Further evaluation and validation of a commercially available competitive ELISA (cELISA) for the detection of antibodies specific to equine arteritis virus (EAV).

The purpose of this study was to further evaluate and validate two commercially available equine arteritis virus (EAV) competitive ELISAs (original and enhanced cELISAs) using archived equine sera from experimentally inoculated animals and field sera submitted for laboratory diagnosis. First, the original and subsequently enhanced cELISAs were compared with the virus neutralisation test (VNT) using a panel of archived serum samples from experimentally inoculated animals. Then, the enhanced cELISA was compared with the VNT using a large panel of archived serum samples. The total number of equine sera tested was 3255, which included sera against 25 different EAV strains. The study confirmed that the enhanced cELISA was more sensitive than the original cELISA. Based on testing sera from experimentally inoculated animals and field sera, the enhanced cELISA had an estimated sensitivity (98.9 percent and 99.6 percent, respectively) and specificity (98.3 percent and 98.7 percent, respectively). The currently marketed enhanced VMRD EAV antibody cELISA test kit (VMRD Inc., Pullman, Washington, USA) has high sensitivity and specificity relative to the VNT. Based on the findings of this study, the authors would propose that the enhanced cELISA should be considered as an alternative approved method to the VNT for the detection of antibodies to EAV.

Single-layer centrifugation reduces equine arteritis virus titre in the semen of shedding stallions.

Several countries have adopted strategies for preventing and/or controlling equine viral arteritis based on vaccination and restricting the breeding activities of carrier stallions. However, in some cases, carrier stallions are only identified after they have transmitted virus to a mare. Therefore, a mechanism for separating virus from spermatozoa in the semen of carrier stallions would facilitate control measures for preventing disease transmission. In this study, the use of several modifications of single-layer centrifugation (SLC, SLC with an inner tube and double SLC) through Androcoll-E, a species-specific colloid were evaluated for their ability to separate spermatozoa from virus in ejaculates from carrier stallions. The three types of SLC significantly reduced the virus titre in fresh semen at 0 h and in stored semen at 24 h (p < 0.001) but did not completely eliminate the virus. Sperm motility parameters such as total motility and progressive motility were significantly increased after colloid centrifugation, whereas curvilinear velocity and amplitude of lateral head deviation were decreased, and the remainder (straight line velocity, average path velocity, straightness, linearity, wobble and beat cross-frequency) were not significantly affected by the processing. Although virus titres were reduced in the SLC samples, significant levels of infectivity still remained, especially in stallions shedding large amounts of virus. It remains to be determined whether SLC-processed sperm samples from stallions shedding low virus titres retain sufficient equine arteritis virus to cause infection in mares through artificial insemination.

Analysis of the pore structure of the influenza A virus M(2) ion channel by the substituted-cysteine accessibility method.

The M(2) ion channel of influenza A virus is a small integral membrane protein whose active form is a homotetramer with each polypeptide chain containing 96-amino-acid residues. To identify residues of the transmembrane (TM) domain that line the presumed central ion-conducting pore, a set of mutants was generated in which each residue of the TM domain (residues 25 to 44) was replaced by cysteine. The accessibility of the cysteine mutants to modification by the sulfhydryl-specific reagents methane thiosulfonate ethylammonium (MTSEA) and MTS tetraethylammonium (MTSET) was tested. Extracellular application of MTSEA evoked decreases in the conductances measured from two mutants, M(2)-A30C and M(2)-G34C. The changes observed were not reversible on washout, indicative of a covalent modification. Inhibition by MTSEA, or by the larger reagent MTSET, was not detected for residues closer to the extracellular end of the channel than Ala-30, indicating the pore may be wider near the extracellular opening. To investigate the accessibility of the cysteine mutants to reagents applied intracellularly, oocytes were microinjected directly with reagents during recordings. The conductance of the M(2)-W41C mutant was decreased by intracellular injection of a concentrated MTSET solution. However, intracellular application of MTSET caused no change in the conductance of the M(2)-G34C mutant, a result in contrast to that obtained when the reagent was applied extracellularly. These data suggest that a constriction in the pore exists between residues 34 and 41 which prevents passage of the MTS reagent. These findings are consistent with the proposed role for His-37 as the selectivity filter. Taken together, these data confirm our earlier model that Ala-30, Gly-34, His-37, and Trp-41 line the channel pore (L. H. Pinto, G. R. Dieckmann, C. S. Gandhi, C. G. Papworth, J. Braman, M. A. Shaughnessy, J. D. Lear, R. A. Lamb, and W. F. DeGrado, Proc. Natl. Acad. Sci. USA 94:11301-11306, 1997).

Cu(II) inhibition of the proton translocation machinery of the influenza A virus M2 protein.

The homotetrameric M2 integral membrane protein of influenza virus forms a proton-selective ion channel. An essential histidine residue (His-37) in the M2 transmembrane domain is believed to play an important role in the conduction mechanism of this channel. Also, this residue is believed to form hydrogen-bonded interactions with the ammonium group of the anti-viral compound, amantadine. A molecular model of this channel suggests that the imidazole side chains of His-37 from symmetry-related monomers of the homotetrameric pore converge to form a coordination site for transition metals. Thus, membrane currents of oocytes of Xenopus laevis expressing the M2 protein were recorded when the solution bathing the oocytes contained various transition metals. Membrane currents were strongly and reversibly inhibited by Cu2+ with biphasic reaction kinetics. The biphasic inhibition curves may be explained by a two-site model involving a fast-binding peripheral site with low specificity for divalent metal ions, as well as a high affinity site (Kdiss approximately 2 microM) that lies deep within the pore and shows rather slow-binding kinetics (kon = 18.6 +/- 0.9 M-1 s-1). The pH dependence of the interaction with the high affinity Cu2+-binding site parallels the pH dependence of inhibition by amantadine, which has previously been ascribed to protonation of His-37. The voltage dependence of the inhibition at the high affinity site indicates that the binding site lies within the transmembrane region of the pore. Furthermore, the inhibition by Cu2+ could be prevented by prior application of the reversible blocker of M2 channel activity, BL-1743, providing further support for the location of the site within the pore region of M2. Finally, substitutions of His-37 by alanine or glycine eliminated the high affinity site and resulted in membrane currents that were only partially inhibited at millimolar concentrations of Cu2+. Binding of Cu2+ to the high affinity site resulted in an approximately equal inhibition of both inward and outward currents. The wild-type protein showed very high specificity for Cu2+ and was only partially inhibited by 1 mM Ni2+, Pt2+, and Zn2+. These data are discussed in terms of the functional role of His-37 in the mechanism of proton translocation through the channel.

Performance of the Kallestad Pathfinder enzyme immunoassay in the diagnosis of respiratory syncytial virus infections.

The Kallestad Pathfinder enzyme immunoassay (EIA) for the rapid detection of respiratory syncytial virus (RSV) antigen was compared with virus culture and direct fluorescent antibody (DFA) to determine the reliability of the EIA. During two consecutive winter respiratory seasons, 270 nasopharyngeal wash specimens were tested. RSV was detected in culture by the presence of cytopathic effect and/or an indirect immunofluorescence assay. The sensitivity of the Pathfinder EIA in comparison with isolation in tube culture was 72% (73 of 101) and the specificity was 99% (167 of 169). During the second year of the evaluation period, DFA was performed on all specimens. The sensitivity of the DFA compared with isolation in tube culture was 94%. This study indicates that the Pathfinder EIA is a very specific test for diagnosis of RSV infections, but lacks sensitivity in comparison with tube culture or direct immunofluorescence.

Isolation of seven respiratory viruses in shell vials: a practical and highly sensitive method.

The isolation of respiratory viruses in shell vials was compared with isolation in tube cultures in order to determine the sensitivity of the former, rapid method. Twenty of 21 influenza virus and 15 of 15 parainfluenza virus isolates were recovered in shell vials. One hundred twenty-seven of 138 respiratory syncytial virus isolates were detected in shell vials, but only 10 of 21 adenovirus isolates were positive by the rapid method. Shell vials are very effective for the diagnosis of respiratory viral infections, except for those caused by adenovirus.

Evaluation of Abbott TestPack RSV for the diagnosis of respiratory syncytial virus infections.

Abbott TestPack RSV, a 20-minute enzyme immunoassay, is available for the rapid diagnosis of respiratory syncytial virus (RSV) infections. We have compared TestPack with a "gold standard" method of virus isolation in traditional tube cultures and shell vials to determine the sensitivity and specificity of this rapid method. Respiratory specimens were collected prospectively from 402 children and assayed by the rapid antigen detection method and isolation in culture. Virus was isolated by inoculation of specimen in a total of eight tubes and 2-3 shell vials. Isolation of RSV was confirmed by characteristic cytopathic effect and immunofluorescence using monoclonal antibodies to RSV. Of the 402 specimens tested, there were only 18 discrepant results (seven TestPack-positive, culture-negative, and 11 TestPack-negative, culture-positive specimens). The sensitivity of TestPack RSV versus culture was 93.6% (162 of 173) and the specificity was 97.0% (222 of 229). Using a very rigorous culture system, we have obtained high values for the sensitivity and specificity of TestPack RSV. This assay is an excellent method for the rapid diagnosis of RSV infections in young children.