Restrictions to the adaptation of influenza a virus h5 hemagglutinin to the human host.

The binding specificities of a panel of avian influenza virus subtype H5 hemagglutinin (HA) proteins bearing mutations at key residues in the receptor binding site were investigated. The results demonstrate that two simultaneous mutations in the receptor binding site resulted in H5 HA binding in a pattern similar to that shown by human viruses. Coexpression of the ion channel protein, M2, from most avian and human strains tested protected H5 HA conformation during trafficking, indicating that no genetic barrier to the reassortment of the H5 surface antigen gene with internal genes of human viruses existed at this level.

Identification of novel expressed sequences, up-regulated in the leucocytes of chronic fatigue syndrome patients.

Background: Chronic fatigue syndrome (CFS) is an increasing medical phenomenon of unknown aetiology leading to high levels of chronic morbidity. Of the many hypotheses that purport to explain this disease, immune system activation, as a central feature, has remained prominent but unsubstantiated. Supporting this, a number of important cytokines have previously been shown to be over-expressed in disease subjects.

A reverse genetics approach for recovery of recombinant influenza B viruses entirely from cDNA.

The recovery of recombinant influenza A virus entirely from cDNA was recently described (9, 19). We adapted the technique for engineering influenza B virus and generated a mutant bearing an amino acid change E116G in the viral neuraminidase which was resistant in vitro to the neuraminidase inhibitor zanamivir. The method also facilitates rapid isolation of single-gene reassortants suitable as vaccine seeds and will aid further investigations of unique features of influenza B virus.

Sequences in influenza A virus PB2 protein that determine productive infection for an avian influenza virus in mouse and human cell lines.

Reverse genetics was used to analyze the host range of two avian influenza viruses which differ in their ability to replicate in mouse and human cells in culture. Engineered viruses carrying sequences encoding amino acids 362 to 581 of PB2 from a host range variant productively infect mouse and human cells.

Electron microscopy may reveal structure of docosahexaenoic acid-rich oil within Schizochytrium sp.

Schizochytrium sp. is an algae-like microorganism utilized for commercial production of docosahexaenoic acid (DHA)-rich oil and dried microalgae for use as a source of DHA in foods, feeds, and nutritional supplements. Electron microscopic analysis of whole cells of Schizochytrium sp.

Identification of a cis-acting replication element within the poliovirus coding region.

The replication of poliovirus, a positive-stranded RNA virus, requires translation of the infecting genome followed by virus-encoded VPg and 3D polymerase-primed synthesis of a negative-stranded template. RNA sequences involved in the latter process are poorly defined. Since many sequences involved in picornavirus replication form RNA structures, we searched the genome, other than the untranslated regions, for predicted local secondary structural elements and identified a 61-nucleotide (nt) stem-loop in the region encoding the 2C protein.

Isolation and characterization of a transfectant influenza B virus altered in RNA segment 6.

This report describes the successful generation of an influenza B transfectant virus altered in RNA segment 6, which encodes the neuraminidase (NA) protein. The procedure for selection of the transfectant virus relies on the use of strain-specific anti-NA monoclonal antibodies to inhibit growth of the helper virus within the system. A transfectant virus has been engineered which has a coding change in the NA protein.

Functional analysis of cell surface-expressed hepatitis C virus E2 glycoprotein.

Hepatitis C virus (HCV) glycoproteins E1 and E2, when expressed in eukaryotic cells, are retained in the endoplasmic reticulum (ER). C-terminal truncation of E2 at residue 661 or 715 (position on the polyprotein) leads to secretion, consistent with deletion of a proposed hydrophobic transmembrane anchor sequence. We demonstrate cell surface expression of a chimeric glycoprotein consisting of E2 residues 384 to 661 fused to the transmembrane and cytoplasmic domains of influenza A virus hemagglutinin (HA), termed E2661-HATMCT.

Phosphorylation of the M2 protein of influenza A virus is not essential for virus viability.

M2 is a minor component of the influenza A virus envelope. The cytoplasmic tail of the M2 protein is posttranslationally modified in the infected cell by palmitylation and phosphorylation. The primary site for phosphorylation of the M2 cytoplasmic tail is serine 64, which is highly conserved yet not required for the activity of the M2 ion channel.

Encapsidation studies of poliovirus subgenomic replicons.

The inclusion of a foreign marker gene, chloramphenicol acetyltransferase (CAT) gene, into the poliovirus genome allows its replication and encapsidation to be easily monitored using a simple enzyme assay. Such poliovirus replicons require the presence of helper virus for their successful propagation and thus are similar to defective interfering (DI) viruses. In genomes containing the CAT gene, the majority of the P1 virus capsid region of the poliovirus genome could be removed without destroying viability.

The N-terminal extension of the influenza B virus nucleoprotein is not required for nuclear accumulation or the expression and replication of a model RNA.

The nucleoprotein (NP) of influenza B virus is 50 amino acids longer at the N-terminus than influenza A virus NP and lacks homology to the A virus protein over the first 69 residues. We have deleted the N-terminal 51 and 69 residues of the influenza B/Ann Arbor/1/66 virus NP and show that nuclear accumulation of the protein is unaffected. This indicates that the nuclear localization signal is not located at the extreme N terminus, as in influenza A virus NP.

Influenza B viruses with site-specific mutations introduced into the HA gene.

We have succeeded in engineering changes into the genome of influenza B virus. First, model RNAs containing the chloramphenicol acetyltransferase gene flanked by the noncoding sequences of the HA or NS genes of influenza B virus were transfected into cells which were previously infected with an influenza B helper virus. Like those of the influenza A viruses, the termini of influenza B virus genes contain cis-acting signals which are sufficient to direct replication, expression, and packaging of the RNA.

Use of a mammalian internal ribosomal entry site element for expression of a foreign protein by a transfectant influenza virus.

The ribonucleoprotein transfection system for influenza virus allowed us to construct two influenza A viruses, GP2/BIP-NA and HGP2/BIP-NA, which contained bicistronic neuraminidase (NA) genes. The mRNAs derived from the bicistronic NA genes have two different open reading frames (ORFs). The first ORF encodes a foreign polypeptide (GP2 or HGP2) containing amino acid sequences derived from the gp41 protein of human immunodeficiency virus type 1.

Expression of a foreign protein by influenza A virus.

In this report we describe the rescue of a transfectant influenza A virus which stably expresses a heterologous protein, bacterial chloramphenicol acetyltransferase (CAT). The foreign sequences encoding CAT are expressed as part of an essential influenza virus segment, that coding for the neuraminidase (NA) protein. The novel way by which this was achieved involved inserting in frame the 16-amino-acid self-cleaving 2A protease of foot-and-mouth disease virus between the CAT and the NA coding sequences.

The 5'-untranslated regions of picornavirus RNAs contain independent functional domains essential for RNA replication and translation.

The role of the 5'-untranslated region (5'UTR) in the replication of enteroviruses has been studied by using a series of poliovirus type 3 (PV3) replicons containing the chloramphenicol acetyltransferase reporter gene in which the 5'UTR was replaced by the 5'UTR of either coxsackievirus B4 or human rhinovirus 14 or composite 5'UTRs derived from sequences of PV3, human rhinovirus 14, coxsackievirus B4, or encephalomyocarditis virus. The results indicate that efficient replication of an enterovirus genome requires a compatible interaction between the 5'-terminal cloverleaf structure and the coding and/or 3'-noncoding regions of the genome. A crucial determinant of this interaction is the stem-loop formed by nucleotides 46 to 81 (stem-loop d).

Introduction of foreign sequences into the genome of influenza A virus.

The ability to apply reverse genetics technologies to influenza virus now allows us to construct novel viruses containing heterologous sequences. We have engineered two neuraminidase (NA) genes of influenza A/WSN/33 virus containing additional sequences which were inserted downstream of the open reading frame of the NA. These NA genes, NA/EMC and NA/EMC-NS1, possess a 546 and a 917 nucleotide (nt) insertion respectively.

A poliovirus replicon containing the chloramphenicol acetyltransferase gene can be used to study the replication and encapsidation of poliovirus RNA.

A poliovirus replicon, FLC/REP, which incorporates the reporter gene chloramphenicol acetyltransferase (CAT) in place of the region encoding the capsid proteins VP4, VP2, and part of VP3 in the genome of poliovirus type 3, has been constructed. Transfection of cells indicates that the FLC/REP replicon replicates efficiently and that active CAT enzyme is produced as a CAT-VP3 fusion protein. The level of CAT activity in transfected cells broadly reflects the level of FLC/REP RNA.

Tryptophan analog resistance mutations in Chlamydomonas reinhardtii.

Forty single gene mutations in Chlamydomonas reinhardtii were isolated based on resistance to the compound 5'-methyl anthranilic acid (5-MAA). In other organisms, 5-MAA is converted to 5'-methyltryptophan (5-MT) and 5-MT is a potent inhibitor of anthranilate synthase, which catalyzes the first committed step in tryptophan biosynthesis. The mutant strains fall into two phenotypic classes based on the rate of cell division in the absence of 5-MAA.

Immunogenicity and antigenicity of chimeric picornaviruses which express hepatitis A virus (HAV) peptide sequences: evidence for a neutralization domain near the amino terminus of VP1 of HAV.

We evaluated the antigenic characteristics of chimeric picornaviruses created by inserting peptide sequences from hepatitis A virus (HAV) capsid proteins into the B-C loop of VP1 of Sabin strain type 1 poliovirus (PV-1). Fifteen viable chimeras were generated. Each retained the ability to be neutralized by polyclonal PV-1 antisera.

The specificity of antibodies induced by infection with rhinovirus type 2.

Mouse monoclonal antibodies (MAbs) against three distinct antigenic sites on rhinovirus type 2 have been obtained and the sites identified. We describe how these MAbs were used in a blocking test to detect antibodies in human sera directed against the same three defined sites. Sera from twelve volunteers were studied.

The time course of the humoral immune response to rhinovirus infection.

The specific humoral immune response of 17 volunteers to infection with human rhinovirus type 2 (HRV-2) has been measured both by neutralization and by ELISA. Six volunteers who had HRV-2-specific antibodies in either serum or nasal secretions before HRV-2 inoculation were resistant to infection and illness. Of the remaining 11 volunteers who had little pre-existing HRV-2-specific antibody, one was immune but 10 became infected and displayed increases in HRV-2-specific antibodies.