A simple immunoperoxidase plaque assay to detect and quantitate Marek's disease virus plaques.

We report an immunoperoxidase-based staining technique that can be used to rapidly and accurately detect and quantitate Marek's disease virus (MDV) plaques. Monolayer cultures were fixed and incubated with a monoclonal antibody specific for MDV. After washing, a second antibody of horseradish peroxidase-conjugated goat anti-mouse IgG was applied, incubated for 1 hr, and washed with phosphate-buffered saline. After the cultures were incubated with diaminobenzidine, CoCl2, and H2O2, the plaques appeared as black spots and were easily seen and counted. Significantly more immunoperoxidase-stained serotype 1 MDV plaques could be counted at 4 days postinoculation than were seen in unstained cultures. With serotype 2 MDV-infected cells, the difference in plaque counts was less dramatic. Nevertheless, at 3 days postinoculation, significantly more stained serotype 2 plaques were seen than unstained plaques. Immunoperoxidase staining of turkey herpesvirus plaques did not increase the sensitivity of viewing plaques. Similar numbers of stained and unstained plaques were seen at 2 days postinoculation. We also demonstrated that we could count serotype-specific MDV plaques in a mixed infection that contained all three serotypes.

Dual-inlet chemical amplifier for atmospheric peroxy radical measurements.

The state-of-the-art of peroxy radical measurements using the technique of chemical amplification has matured in recent years. The NCAR chemical amplifier has been improved over previous versions by employing a dual-inlet system that allows more rapid switching between measurement and background modes. Further improvement is realized by the use of two NO(2) detectors. The dual-inlet chemical amplifier (DICHAMP) employs two identical glass inlet reactor-NO(2) detector combinations, one of which is operated in the background, while the other operates in the radical measurement mode. This instrument is less sensitive to fluctuations in ambient ozone than the single-channel version because of the continuous monitoring of the background and background plus radical signals. The single-inlet, dual-inlet with single-detector, and DICHAMP instruments are compared through theoretical calculations of the effect of noise at a given frequency and amplitude on retrieved radical levels. Laboratory experiments were conducted to support the theoretical results. Ambient radical concentrations were determined using these configurations to evaluate the performance under actual measurement conditions.

Partial inhibition by turkey herpesvirus of serotype 2 Marek's disease virus plaque formation and in vivo infectivity.

The increased use of serotype 2 Marek's disease virus (MDV) and serotype 3 turkey herpesvirus (HVT) as components of effective bivalent vaccines against Marek's disease (MD) prompted studies on the possible interactions of these two viruses in vitro and in vivo. The replication of the SB-1 strain of MDV was compared with replication of the FC126/2 strain of HVT in chickens and cell cultures infected with one or both viruses. Replication of MDV was reduced in the presence of HVT in both in vitro and in vivo systems. MDV plaque counts in dually infected chicken embryo fibroblast cultures inoculated with tissue-culture-propagated viruses were reduced by up to 91%; however, no inhibition was noted when inocula consisted of virus-infected buffy-coat cells. Plaque formation by MDV in chicken embryo fibroblast cultures was inhibited by virus-free conditioned medium from HVT-infected cultures. This conditioned medium also inhibited growth of vesicular stomatitis virus in a standard interferon assay. In chickens inoculated with both MDV and HVT, MDV viremia titers were lower and the dose required to infect 50% of susceptible chickens was increased 13-fold compared with chickens inoculated with MDV alone. In spite of these findings, there was no evidence that high concentrations of HVT interfered with either the ability of MDV to induce protective synergism in vivo or the protective efficacy of bivalent vaccines. No reciprocal inhibitory effects of MDV on the replication of HVT in vivo or in vitro were noted.

An immunoperoxidase plaque assay for reticuloendotheliosis virus and its application to a sensitive serum neutralization assay.

A rapid assay for the enumeration of reticuloendotheliosis virus (REV) is described. Chicken embryo fibroblast monolayer cultures were infected with REV and incubated 6 days under an agar overlay. After removal of the overlay, cells were fixed with acetone/ethanol. Foci of infection (hereafter referred to as plaques) were detected using either an anti-REV envelope monoclonal antibody or convalescent chicken serum as the primary antibody; the secondary antibody was either horseradish peroxidase-conjugated goat anti-mouse IgG (for use with monoclonals) or goat anti-chicken IgG (for use with chicken serum). Staining with a substrate solution containing diaminobenzidine, CoCl2, and H2O2 revealed individual dark plaques on a light gray background. This method worked equally well for the SNV, CSV, and REV-T strains of REV; further, it detected all six field isolates tested. Results indicate that this immunoperoxidase technique is a rapid and reliable method for detection and titration of REV as well as for the assay of neutralizing antibody in chicken serum.

Achieving acceptable air quality: some reflections on controlling vehicle emissions.

Motor vehicle emissions have been and are being controlled in an effort to abate urban air pollution. This article addresses the question: Will the vehicle exhaust emission control and fuel requirements in the 1990 Clean Air Act Amendments and the California Air Resources Board regulations on vehicles and fuels have a significant impact? The effective control of in-use vehicle emissions is the key to a solution to the motor vehicle part of the urban air pollution problem for the next decade or so. It is not necessary, except perhaps in Southern California, to implement extremely low new car emission standards before the end of the 20th century. Some of the proposed gasoline volatility and composition changes in reformulated gasoline will produce significant reductions in vehicle emissions (for example, reduced vapor pressure, sulfur, and light olefin and improved high end volatility), whereas others (such as substantial oxygenate addition and aromatics reduction) will not.

Fowlpox virus recombinants expressing the envelope glycoprotein of an avian reticuloendotheliosis retrovirus induce neutralizing antibodies and reduce viremia in chickens.

Eight stable fowlpox virus (FPV) recombinants which express the envelope glycoprotein of the spleen necrosis virus (SNV) strain of reticuloendotheliosis virus (REV), an avian retrovirus, were constructed. These recombinants differ in the genomic location of the inserted genes, in the orientation of the insert relative to flanking viral sequences, and in the promoter used to drive expression of the env gene. Of these variables, promoter strength seems to be the most crucial. The P7.5 promoter of vaccinia virus, which is commonly used in the construction of both vaccinia virus and FPV recombinants, resulted in lower levels of expression of the envelope antigen in infected chicken cells compared with a strong synthetic promoter, as determined by immunofluorescence and enzyme-linked immunosorbent assay. Two peptides encoded by the env gene, the 21-kDa transmembrane peptide and a 62-kDa precursor, were detected by immunoprecipitation of labeled proteins from cells infected with recombinant FPVs, using monoclonal antibodies against REV. These peptides comigrated with those precipitated from REV-infected cells. One of the recombinants (f29R-SNenv) was used for vaccination of 1-day-old chickens. Vaccinated chicks developed neutralizing antibodies to SNV more rapidly than did unvaccinated controls following SNV challenge and were protected against both viremia and the SNV-induced runting syndrome.

Insertional inactivation of a fowlpox virus homologue of the vaccinia virus F12L gene inhibits the release of enveloped virions.

Insertion of the Escherichia coli lacZ gene into a ClaI restriction enzyme site of a 5.7 kb HindIII fragment of the fowlpox virus (FPV) genome resulted in the generation of stable recombinants. These recombinants produced plaques that were significantly smaller than those produced by parental FPV or by FPV recombinants containing the lacZ gene at other non-essential sites. Insertion of foreign DNA into the ClaI site disrupts a previously unidentified open reading frame (ORF) which potentially encodes a 74K polypeptide. The predicted amino acid sequence of this FPV ORF has 24% identity with the F12L ORF of vaccinia virus, the function of which is not currently known. Production of intracellular FPV was similar in cells infected with recombinant or parental viruses, but the number of infectious extracellular virions released into the medium by the recombinant was about 20% of that released by the parental virus. Likewise, the release of FPV particles, which were labelled in vivo with [3H]thymidine, was significantly lower in recombinant FPV-infected cells. These results suggest that the FPV homologue of the vaccinia virus F12L ORF is involved in the envelopment or release of infectious extracellular virions.

Identification and functional analysis of the fowlpox virus homolog of the vaccinia virus p37K major envelope antigen gene.

A fowlpox virus (FPV) gene with homology to the vaccinia virus p37K major envelope antigen gene was identified and sequenced. The predicted product has a molecular weight of 43,018 Da (p43K). The FPV p43K gene has 37.5% identity with its vaccinia counterpart and higher homology with a molluscum contagiosum virus gene (42.6% identity). Based on upstream sequences, p43K appears to be regulated as a late gene. Recombinant FPV were generated in which a large portion of p43K was replaced by the Escherichia coli lacZ gene. These recombinants failed to produce visible plaques under standard conditions. After prolonged incubation the microplaques developed into small macroscopic plaques. Plaques were purified on the basis of lacZ expression. Single-cycle growth curves comparing the p43K-deleted recombinant (designated fJd43Z) with parental FPV showed that the two viruses produce identical amounts of intracellular virions, but that fJd43Z released 20-fold fewer infectious particles into the medium. CsCl gradient centrifugation of [3H]thymidine-labeled virus was employed to examine differences in the production of physical particles. The two viruses produced equivalent levels of intracellular virions, but fJd43Z failed to produce detectable levels of released particles. FPV p43K is therefore involved in the release of virions from infected cells.

Characterization of the VPg-dsRNA linkage of infectious pancreatic necrosis virus.

By the use of strong denaturing agents, a genome-linked protein (VPg)-RNA complex was purified from infectious pancreatic necrosis virus. Ribonuclease treatment of 125I-labelled VPg-RNA released a 90K polypeptide identical to the minor structural polypeptide VP1 (the putative RNA polymerase), as determined by peptide mapping. The polypeptide is linked to the RNA by a serine-5' GMP phosphodiester bond. The results identify birnaviruses as the only dsRNA viruses with a VPg, the size of which is the largest of the VPgs of RNA viruses.

Effects of the copper chelators diethyldithiocarbamate and bathocuproine sulfonate on interferon and its antiviral state.

Treatment of mouse L cells with two structurally unrelated chelators of copper ions, diethyldithiocarbamate (DDC) or bathocuproine sulfonate (BCS), completely inhibited the ability of interferon (IFN) to inhibit mengovirus growth. However, mengovirus virions were inactivated by incubation with DDC, and DDC induced a generalized inhibition of cellular RNA and protein synthesis, possibly combined with the inactivation of one or more specific enzymatic activities involved in the establishment or maintenance of the antiviral state. In contrast, BCS had no effect on either cell or virus growth. BCS combined with trace copper ions in the growth medium and the resulting complex prevented IFN from interacting properly with cellular receptors, apparently by binding noncovalently to the IFN molecules. In some cases, IFN was irreversibly inactivated, probably due to oxidation of essential cystein, tyrosine, or tryptophan residues by the (BCS)2Cu2+ complex. BCS was at least as effective as anti-IFN antibody at neutralizing cell-bound IFN, and has a number of advantages over it.

Use of the AFCRL line parameters compilation to test the performance of an infrared spectrometer system.

The resolution and the frequency accuracy of a spectrum and the abundance of an absorbing gas can be obtained by ratioing observed and calculated spectra. Line positions (to +/-0.002 cm(-1)), spectral resolution (0.075 +/- 0.003 cm(-1)), and a CO abundance [(2.35 +/- 0.23) x 10(-2)atm-cm (STP)] have been found by comparing an observed CO spectrum with a spectrum calculated from the AFCRL line parameter listing.

A study of the N2O5-SO2-O3 reaction system.

Infrared spectroscopy was used to follow the rates of the chemical changes in gaseous N(2)O(5)-SO(2) and N(2)O(5)-SO(2)-O(3) mixtures. Several results of interest to atmospheric scientists were obtained. (I) SO(3) was not a detectable product of these reaction systems, and no significant SO(2) removal occurred. From the kinetic treatment of these results, estimates were derived for the upper limits of the rate constants of the reactions 1 and 2: NO(3) + SO(2) leads to NO(2) + SO(3) (1); N2O5 +SO2 leads to N(2)O(4) + SO(3) (2); k(1) less than or equal to 4.2 1. mole-minus 1sec-minus 1; k(2) less than or equal to 2.5 x 10-minus 2 1. mole-minus1sec-minus 1 at 30 degrees C. These data suggest that reactions 1 and 2 are not important removal paths for SO(2) in the sunlight irradiated, NO(x)hydrocarbon polluted atmospheres. (II) The near ultraviolet absorption spectrum of pure N(2)O(5) has been determined. From these results and estimates of the actinic irradiance, it was shown that the rate of photochemical decomposition of N(2)O(5) by the absorption of solar light in the urban atmosphere is an unimportant factor among the reactions which establish the N(2)O(5) and NO(3) concentrations. (III) It has been observed that gaseous SO(3) and NO(2) react rapidly to form a relatively nonvolatile white solid. Preliminary data suggest a 1:1 mole ratio for this adduct. The significance, if any, of this and related compounds in urban aerosol formation must be evaluated.