Caroline R. Astell

Human Illness from Avian Influenza H7N3, British Columbia

Avian influenza that infects poultry in close proximity to humans is a concern because of its pandemic potential. In 2004, an outbreak of highly pathogenic avian influenza H7N3 occurred in poultry in British Columbia, Canada. Surveillance identified two persons with confirmed avian influenza infection. Symptoms included conjunctivitis and mild influenzalike illness.

Sequence of the gene for iso-l-cytochrome c in saccharomyces cerevisiae

Abstract The complete sequence of the iso-1-cytochrome c gene of yeast has been determined. The coding region of the gene contains no intervening sequences. The coding strand of the DNA immediately upstream from the coding region contains many fewer G residues than the rest of the coding strand both within and beyond the carboxy terminus of the coding region. One consequence of the reduced number of G residues in this region is the absence of the sequence ATG for 122 nucleotides upstream from the initiating ATG. Together with previous studies on the mRNA and the genetics of yeast iso-1-Cytochrome c, the sequence supports a model in which translation starts at the first AUG down-stream from the 5′ terminus of the mRNA, with no other sequence requirements. It also is evident that iso-1-cytochrome c is synthesized directly and not through an intermediary, longer precursor protein, as is often the case for proteins that interact with membranes. The DNA upstream and downstream from the coding region contains sequences which are potential transcription start and stop signals. The sequence confirms the assignments of non-sense and missense mutations throughout the coding region of the gene and provides a rationale for some mutational characteristics of the gene. Part of the sequence was determined using two new strategies for the Sanger terminator method, both of which obviate the need for restriction fragment isolation and template strand separation.

Novel Avian Influenza H7N3 Strain Outbreak, British Columbia

Genome sequences of chicken (low pathogenic avian influenza [LPAI] and highly pathogenic avian influenza [HPAI]) and human isolates from a 2004 outbreak of H7N3 avian influenza in Canada showed a novel insertion in the HA0 cleavage site of the human and HPAI isolate. This insertion likely occurred by recombination between the hemagglutination and matrix genes in the LPAI virus.

In vitro identification of a B19 parvovirus promoter

The nucleotide sequence of the B19-Wi isolate of human parvovirus was determined and compared throughout the open reading frames and putative transcription signals with the sequence of the closely related B19-Au isolate. In vitro run off transcription assays, using B19-Wi DNA as the template, indicated that there is a strong promoter between m.u. 5 and 7. Deletion clones show that a region between nt 258 and 321 is necessary for in vitro transcriptional activity. Primer extension studies identified the start site at 31-32 nucleotides downstream of the sequence TATATATA. The strength of this left-hand promoter is unusual among parvovirus promoters characterized to date, and the possibility of an upstream enhancer element is discussed.

Expression of minute virus of mice major nonstructural protein in insect cells: Purification and identification of ATPase and helicase activities

The gene encoding the major nonstructural (NS-1) protein of minute virus of mice (MVM) has been expressed in insect cells using a baculovirus expression system. This 83-kDa polypeptide was found to be localized in the soluble (cytosolic) fraction in insect cells, in contrast with the nuclear localization of NS-1 expressed in MVM-infected mouse LA-9 cells. The protein was purified by immunoaffinity chromatography using a monoclonal antibody (MAb) prepared to an NS-1 fusion peptide [(Yeung et al., Virology 185, 35-45 (1991)]. Recombinant NS-1 was eluted using either low pH or a synthetic peptide corresponding to the epitope of the MAb. The peptide-eluted material is greater than 95% pure and biologically active in that it has ATPase activity and ATP-dependent helicase activity as determined by a strand displacement assay.

Structural and Functional Homology of Parvovirus and Papovavirus Polypeptides

We have compared the sequences of the putative polypeptides of the human pathogenic B19 parvovirus with protein sequences in the National Bethesda Research Foundation Library, and have discovered a significant homology between a B19 parvovirus non-structural (NS) protein and the T antigens of polyomaviruses and simian virus 40 (SV40) and the putative E1 proteins of papillomaviruses. The region of highest homology with the papovavirus proteins corresponds to the region that is most highly conserved in the NS1 proteins of several other parvoviruses. Studies with the T antigen of both polyomaviruses and SV40 have implicated this region as having an ATPase activity and nucleotide-binding function.

Monoclonal antibodies to the major nonstructural nuclear protein of minute virus of mice

Monoclonal antibodies were raised against a bacterial fusion protein containing amino acids 364 to 623 of the major nonstructural protein, NS-1, of minute virus of mice (MVMp), an autonomous parvovirus. By immunoblot analyses, these antibodies all recognized an 83-kDa protein in MVM-infected mouse fibroblast cells. Indirect immunofluorescence studies showed that five of the six react against a nuclear protein in MVM-infected mouse cells resulting in discrete foci of fluorescence. These foci do not correspond with the nucleoli, the site of MVM DNA replication. The epitopes of the antibodies were mapped using carboxy-terminal deleted bacterial fusion proteins derived from the plasmid encoding the original antigen and showed that four distinct epitopes were recognized by the different antibodies. A 25-amino-acid peptide was used in competition ELISAs to confirm the location of the epitope recognized by two antibodies CE10 and AC6. Preliminary characterization of an NS-1/NS-2 fusion protein synthesized in insect cells using a baculovirus expression vector showed that this fusion protein is also localized within the nucleus; however, in contrast, the full-length NS-1 polypeptide is located within the cytoplasm.

Analysis of splice junctions and in Vitro and in Vivo translation potential of the small, abundant B19 parvovirus RNAs

Two parvovirus B19 cDNA libraries have been constructed; one from COS-7 cells transfected with a B19/pSVOd hybrid vector and the other from B19-infected human erythroid leukemic cells. We have used these libraries to investigate the expression of the abundant classes of polyadenylated B19 RNAs; the 700- and 800-nt class which terminates in the middle of the genome and the 500- and 600-nt class which contains an ORF from the extreme right-hand end of the genome. The 700- and 800-nt RNA species were not found in the COS cell library, suggesting that a variant polyadenylation signal (ATTAAA or AATAAC) in the middle of the genome is not efficiently recognized in these cells. In contrast, the 700- and 800-nt class was highly represented in the human library, confirming the use of this variant polyadenylation signal in the normal host cell of the virus. In COS cells the middle exon of the 500- and 600-nt class of RNA exhibited variability in both splice donor and acceptor sites. However, in human cells there were only two splice acceptor sites nt 1910 and 2030, and a single splice donor site nt 2183 for this exon. Antisera, prepared against a peptide derived from the 94-aa potential protein encoded by the 500- and 600-nt class of RNA, recognized, on a Western blot, a polypeptide of approximately 11 kDa that was translated in vitro from these cDNAs and in vivo in pSVOd/B19 transfected COS cells. Immunoprecipitation revealed that two proteins were made from this ORF, suggesting the use of internal translation initiation site(s). Another antisera, raised against a second peptide corresponding to an antigenic region of the potential protein encoded by the 700- and 800-nt class of RNA, failed to detect a 15-kDa protein by Western blotting or immunoprecipitation of labeled proteins both in vitro and in vivo in COS cells.

Recombinant Leishmania surface glycoprotein GP63 is secreted in the baculovirus expression system as a latent metalloproteinase

A gene encoding the Leishmania surface metalloproteinase, GP63, was modified using the polymerase chain reaction to obtain effective secretion of recombinant GP63 (reGP63) in the baculovirus insect cell expression system. The coding region for the N-terminal signal peptide (SP) of GP63 was modified to resemble the SP for the GP67 envelope protein from the budded virus form of Autographa californica nuclear polyhedrosis virus. To prevent processing at the C-terminus with a glycosyl phosphatidylinositol anchor and the subsequent membrane anchoring of reGP63 in insect cells, the coding region for a putative SP at the C-terminus of GP63 was deleted. The reGP63 protein was glycosylated and secreted as a latent metalloproteinase in the baculovirus expression system. The reGP63 protein was purified from serum-free medium using concanavalin A lectin affinity chromatography, with a yield of 1 mg/l. The purified Leishmania reGP63 was secreted as a latent proteinase. Treatment of reGP63 with HgCl2 resulted in activation of full proteinase activity and a concomitant decrease in M(r). The mechanism of the activation of Leishmania reGP63 is consistent with that of other members of the family of matrix-degrading metalloproteinases.

An Outbreak of Human Coronavirus OC43 Infection and Serological Cross-Reactivity with SARS Coronavirus

BACKGROUND In summer 2003, a respiratory outbreak was investigated in British Columbia, during which nucleic acid tests and serology unexpectedly indicated reactivity for severe acute respiratory syndrome coronavirus (SARS-CoV). METHODS Cases at a care facility were epidemiologically characterized and sequentially investigated for conventional agents of respiratory infection, SARS-CoV and other human CoVs. Serological cross-reactivity between SARS-CoV and human CoV-OC43 (HCoV-OC43) was investigated by peptide spot assay. RESULTS Ninety-five of 142 residents (67%) and 53 of 160 staff members (33%) experienced symptoms of respiratory infection. Symptomatic residents experienced cough (66%), fever (21%) and pneumonia (12%). Eight residents died, six with pneumonia. No staff members developed pneumonia. Findings on reverse transcriptase-polymerase chain reaction assays for SARS-CoV at a national reference laboratory were suspected to represent false positives, but this was confounded by concurrent identification of antibody to N protein on serology. Subsequent testing by reverse transcriptase-polymerase chain reaction confirmed HCoV-OC43 infection. Convalescent serology ruled out SARS. Notably, sera demonstrated cross-reactivity against nucleocapsid peptide sequences common to HCoV-OC43 and SARS-CoV. CONCLUSIONS These findings underscore the virulence of human CoV-OC43 in elderly populations and confirm that cross-reactivity to antibody against nucleocapsid proteins from these viruses must be considered when interpreting serological tests for SARS-CoV.