Carol A. Hartley

Complement-dependent neutralization of influenza virus by a serum mannose-binding lectin

The nature of the beta inhibitor in guinea-pig serum and its mechanism of neutralization of influenza virus have been investigated. This inhibitor was shown to be a mannose-binding lectin serologically related to human serum mannose-binding protein. Ca(2+)-dependent binding of the guinea-pig lectin to influenza virus or to mannan could be detected with polyclonal or monoclonal antibodies against human mannose-binding protein in an ELISA. Furthermore, the monoclonal antibody inhibited both the haemagglutination-inhibiting and virus-neutralizing activities of the guinea-pig lectin. The lectin was active against influenza viruses of both type A and type B. In haemagglutination inhibition it acts independently of complement, apparently by sterically hindering access to the receptor-binding site on the viral haemagglutinin through binding of the lectin to carbohydrate side-chains in the vicinity of this site. Neutralization by the lectin, however, was shown to require activation of the classical complement pathway. To our knowledge, the neutralization of influenza virus by a serum lectin plus complement represents a previously unrecognized mechanism of complement-dependent viral inactivation that may be important in first-line host defence against a variety of enveloped viruses.

Attenuated Vaccines Can Recombine to Form Virulent Field Viruses

Problems can arise when vaccines and wild strains of a chicken herpesvirus recombine. Recombination between herpesviruses has been seen in vitro and in vivo under experimental conditions. This has raised safety concerns about using attenuated herpesvirus vaccines in human and veterinary medicine and adds to other known concerns associated with their use, including reversion to virulence and disease arising from recurrent reactivation of lifelong chronic infection. We used high-throughput sequencing to investigate relationships between emergent field strains and vaccine strains of infectious laryngotracheitis virus (ILTV, gallid herpesvirus 1). We show that independent recombination events between distinct attenuated vaccine strains resulted in virulent recombinant viruses that became the dominant strains responsible for widespread disease in Australian commercial poultry flocks. These findings highlight the risks of using multiple different attenuated herpesvirus vaccines, or vectors, in the same populations.

Changes in the hemagglutinin molecule of influenza type A (H3N2) virus associated with increased virulence for mice

SummaryThe H3N2 influenza virus A/Philippines/82 (Phil82) and its bovine serum-resistant mutant, Phil82/BS, were used to investigate factors that influence virulence of influenza virus for mice. Phil82/BS, which lacks the high-mannose oligosaccharide at residue 165 of the hemagglutinin (HA) molecule, was found to replicate to a much higher titer in mouse lung than the parent Phil82, and had acquired lethality for mice. Further adaptation of Phil82/BS by sequential lung passage in mice yielded a strain of greater virulence, Phil82/BS/ML10, in which a change at residue 246 of HA resulted in loss of a second potential glycosylation site. Phil82 is highly sensitive to neutralization in vitro by murine serum- and lung-associated mannose-binding lectins (collectins). Characterization of the two mutant viruses indicated that resistance to murine collectins can account for the enhanced virulence of Phil82/BS but not for the further increase in virulence of Phil82/BS/ML10. Evidence is presented that residue 246 is not in fact glycosylated in Phil82/BS HA, nor presumably in the parent Phil82 virus. The HA molecule of Phil82/BS/ML10 displayed functional differences from Phil82/BS, including a change in the optimum pH of fusion and a minor change in receptor-binding specificity, which may allow improved efficiency of replication in the mouse lung.

Outbreak of equine herpesvirus type 1 myeloencephalitis: new insights from virus identification by PCR and the application of an EHV-1-specific antibody detection ELISA.

Five of 10 pregnant, lactating mares, each with a foal at foot, developed neurological disease. Three of them became recumbent, developed complications and were euthanased; of the two that survived, one aborted an equine herpesvirus type 1 (EHV-1)-positive fetus 68 days after the first signs were observed in the index case and the other gave birth to a healthy foal on day 283 but remained ataxic and incontinent. The diagnosis of EHV-1 myeloencephalitis was supported by postmortem findings, PCR identification of the virus and by serological tests with an EHV-1 -specific ELISA. At the time of the index case, the 10 foals all had a heavy mucopurulent nasal discharge, and PCR and the ELISA were used to detect and monitor EHV-1 infection in them. The status of EHV-1 infection in the five in-contact mares was similarly monitored. Sera from three of the affected mares, taken seven days after the index case were negative or had borderline EHv-1 -specific antibody titres. In later serum samples there was an increase in the titres of EHv-1 -specific antibody in two of the affected mares. In contrast, sera from the five unaffected in-contact mares were all EHv-1 -antibody positive when they were first tested seven or 13 days after the index case.

Evaluation of immunological responses to a glycoprotein G deficient candidate vaccine strain of infectious laryngotracheitis virus.

Infectious laryngotracheitis virus (ILTV), an alphaherpesvirus, causes severe respiratory disease in poultry. Glycoprotein G (gG) is a virulence factor in ILTV. Recent studies have shown that gG-deficient ILTV is an effective attenuated vaccine however the function of ILTV gG is unknown. This study examined the function and in vivo relevance of ILTV gG. The results showed that ILTV gG binds to chemokines with high affinity and inhibits leukocyte chemotaxis. Specific-pathogen-free (SPF) chickens infected with gG-deficient virus had altered tracheal leukocyte populations and lower serum antibody levels compared with those infected with the parent virus. The findings suggest that the absence of chemokine-binding activity during infection with gG-deficient ILTV results in altered host immune responses.

First complete genome sequence of infectious laryngotracheitis virus.

BackgroundInfectious laryngotracheitis virus (ILTV) is an alphaherpesvirus that causes acute respiratory disease in chickens worldwide. To date, only one complete genomic sequence of ILTV has been reported. This sequence was generated by concatenating partial sequences from six different ILTV strains. Thus, the full genomic sequence of a single (individual) strain of ILTV has not been determined previously. This study aimed to use high throughput sequencing technology to determine the complete genomic sequence of a live attenuated vaccine strain of ILTV.ResultsThe complete genomic sequence of the Serva vaccine strain of ILTV was determined, annotated and compared to the concatenated ILTV reference sequence. The genome size of the Serva strain was 152,628 bp, with a G + C content of 48%. A total of 80 predicted open reading frames were identified. The Serva strain had 96.5% DNA sequence identity with the concatenated ILTV sequence. Notably, the concatenated ILTV sequence was found to lack four large regions of sequence, including 528 bp and 594 bp of sequence in the UL29 and UL36 genes, respectively, and two copies of a 1,563 bp sequence in the repeat regions. Considerable differences in the size of the predicted translation products of 4 other genes (UL54, UL30, UL37 and UL38) were also identified. More than 530 single-nucleotide polymorphisms (SNPs) were identified. Most SNPs were located within three genomic regions, corresponding to sequence from the SA-2 ILTV vaccine strain in the concatenated ILTV sequence.ConclusionsThis is the first complete genomic sequence of an individual ILTV strain. This sequence will facilitate future comparative genomic studies of ILTV by providing an appropriate reference sequence for the sequence analysis of other ILTV strains.

Equine rhinitis A virus: structural proteins and immune response.

Equine rhinitis A virus (ERAV) is a picornavirus that has been reclassified as a member of the Aphthovirus genus because of its resemblance to foot-and-mouth disease virus at the level of nucleotide sequence and overall genomic structure. The N-terminal amino acid sequence of three of the four capsid proteins of ERAV was determined and showed that the proteolytic cleavage sites within the precursor P1 polypeptide occur exactly as those predicted for an aphthovirus-like 3C protease, which generates the capsid proteins VP1 and VP3. However, the autocatalytic cleavage site between VP4 and VP2, which is independent of 3C protease cleavage, was different from that predicted previously. ERAV.393/76 antisera from horses and rabbits showed different reactivity to the viral structural proteins in both serum neutralization assays and Western blots. High neutralizing antibody titres appeared to correlate with strong reactivity to VP1 in Western blots.

Evidence that Equine Rhinitis A Virus VP1 Is a Target of Neutralizing Antibodies and Participates Directly in Receptor Binding

ABSTRACT Equine rhinitis A virus (ERAV) is a respiratory pathogen of horses and is classified as anAphthovirus, the only non-Foot-and-mouth disease virus (FMDV) member of this genus. In FMDV, virion protein 1 (VP1) is a major target of protective antibodies and is responsible for viral attachment to permissive cells via an RGD motif located in a distal surface loop. Although both viruses share considerable sequence identity, ERAV VP1 does not contain an RGD motif. To investigate antibody and receptor-binding properties of ERAV VP1, we have expressed full-length ERAV VP1 in Escherichia coli as a glutathioneS-transferase (GST) fusion protein (GST-VP1). GST-VP1 reacted specifically with antibodies present in serum from a rabbit immunized with purified ERAV virions and also in convalescent-phase sera from horses experimentally infected with ERAV. An antiserum raised in rabbits to GST-VP1 reacted strongly with viral VP1 and effectively neutralized ERAV infection in vitro. Using a flow cytometry-based binding assay, we found that GST-VP1, but not other GST fusion proteins, bound to cell surface receptors. This binding was reduced in a dose-dependent manner by the addition of purified ERAV virions, demonstrating the specificity of this interaction. A separate cell-binding assay also implicated GST-VP1 in receptor binding. Importantly, anti-GST-VP1 antibodies inhibited the binding of ERAV virions to Vero cells, suggesting that these antibodies exert their neutralizing effect by blocking viral attachment. Thus ERAV VP1, like its counterpart in FMDV, appears to be both a target of protective antibodies and involved directly in receptor binding. This study reveals the potential of recombinant VP1 molecules to serve as vaccines and diagnostic reagents for the control of ERAV infections.

Comparison of the safety and protective efficacy of vaccination with glycoprotein-G-deficient infectious laryngotracheitis virus delivered via eye-drop, drinking water or aerosol

Infectious laryngotracheitis virus (ILTV), an alphaherpesvirus, causes respiratory disease in chickens and is commonly controlled by vaccination with conventionally attenuated virus strains. These vaccines have limitations due to residual pathogenicity and reversion to virulence. To avoid these problems and to better control disease, attention has recently turned towards developing a novel vaccine strain that lacks virulence gene(s). Glycoprotein G (gG) is a virulence factor in ILTV. A gG-deficient strain of ILTV has been shown to be less pathogenic than currently available vaccine strains following intratracheal inoculation of specific pathogen free chickens. Intratracheal inoculation of gG-deficient ILTV has also been shown to induce protection against disease following challenge with virulent virus. Intratracheal inoculation, however, is not suitable for large-scale vaccination of commercial poultry flocks. In this study, inoculation of gG-deficient ILTV via eye-drop, drinking water and aerosol were investigated. Aerosol inoculation resulted in undesirably low levels of safety and protective efficacy. Inoculation via eye-drop and drinking water was safe, and the levels of protective efficacy were comparable with intratracheal inoculation. Thus, gG-deficient ILTV appears to have potential for use in large-scale poultry vaccination programmes when administered via eye-drop or in drinking water.

Sequence Conservation and Antigenic Variation of the Structural Proteins of Equine Rhinitis A Virus

ABSTRACT The nucleotide and deduced amino acid sequences of the P1 region of the genomes of 10 independent equine rhinitis A virus (ERAV) isolates were determined and found to be very closely related. A panel of seven monoclonal antibodies to the prototype virus ERAV.393/76 that bound to nonneutralization epitopes conserved among all 10 isolates was raised. In serum neutralization assays, rabbit polyclonal sera and sera from naturally and experimentally infected horses reacted in a consistent and discriminating manner with the 10 isolates, which indicated the existence of variation in the neutralization epitopes of these viruses.