 isolated in Newcastle. This is the first sequence reported for a representative of this virus family. We find one open reading frame which terminates 83 bases from a poly A tail. The 3' non-coding-region has similarities to some picornavirus termini. However the amino acids specified by the coding region have no significant homology to the picornavirus protein 3D, encoded at the 3' terminus of these viruses. Northern blot analysis of intracellular virus-specific RNAs revealed one size of transcript which corresponded to full-length virus RNA. Available data thus indicate that astroviruses may resemble picornaviruses in replication strategy.

Defective translation of measles virus matrix protein in a subacute sclerosing panencephalitis cell line.

Subacute sclerosing panencephalitis (SSPE) is a slowly progressing fatal human disease of the central nervous system (CNS) that is associated with measles virus persistence. Virus nucleocapsids are present in the brain and the patient is in a state of hyperimmunization towards this agent. However, although all other structural polypeptides are recognized by the immune system, there is a markedly decreased antibody response towards virus matrix or membrane protein. Matrix protein has not been detected in brain cells and infectious virus is not present. The absence of this virus structural polypeptide is thought to account for the apparent restriction in virus maturation both in vivo and in vitro. SSPE viruses can only rarely be rescued from brain tissue by co-cultivation or cell fusion techniques using tissue culture cell lines susceptible to measles virus infection. Often this procedure fails to yield a lytic budding virus but produces instead a carrier cell line in which the agent is cell associated. These lines (known as SSPE cell lines) also do not contain matrix protein. However, the reason for this deficiency is unknown. We have therefore now examined an SSPE cell line which does not yield infectious virus in order to define this process further. We found that although messenger RNA for membrane protein was present, it was unable to form normal matrix protein in translation reactions.

A dot-blot hybridization procedure for the detection of astrovirus in stool samples.

We have developed a nucleic acid dot-blot hybridization test for the detection of astroviruses in stool samples. The test was not as sensitive as electron microscopy for the detection of low numbers of well preserved astrovirus particles, but was able to identify astroviruses in stools containing particles of indistinct morphology. In total, this procedure identified astroviruses in more samples than did electron microscopy, and the data indicate that the incidence of astroviruses may be substantially underestimated.

Growth and characterisation of human faecal astrovirus in a continuous cell line.

We report conditions for the growth of human faecal astrovirus in a continuous colonic carcinoma cell line (CaCo-2). Purified particles contained three polypeptides, one of which (24k) appeared loosely held on the exterior.

Restriction enzyme analysis of faecal adenoviruses in Newcastle upon Tyne.

Adenovirus DNA was isolated directly from virus-containing stools and digested with restriction endonucleases. The resulting fragments were separated by polyacrylamide gel electrophoresis (PAGE) and visualized by silver staining. This enabled us to assign most of the viruses detected to subgenus, serotype and, sometimes, unique strains. Although less sensitive than electron microscopy, the method allowed more information about the infecting virus to be obtained and no cultivation was necessary. Comparison with culture also allowed dual infections to be recognized. A 2-year survey of faecal adenoviruses in Newcastle upon Tyne showed that type 41 (strain 41a) was the predominant type and strain 41p was not recorded. Heterogeneity in strain 41a was also noted as found elsewhere. Adenovirus type 40 was common prior to 1985 but was absent during the last 2 years.

Respiratory syncytial virus glycoprotein expression in human and simian cell lines.

Glycoproteins synthesized in both human (HeLa and HEp-2) and simian (Vero and BS-C-1) cell lines following infection with two different strains of respiratory syncytial virus (A2 and Long) were analysed by SDS-PAGE following immunoprecipitation with monoclonal antibodies. Minor virus strain-dependent differences in the large glycoprotein, G, and the fusion protein polypeptides F1 and F2 were observed together with minor cell line-dependent differences in the size of the F2 polypeptide. Major quantities of two glycoproteins, termed Ga (50K) and Gb (45K), were detected in A2 strain-, and to a lesser extent in Long strain-, infected simian cells. These proteins were also present in infected human cells, but in much reduced amounts. Immunoprecipitation with anti-G monoclonal antibodies demonstrated that Ga and Gb shared different epitopes with G.

Cell culture adaptation of astrovirus involves a deletion.

Astroviruses have been adapted to culture by serial blind passage in primary human embryo cells. All viruses thus adapted possess a 45-nucleotide deletion relative to fecal viruses or isolates made in CaCo-2 cells; this deletion may be responsible for the change in host cell range.

The complete sequence of a human astrovirus.

We have determined the complete genomic sequence of human astrovirus serotype 1 isolated in Newcastle upon Tyne. The genome is 6813 nucleotides long and contains three sequential open reading frames (ORFs). The two closest to the 5' end are linked by a ribosomal frameshifting motif and contain sequence motifs indicative of non-structural virus proteins: serine protease and RNA-dependent RNA polymerase. A nuclear addressing sequence is also located here. The 3' ORF encodes the virion structural polypeptides as a polyprotein precursor. This genomic organization resembles that of the plant virus family Luteoviridae.

Prevalence of human astrovirus serotype 4: capsid protein sequence and comparison with other strains.

Astrovirus serotype 4 has increased in relative prevalence in the Oxford, UK area in 1993. The structural gene of human astrovirus serotype 4 has been sequenced and the results indicate that this protein differs substantially from serotypes 1 and 2. In particular, conservation at the C terminus is greatly reduced. However, amino acid substitutions in this region show a strong conservation in character suggesting that structural or functional constraints operate in this region.

Seroprevalence of astrovirus types 1 and 6 in London, determined using recombinant virus antigen.

We have developed a microimmunofluorescence test (IF) which uses cells infected with a recombinant baculovirus which expresses the capsid proteins of astrovirus types 1 or 6. The IF test was sensitive and specific and the results for human astrovirus type 1 (HAst-1) were comparable to those obtained by immune electronmicroscopy and radioimmunoassay. Application of the test to a panel of 273 sera collected from patients and staff at two childrens hospitals in London showed that over 50% of the population were infected by HAst-1 between the age of 5 and 12 months rising to 90% by 5 years, whereas human astrovirus type 6 (HAst6) was relatively uncommon (10-30%) in all age groups.

Application of electronmicroscopy, enzyme immunoassay, and RT-PCR to monitor an outbreak of astrovirus type 1 in a paediatric bone marrow transplant unit.

During 1997, an extensive outbreak of astrovirus occurred in a unit where paediatric patients were being treated for leukaemias and inherited immune deficiency disorders. Prolonged shedding of virus for many months following infection was demonstrated in three patients who had undergone bone marrow transplantation. Comparison of reverse transcription-polymerase chain reaction (RT-PCR), enzyme immunoassay (EIA), and electronmicroscopy (EM) to monitor the outbreak showed that many subclinical infections, mainly in children aged > 3 years could only be detected by RT-PCR. Use of RT-PCR revealed that several patients were infected earlier and shed virus for longer than by using EM or EIA. The virus responsible for the outbreak was identified as HAstV-1 and was shown to have a sequence that differed from a strain obtained in 1988.

Relationships between monoclonal antibody-binding sites on the measles virus haemagglutinin.

Twenty-one monoclonal antibodies directed against the measles virus haemagglutinin have recently been obtained. These were known to fall into five groups, each defined by its effects on the biological functions of the H protein. A representative of each group was selected and examined by competitive radioimmunoassay in an attempt to deduce the relationships between antibody-binding sites on the antigen. It was found that these five antibodies formed three binding groups which recognized different but overlapping areas of the molecule. These three areas formed a series of sites which traversed the active region of the H polypeptide. A haemagglutinin-directed monoclonal antibody which displayed a haemolysin-inhibiting activity was also examined. This antibody fitted into the binding group scheme determined here and there was no additional binding site for this molecule on the F protein itself.

Comparison of lytic and persistent measles virus matrix proteins by competition radioimmunoassay.

Measles virus matrix (M) proteins were compared by competitive monoclonal antibody-binding studies. Three strains of measles and of subacute sclerosing panencephalitis viruses were found to be identical in this way. The matrix protein formed by Edmonston strain virus during a persistent infection could be distinguished from that made in the lytic virus infection. It is concluded that structural alterations in the M peptide can occur during persistence.

The nucleotide sequence of sacbrood virus of the honey bee: an insect picorna-like virus.

We have determined the nucleotide sequence of sacbrood virus (SBV), which causes a fatal infection of honey bee larvae. The genomic RNA of SBV is longer than that of typical mammalian picornaviruses (8832 nucleotides) and contains a single, large open reading frame (179-8752) encoding a polyprotein of 2858 amino acids. Sequence comparison with other virus polyproteins revealed regions of similarity to characterized helicase, protease and RNA-dependent RNA polymerase domains; structural genes were located at the 5' terminus with non-structural genes at the 3' end. Picornavirus-like agents of insects have two distinct genomic organizations; some resemble mammalian picornaviruses with structural genes at the 5' end and non-structural genes at the 3' end, and others resemble caliciviruses in which this order is reversed; SBV thus belongs to the former type. Sequence comparison suggested that SBV is distantly related to infectious flacherie virus (IFV) of the silk worm, which possesses an RNA of similar size and gene order.

Strain variation of respiratory syncytial virus.

Western blotting and immunoperoxidase staining with monoclonal antibodies were employed to analyse epitopic and polypeptide molecular weight variation among isolates of respiratory syncytial (RS) virus collected in Newcastle between 1965 and 1986. One group of isolates resembled the A2 and Long prototype subgroup A strains of RS virus in possessing a P protein of Mr 34,000. Isolates in this subgroup showed two patterns of reactivity with subgroup A-specific monoclonal antibodies to the G glycoprotein and 22K protein. Isolates with both reactivity patterns were isolated throughout the period studied. Isolates in the second group resembled the 8/60 subgroup B prototype strain in their lack of reactivity to subgroup A-specific monoclonal antibodies but were heterogeneous in P protein molecular weight. The earliest isolate only, made in 1965, possessed a P protein of Mr 31,000 resembling the prototype strain. All subsequent subgroup B isolates possessed a higher Mr, 33,000, P protein. Overall, subgroup A viruses were isolated most frequently although subgroup B strains may have predominated in some epidemics.