- Volume 69, Issue 5, 1988
Volume 69, Issue 5, 1988
- Articles
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ANNOUNCEMENTS
Genetics and Pathogenicity of Negative Strand Viruses
Dinard, France September 18–23 1988
The seventh triennial international conference on negative strand viruses will be organized by Anne Flamand, Dan Kolakofsky and Brian Many. For information please contact:
Dr Brian Mahy
AFRC Institute for Animal Health
Pirbright Laboratory
Woking
Surrey GU24 0NF
U.K.
Tel: +44 483 232442 Telex 859137 AVRI G
First Asia-Pacific Congress of Medical Virology
Singapore 6–11 November 1988
The scientific programme has been planned to cater for the interests of both the virologist and the clinician. Eighteen distinguished scientists will talk on AIDS, viral vaccines, viruses and cancers, viral hepatitis, persistent viral infections and viral haemorrhagic fevers. Participants are invited to submit papers for the colloquia and poster sessions. An interesting social programme for accompanying persons as well as post-congress tours in South-East Asia are available. For further information please contact:
The Congress Secretariat
c/o Department of Microbiology
National University of Singapore
Lower Kent Ridge Road
Singapore 0511
Tel: +65 7723276 Telex 33943 UNISPO RS
Electron Microscopy in Applied Plant Pathology
Konstanz, F.R.G. 19–22 September 1989
Organized by Kurt Mendgen, University of Konstanz and Dietrich-E. Lesemann, Biologische Bundesanstalt für Land- und Forstwirtschaft, Braunschweig.
The scope of this symposium is to provide up-to-date reviews of a broad spectrum of electron microscopical techniques currently in use as well as to demonstrate their application to the study of various groups of plant pathogens and of host-pathogen (symbiont) interactions. In plenary sessions invited speakers of different disciplines will review their field of research. Offered papers will be presented in poster sessions. For further information please contact:
Prof. Dr Kurt Mendgen
University of Konstanz
Lehrstuhl für Phytopathologie
D-7750 Konstanz
F.R.G.
Tel: +49 7531 882105 Telex 073359 UNIV D
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- Bacterial
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Formation of Complexes between Long Tail Fibres and Substructural Elements of Phage T4D
More LessSummaryComplexes of substructural elements of bacteriophage T4 (baseplates, baseplate-core complexes) with long tail fibres were obtained for the first time by complementation in vitro. A study of the organization of the complexes was carried out by PAGE, electron microscopy and sedimentation analysis. About 90% of baseplates and baseplate-core complexes were combined with fibres. However, the number of the attached fibres varied from one to six. On the basis of the data obtained, we proposed that the attachment of long tail fibres can occur before the assembly of the whole bacteriophage.
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- Animal
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Biochemical and Serological Comparisons of Australian Bunyaviruses Belonging to the Simbu Serogroup
More LessSummaryComparative analysis of the structural and possible non-structural proteins of seven Simbu serogroup bunyaviruses isolated in Australia revealed them all to be similar in size to those of Bunyamwera virus, the prototype of the Bunyavirus genus. The molecular weights of the structural proteins for these bunyaviruses (Akabane, Aino, Tinaroo, Douglas, Peaton, Facey’s Paddock and Thimiri viruses) were 193K to 205K (L), 103K to 125K (G1), 33K to 37K (G2) and 25K to 26K (N). Analysis of the virion RNA of three viruses (Akabane, Douglas and Facey’s Paddock) showed them all to be similar to Bunyamwera virus RNA, apparent M r values being 2·6 × 106 (L), 1·4 × 106 to 1·9 × 106 (M) and 0·24 × 106 to 0·42 × 106 (S). Host cell protein synthesis was switched off late during infection, revealing four structural proteins L, G1, G2 and N. Comparative analysis of these protein profiles in infected Vero cells showed each virus, although similar, to be unique and easily identified; this method of comparison was efficient and rapid compared to the difficulty in obtaining adequate amounts of purified virus for analysis. Additionally, for all viruses except Douglas, two to four possible non-structural proteins were identified, with an M r range from 12K to 30K. The viruses Akabane and Tinaroo, which have previously been shown to cross-react by plaque inhibition virus neutralization tests, were readily distinguished in migration of the G1 glycoprotein and by analysis of plaque reduction virus neutralization data using linear regression analysis of the dose—response curves. Using these same analyses, the differences between Aino and Douglas viruses, also related by plaque inhibition, were even greater. Application of the biochemical analysis of virus-specified proteins and some serological comparisons identified a mixed pool of different viruses in two unknown isolates grouped as Simbu serogroup viruses, and further identified a potential teratogenic strain in one of the two pools.
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Protective Effects of Monoclonal Antibodies against Parainfluenza Virus Type 3-induced Brain Infection in Hamsters
More LessSummaryA collection of monoclonal antibodies (MAbs) against the haemagglutinin—neuraminidase (HN) and fusion (F) glycoproteins of parainfluenza virus type 3 (PIV3) was used for passive immunization of intracerebrally infected newborn hamsters. A significant degree of protection was obtained with MAbs against both the HN and F viral proteins, but no individual antibody conferred complete protection against disease. MAbs against different epitopes provided different degrees of protection. A MAb to the Sendai virus HN protein was found to confer protection against PIV3 brain infection.
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Sequence Analysis of an 11.2 Kilobase, Near-terminal, BamHI Fragment of Fowlpox Virus
More LessSummaryThe nucleotide sequence of an 11·2 kilobase fragment of the fowlpox virus genome is presented. The fragment comes from near one end of the genome and contains part of the terminal inverted repeat. Twenty open reading frames (ORFs) are predicted from the sequence and are classified into 13 major and seven minor ORFs. The 100 base pairs immediately upstream of each ORF are up to 83% AT-rich, with some motifs similar to those seen in vaccinia virus early gene promoters. The TTTTTNT element which has been identified as a termination signal for vaccinia virus early genes is also found downstream of several ORFs. Three ORFs are predicted to specify polypeptides with significant homology to proteins coded by genes near termini of orthopoxvirus genomes: the vaccinia virus 42K early gene and 32·5K host range gene, and the cowpox virus 38K red pock gene. In addition, there are two families of ORFs within the fragment which potentially encode related polypeptides. One of these, family B, contains three ORFs which are related to those of chicken and rat hepatic lectins.
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Identification of the Coronavirus MHV-JHM mRNA 4 Product
More LessSummaryA bacterial expression vector was constructed to encode a fusion protein which had, at its carboxy terminus, a polypeptide encoded within the 5′ proximal open reading frame of the coronavirus MHV-JHM mRNA 4. This polypeptide was isolated and used to produce an antiserum. The antiserum reacted specifically with a 15000 M r polypeptide synthesized in MHV-JHM-infected cells, or in vitro translations of infected cell poly(A) RNA enriched for mRNA 4. These results demonstrate the translational activity of mRNA 4 during infection, identify conclusively the translation product and provide a means to investigate the synthesis and function of this protein.
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Epstein—Barr Virus Gene Expression in Nasopharyngeal Carcinoma
More LessSummaryEpstein—Barr virus (EBV), an agent with growth transforming potential for human B cells, is associated with certain B cell lymphomas in man and also with an epithelial tumour, undifferentiated nasopharyngeal carcinoma (NPC). Since B cell growth transformation is associated with the constitutive expression of a small number of EBV-coded latent proteins, the nuclear antigens EBNA 1, EBNA 2, EBNA 3 and EBNA-LP and the latent membrane protein (LMP), the present work sought to determine whether this same pattern of virus gene expression occurred in NPC. Tumour biopsies were taken from NPC patients from three areas of differing tumour incidence (Kenya, Algeria, Britain) and immediately snap-frozen, as were biopsies of non-EBV-related carcinomas for controls. Immunoblotting of PAGE-separated proteins with selected human sera identified 24 NPC biopsies clearly expressing EBNA 1. When the analysis was extended using selected human sera with antibodies against the other EBNAs, there was no detectable expression of EBNA 2, EBNA 3 or EBNA-LP in any of these 24 biopsies; their EBNA 2-negative status was confirmed using a monoclonal antibody (MAb) PE2 which was reactive in immunoblotting and in immunoprecipitation with EBNA 2A and EBNA 2B proteins. Similar experiments with two different LMP-specific MAbs, CS1 to 4 and S12, revealed heterogeneity between NPC biopsies; 9/24 biopsies were demonstrably LMP-positive, the degree of expression varying considerably between individual tumours in a manner which was not related to the level of EBNA 1 expression. None of the 24 NPC biopsies expressed detectable amounts of EBV lytic cycle antigens. A nude mouse-passaged NPC cell line, C15, likewise expressed EBNA 1 and LMP but none of the other EBV latent proteins nor lytic cycle antigens. This work identifies a novel type of EBV-cell interaction in NPC cells which is distinct from that seen in in vitro transformed B cell lines and from that seen to date in EBV-positive B cell lymphomas.
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Effect of Immunocompetence on the Establishment and Maintenance of Latency with Marek’s Disease Herpesvirus
More LessSummaryMarek’s disease virus (MDV) infections normally have an early cytolytic phase in lymphoid organs at 3 to 6 days post-infection followed by a period of latent infection. Little is known about the mechanisms that govern latency with herpesvirus infections, including Marek’s disease (MD). To investigate the importance of immunocompetence for either the establishment or the maintenance of latency in MD, immunosuppressive treatments were applied prior to infection with MDV or after latency was established. These included cyclosporin (Cs) or betamethasone (BM) treatments, neonatal thymectomy plus cyclophosphamide treatment (Tx/Cy), and infection at a young age before full competence. The effect of all the treatments was determined by examining tissues and spleen cells for evidence of virus replication before and after cultivation in vitro. Induced (Cs or Tx/Cy treatments) or natural (infection at a young age) incompetence resulted in prolonged and more widespread early cytolytic infection. Immunosuppression by Cs after latency had developed resulted in a reappearance of cytolytic infection in the spleen and it enhanced the cytolytic infection in the thymus and the bursa of Fabricius. After immunosuppression with Cs, cytolytic infection was found mostly in T cells, although many of the virus-positive cells did not have markers for either T cells or B cells. Immunosuppression by BM after latency had developed also resulted in the reappearance of cytolytic infection in the spleen but only at a very low level. These results suggest that immunocompetence is required for the establishment and maintenance of latency.
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Enhanced in vitro Reactivation of Herpes Simplex Virus Type 2 from Latently Infected Guinea-pig Neural Tissues by 5-Azacytidine
More LessSummary5-Azacytidine (5-AZC) reduces cytosine methylation in DNA and has been reported to activate quiescent virus genes. Treatment of explant cultures of latently herpes simplex virus type 2 (HSV-2)-infected guinea-pig dorsal root ganglia and spinal cords in vitro with 5-AZC significantly enhanced the rate of HSV recovery. Both the number of isolates from ganglia (P < 0.001) and the rate of recovery (P < 0.001) were significantly increased with the addition of 50 µM-5-AZC to explant cultures. Increased virus recovery appeared to be due to the induction of reactivation of latent virus, rather than an increase in replication, since 5-AZC inhibited HSV replication. These data support a role for methylation in HSV latency and reactivation.
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Identification of Antigenic Differences Between the Diabetogenic and Non-diabetogenic Variants of Encephalomyocarditis Virus Using Monoclonal Antibodies
SummaryThe M variant of encephalomyocarditis (EMC) virus consists of two biologically distinct variants: one, diabetogenic D variant (EMC-D) and the other, non-diabetogenic B variant (EMC-B). These two variants cannot be distinguished by hyperimmune sera. Monoclonal antibodies were generated against EMC-D or EMC-B to identify antigenic differences between these two variants. Fourteen independent hybrid cell lines, selected from seven separate fusions of mouse myeloma cells to spleen cells isolated from mice immunized with EMC-D, consisted of 12 hybrids which produced monoclonal antibodies that neutralized both EMC-D and EMC-B, and two hybrids (ED-HJ-23 and ED-HJ-31) which produced monoclonal antibodies that neutralized EMC-D but not EMC-B. Similarly, 16 independent hybrid cell lines, selected from eight separate fusions using spleen cells prepared from mice immunized with EMC-B, consisted of 15 hybrids which produced monoclonal antibodies neutralizing both EMC-D and EMC-B, and one hybrid (EB-48A-F1) which produced antibody that neutralized EMC-B, but not EMC-D. The specificities of these monoclonal antibodies (ED-HJ-23, ED-HJ-31, EB-48A-F1) were further confirmed using an immunofluorescent technique. The D variant-specific monoclonal antibodies reacted with cells infected with EMC-D but not EMC-B. In contrast, the B variant-specific monoclonal antibody reacted with the cells infected with EMC-B but not EMC-D. It is concluded that the EMC-D- and EMC-B-specific monoclonal antibodies are able to identify antigenic differences between diabetogenic and non-diabetogenic variants of EMC virus which cannot be distinguished by hyperimmune sera.
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The Effect of Sequences in the 5′ Non-coding Region on the Replication of Polioviruses in the Human Gut
More LessSummaryThe genomic sequences of a portion of the 5′ non-coding region of polioviruses excreted by recipients of the Sabin vaccine strains of poliovirus were examined. It was found that in all three types a residue changed in the course of excretion to give a sequence representing the consensus of poliovirus sequences. For type 1 this residue was at position 481, which altered in half the vaccinees, for type 2 it was at position 480, which altered in all vaccinees and for type 3 it was at position 472 in all vaccinees, as previously reported. Other vaccine strains unrelated to the Sabin strains but used in limited vaccination programmes did not have such changing residues. It was concluded that these regions indicated sequences that were strongly selected during replication in the human gut. Such sequences were further defined by examination of strains of poliovirus unrelated to the vaccine strains, the sequences of which may be expected to vary at less stringently defined regions.
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Virus-specific Polypeptides of Caprine Arthritis-Encephalitis Virus Recognized by Monoclonal Antibodies to Virion Proteins p24 and p14
More LessSummaryThe proteins of an isolate of caprine arthritis-encephalitis virus (CAEV) were analysed by SDS-PAGE and Western blotting. Monoclonal antibodies (MAbs) produced to the main core protein p24 and the small structural protein p14 also recognized two major polypeptides of M r 41K and 55K in infected cell material, consistent with a precursor role for these gag polypeptides. In addition, the p24 MAbs detected a 33K polypeptide in extracellular virus preparations, while the p14 MAbs reacted strongly with a polypeptide of 18K (corresponding to structural protein p18) and weakly with another of 21K. The use of these MAbs in an indirect fluorescent antibody method revealed an intracytoplasmic location of these viral antigens in both mononucleated and multinucleated (syncytial) infected cells. Cross-reactivity with several other isolates indicated that these MAbs may be useful for diagnosis of CAEV infection.
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Antigenic Structure of the Murray Valley Encephalitis Virus E Glycoprotein
More LessSummaryTo complement our battery of St Louis encephalitis (SLE) virus monoclonal antibodies (MAbs), we isolated and characterized MAbs reactive with another member of the SLE virus serocomplex, Murray Valley encephalitis (MVE) virus. From 40 fusion products, we isolated 10 stable hybridomas. The combination of SLE and MVE virus MAbs defined eight epitopes on the MVE envelope (E) glycoprotein. Six of these epitopes (E-1a, E-1c, E-1d, E-3, E-4a, E-4b) were identical to those previously demonstrated on SLE virus. Two new epitopes (E-5 and E-6) were also identified. As with SLE virus, the MVE E-1c epitope elicited the most potent virus-neutralizing and protective MAb. Unlike SLE virus, however, one of the cross-reactive epitopes (E-5) elicited neutralizing antibody and protected animals from MVE virus challenge. These results indicate that, while the antigenic domains on viruses within the SLE virus serocomplex are quite similar, epitopes involved in virus neutralization or protection from virus challenge may vary and can be topologically distinct.
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The Global Spread and Replacement of Canine Parvovirus Strains
SummaryCanine parvovirus type 2 (CPV-2) became widespread during 1978 and was reported in many countries during 1978 and 1979. Earlier studies showed that CPV-2 was replaced in the U.S.A. around 1980 by an antigenically and genetically variant virus (CPV-2a). Here we show that CPV-2 was present in the U.S.A., Japan, Belgium and Australia prior to 1980, but that between 1979 and 1982 CPV-2 was replaced by CPV-2a in all of those countries as well as in France and Denmark. Examination of sera collected between 1979 and 1984 from wild coyotes (Canis latrans) in the U.S.A. by an agar gel precipitin assay indicated that the coyotes were originally infected by CPV-2, but that after 1980 the juvenile coyotes were being infected with CPV-2a. The natural global replacement of CPV-2 by CPV-2a over a period of 2 to 3 years indicates that CPV-2a has a strong epidemiological advantage over CPV-2, although the mechanism involved remains to be defined.
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- Plant
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Characterization of Maize Streak Virus Isolates from Different Plant Species by Polyclonal and Monoclonal Antibodies
More LessSummaryRabbit polyclonal antisera raised against six different isolates of maize streak virus (MSV) were used to determine the serological relationships among a range of MSV isolates from different plant species originating from various African countries and from Vanuatu. Their reactions indicated a great serological diversity among these isolates. Fifteen monoclonal antibodies (MAbs) raised against a Nigerian maize isolate, MSV-M(N)M, were characterized according to their reactivity with the various MSV isolates in indirect ELISA. All the MAbs reacted with the virus both in an antigen-coated plate and in a double antibody sandwich ELISA. None of the MAbs reacted with all isolates, and some isolates most distantly related to MSV-M(N)M according to results obtained with polyclonal antisera were not recognized by any of the MAbs. Some isolates which were shown to be closely related using the polyclonal antisera could be distinguished by several of the MAbs. The results obtained both with the polyclonal antisera and the MAbs suggested that several of the isolates were a mixture of a number of different serotypes.
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Detection of Potato Virus X by One Incubation Europium Time-resolved Fluoroimmunoassay and ELISA
More LessSummaryA time-resolved fluorimmunoassay (TRFIA) with europium-labelled monoclonal antibodies (MAbs) was used to detect potato virus X (PVX) at a concentration of 5 pg/ml, in potato tuber extracts diluted up to 7 × 104-fold and leaf extracts diluted up to 2 × 107-fold. When mixed with potato leaf sap, PVX was detected at 100 pg/ml. TRFIA with simultaneous incubation of antigen and labelled antibody (one incubation) was two orders of magnitude more sensitive for PVX detection than the conventional double antibody sandwich ELISA. The fluorescence signal in TRFIA with separate incubations of antigen and labelled antibody (two incubations) with MAbs was linearly related to the virus concentration between 1.6 ng/ml and 1000 ng/ml; the method can therefore be used for the quantification of PVX. PVX detection in plant specimens by one incubation ELISA with MAbs was generally 10 times more sensitive than the standard two incubation procedure. Europium TRFIA with MAbs to potato viruses was found to be a very sensitive method for the detection of PVX, potato virus M, potato virus S, potato virus Y and potato leaf roll virus.
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Isolation of Defective Interfering Particles of Sonchus Yellow Net Virus from Chronically Infected Plants
More LessSummaryNicotiana edwardsonii plants were examined by electron microscopy 5 months after inoculation with Sonchus yellow net virus (SYNV). No virions were observed in leaf or root cells, but cells in sections of calyx tissue contained large numbers of virus particles. Most of these particles were only 73 to 86% as long as particles of standard SYNV. Nicotiana edwardsonii inoculated with sap extracted from chronically infected calyx became systemically infected but exhibited chlorotic mottling, instead of the normal vein-clearing symptoms. Most virus particles in these plants were short, and when purified, sedimented more slowly than standard SYNV. Purified short particles were not infective, but plants inoculated with a mixture of short and standard particles developed mottling symptoms and yielded predominantly short particles. Proteins from short particles were electrophoretically and antigenically identical to those from standard virus. RNA from short particles was 77% the size of RNA from standard SYNV and hybridized to cloned SYNV cDNA. These short particles have all the characteristics of defective interfering particles.
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Analysis of in vitro Translation of Bean Yellow Mosaic Virus RNA: Inhibition of Proteolytic Processing by Antiserum to the 49K Nuclear Inclusion Protein
More LessSummaryIn vitro translation of bean yellow mosaic virus (BYMV) RNA in rabbit reticulocyte lysate without dithiothreitol (DTT) resulted in the accumulation of high M r products. These were readily processed to low M r mature proteins after the addition of DTT followed by a 2 h incubation. Immunoprecipitation analyses of the partially processed high M r products with antisera to the 49K and 54K nuclear inclusion (NI) proteins, cylindrical inclusion (CI) protein, tobacco vein mottling virus helper component (HC) protein and capsid protein (CP) resolved the following BYMV RNA genome map: 5′ end, 32K unidentified protein, 48K HC, 42K unidentified protein, 73K CI, 49K NI, 54K NI, 32K CP, 3′ end. The addition of the 49K NI protein antiserum inhibited the proteolytic processing of the high M r translation products. The inhibition of processing was diminished by diluting the 49K NI antiserum or by adding purified 49K NI protein. Preimmune serum or the antisera to the 54K NI, CI and capsid proteins were not as effective in inhibiting the proteolytic processing. These results provide evidence that the 49K NI protein or a related protein may have a protease function in the processing of the potyvirus polyproteins.
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- Fungal
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Relationships and Functions of Virus Double-stranded RNA in a P4 Killer Strain of Ustilago maydis
More LessSummaryDouble-stranded RNA (dsRNA) from virus particles isolated from a P4 killer strain (isolate 77) of Ustilago maydis, which secretes a protein toxin of apparent M r approx. 10000 (10K) was resolved by PAGE into eight segments with sizes (kbp) 6·7 (H1), 4·5 (H2), 3·2 (H3a), 3·1 (H3b), 2·6 (H4), 0·98 (M2-4), 0·92 (M3-4) and 0·36 (L1). A subculture, designated 77-3A, was found to have lost the H2 and M3-4 segments, but still produced virus particles and protein toxin. Northern hybridization showed that dsRNA segments M2-4 and L1 are related but no homology was detected between H1, H3a, H3b and H4, or between any of these H segments and M2-4 or L1. When individual denatured dsRNA segments were translated in vitro in a reticulocyte lysate system H1, H3a, H3b and H4 each gave rise to polypeptides in the M r range 100K to 128K. Polypeptides of apparent M r 108K and 128K, encoded by H3b, and 100K, encoded by H4, were immunoprecipitated by antiserum to purified virus particles from subculture 77-3A. In vitro translation of denatured M2-4 gave rise to polypeptides of apparent M r 26K and 13K which were immunoprecipitated by antiserum to the secreted protein toxin. No in vitro translation product was detected from denatured L1.
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- Addendum
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An Antigenic Analysis of the Adenovirus Type 2 Fibre Polypeptide
By G. Watson, M. G. Burdon and W. C. Russel
Journal of General Virology (1988), 69, 525–535
The authors of this paper wish to emphasize that all the work described was performed at St Andrews before the first author moved to her present address.
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