What's hot in animal biosafety?

In recent years, the emergence or re-emergence of critical issues in infectious disease and public health has presented new challenges and opportunities for laboratory animal care professionals. The re-emergence of bioterrorism as a threat activity of individuals or small groups has caused a heightened awareness of biosecurity and improved biosafety. The need for animal work involving high-risk or high-consequence pathogens and for arthropod-borne diseases has stimulated renewed interest in animal biosafety matters, particularly for work in containment. Application of these principles to animals retained in outdoor environments has been a consequence of disease eradication programs. The anticipated global eradication of wild poliovirus has prompted the promulgation of new biosafety guidelines for future laboratory and animal work. Increased concern regarding the use of biologically derived toxins and hazardous chemicals has stimulated a new categorization of facility containment based on risk assessment. Recognition that prion disease agents and other high-consequence pathogens require safe handling and thorough destruction during terminal decontamination treatment has led to the development of new biosafety guidelines and technologies. The implementation of these guidelines and technologies will promote state-of-the-art research while minimizing risk to laboratory animals, researchers, and the environment.

Hantaviral biosafety issues in the autopsy room and laboratory: concerns and recommendations.

The need to perform autopsies on and examine laboratory specimens from patients with hantavirus pulmonary syndrome (HPS) has raised questions about biosafety. Human illness associated with hantaviruses is usually the result of exposure to infectious aerosols from saliva or excreta of wild rodents. It is unclear whether or nor certain autopsy and laboratory procedures can also generate similar potentially infectious aerosols. As the biosafety information developed for the HPS agent is limited and the consequences of infection are serious we recommend a cautious approach. Autopsy prosectors should use N-95 particulate respirators as a minimum standard. If aerosols will be generated they should use N-100 particulate respirators or powered air purifying respirators with high-efficiency particulate air (HEPA) filters. Centrifugation and cytocentrifugation of blood or body fluid samples should be performed in bio-contained systems and these specimen containers should be opened in a class II biological safety cabinet.

Biosafety in the clinical mycobacteriology laboratory.

This article presents an expanded agent summary statement for laboratorians working with Mycobacterium tuberculosis. It focuses on reducing the serious risk of infection in clinical laboratories that process specimens from tuberculosis patients or that work with purified cultures of tubercle bacilli. Administrative and engineering controls, practices and procedures, and personal protective equipment are discussed. Guidelines for packaging specimens for transfer to another laboratory also are presented.

Microassay for interferon, using [3H]uridine, microculture plates, and a multiple automated sample harvester.

A microassay for interferon is described which uses target cells grown in microculture wells, [3H]uridine to measure vesicular stomatitis virus replication in target cells, and a multiple automated sample harvester to collect the radioactively labeled viral ribonucleic acid onto glass fiber filter disks. The disks were placed in minivials, and radioactivity was counted in a liquid scintillation spectrophotometer. Interferon activity was calculated as the reciprocal of the highest titer which inhibited the incorporation of [3H]uridine into viral ribonucleic acid by 50%. Interferon titers determined by the microassay were similar to the plaque reduction assay when 100 plaque-forming units of challenge vesicular stomatitis virus was used. However, it was found that the interferon titers decreased approximately 2-fold for each 10-fold increase in the concentration of challenge vesicular stomatitis virus when tested in the range of 10(2) to 10(5) plaque-forming units. Interferon titers determined by the microassay show a high degree of repeatability, and the assay can be used to measure small and large numbers of interferon samples.

Characterization of a foot-and-mouth disease mutant temperature-sensitive for viral RNA synthesis.

A temperature-sensitive (ts) mutant of foot-and-mouth disease virus (FMDV) did not produce RNA polymerase activity nor synthesize viral RNA when incubated in cells solely at the nonpermissive temperature (38.5 degrees C). Infected cells initially incubated at 38.5 degrees C and then shifted down to 33 degrees C synthesized increased amounts of viral RNA at earlier times compared to infected cells kept at 33 degrees C throughout, indicating that RNA polymerase precursors were synthesized at 38.5 degrees C. In cells shifted up to 38.5 degrees C from 33 degrees C, the total amount of viral RNA synthesized after infection increased sharply for about 15 minutes and then rapidly increased over the next 2 hours. RNA polymerase activity presented a similar pattern in its initial twofold increase and subsequent rapid decrease. Pulse labeling experiments showed that mutant viral RNA synthesis continued at a diminishing rate for 2 hours in cells shifted up to 38.5 degrees C. The data from temperature after shift-up was degraded. The FMDV ts mutant is apparently additionally defective in being unable to protect viral RNA synthesized after shift-up to 38.5 degrees C.

Viral interference phenomena induced by foot-and-mouth disease temperature-sensitive mutants in bovine kidney cells.

Cultures of bovine kidney (BK) cells infected with temperature-sensitive (ts) mutants of foot-and-mouth disease virus (FMDV) were incubated at 38.5 degrees C, a temperature nonpermissive for mutant virus growth and RNA synthesis. The cells were subsequently resistant to viral growth and RNA synthesis when superinfected with wild-type FMDV and with heterologous fowl plague virus. The extent of interference was proportional to the multiplicity of infection of the ts mutant. It increased with time elapsed between infection with mutant and challenge infection, becoming greater than 99 percent after 24 hours. Interference was not proportional to decreased levels of cellular protein synthesis. The interference could be produced in the presence of actinomycin D, and thus was apparently mostly caused by the ts mutant itself rather than by interferon. The interference could not be produced in other less susceptible cell lines. Supernatant fluids from the BK cells infected with ts mutant virus interfered with wild-type FMD viral growth and RNA synthesis in fresh BK cells, and also showed low levels of activity in a vesicular stomatitis virus-plaque reduction assay. The properties of the supernatant fluid-interfering agent resembled to some extent those of an interferon. The ts mutant-mediated interference factor was apparently not able to diffuse into the supernatant fluid.

Preadsorption of boar semen with kaolin: increased efficiency of foot-and-mouth disease virus detection.

The boar semen-associated cytotoxic factor(s), but not the antiviral activity, were removed by adsorption with kaolin. Although foot-and-mouth disease virus was efficiently removed from medium by kaolin or kieselguhr, the virus was not removed from semen-virus mixtures. Because the cytotoxicity induced by boar semen apparently altered the ability of tissue culture cells to support virus replication, preadsorption with kaolin increased the probability of detecting this virus in semen samples.

Evidence for a mouse pathogenicity locus in certain temperature-sensitive mutants of foot-and-mouth disease virus.

Serial tissue culture passaging of three foot-and-mouth disease temperature-sensitive mutants demonstrated the stability of their temperature sensitivity and mouse avirulence characteristics. Recovery of mouse-virulent temperature-sensitive viruses after passage of the mutants in mice suggested that these were not covariant expressions of the same locus, but were under the control of different genes.

Interferon inducers and foot-and-mouth disease vaccines: influence of two synthetic polynucleotides on antibody response and immunity in guinea pigs and swine.

Polyriboadenylic-polybouridylic acid enhanced the immunological response of guinea pigs to aqueous foot-and-mouth disease virus vaccine. Polyriboninosinic-polyribocytidylic acid enhanced the early antibody production of swine to oil emulsified foot-and-mouth disease virus vaccine. Polyriboninosinic-polyribocytidylic acid alone did not stimulate resistance to foot-and-mouth disease in swine.

Further studies of the physical and metabolic properties of foot-and-mouth disease virus temperature-sensitive mutants.

Three temperature-sensitive (ts) mutants of foot-and-mouth disease virus were classified as ribonucleic acid negative and as belonging to the same complementation group when measured by virus yields and [3H] uridine incorporation in paired, mixed infections at the nonpermissive temperature (38.5C). Mutant ts-22, the only mutant able to produce plaques at 38.5 C, was more sensitive to acid than were the parental wild-type or other mutant viruses. Diethylaminoethyl-dextran did not enhance the plaque-forming ability of the mutant viruses at 38.5C. All of the viruses inhibited host cell protein syntehsis at both permissive (33C) and nonpermissive (38.5C) temperatures.

Production, isolation, and partial characterization of three foot-and-mouth disease virus temperature-sensitive mutants.

Three high temperature-sensitive (ts) mutants of foot-and-mouth disease virus were characterized by their relative abilities to grow at 33 or 38.5 C, to kill infant mice, to infect guinea pigs, and to produce foot-and-mouth disease in steers. Mutants ts-24 and ts-42 did not grow at 38.5 C; both may have produced considerable quantities of noninfectious virus particles at 33 C. A third mutant, ts-22, appeared "leaky" because it multiplied to a limited extent during prolonged incubation at the nonpermissive temperature. Mutant ts-42 was pathogenic for infant mice, whereas ts-22 and ts-24 were essentially apathogenic. Mice were protected against the lethal effects of the wild-type (wt) virus if injected 1 week earlier with ts-22, apparently as a result of specific antibody development. One-half of the guinea pigs injected with the mutant viruses showed lesion development, but only at the site of injection. Antibody development was also much less than in those animals injected with the wt virus. The onset of FMD in steers was delayed and the severity of the disease was diminished after intradermalingual injection of the mutant viruses.

Enhancement of primary antigen-specific resistance in infant mice with divinyl ether-maleic anhydride.

The early response of infant mice to foot-and-mouth disease viral antigens, measured by greater resistance and increased serum-neutralizing antibody, was enhanced by divinyl ether-maleic anhydride (DVE/MA). Preparations of known antigen concentration were used to study various parameters influencing this enhancement. Resistance and antibody levels were increased when DVE/MA was administered with aqueous or oil-emulsified antigens, whether given separately or mixed with the antigens, but was dependent on the dose and route of the DVE/MA and antigen. Enhanced resistance developed rapidly, was antigen-specific, and may have been related to the level of neutralizing antibody.

Observations of cattle, goats and pigs after administration of synthetic interferon inducers and subsequent exposure to foot and mouth disease virus.

Polyriboinosinic-polyribocytidylic acid (poly [rI.rC]) was administered intravenously to 11 cattle and 13 goats in doses of 0.25 to 4.0, and 1.0 to 5.0 mg/kg, respectively. Subsequent exposure of these and untreated control animals to foot and mouth disease virus (FMDV) failed to demonstrate any differences in either the course or severity of the disease. Serum interferon was detected in cattle one hour after the intravenous administration of poly (rI.rC). Six pigs given 4, 20, or 100 mg/kg of itaconic-acrylic acid copolymer (IAA, HMW) intraperitoneally reacted clinically the same as six untreated control pigs after contact exposure to FMDV. Three pigs given 50, 100, or 200 mg/kg of divinyl ether-maleic anhydride copolymer (DVE/MA, pyran) intraperitoneally similarly failed to show any difference in clinical reaction from three untreated control pigs after intranasal instillation of FMDV. Three pigs given 100, 200 or 400 mg/kg of DVE/MA intraperitoneally developed rapid diffuse peritonitis causing the death of one in 48 hours.

Enhancement, by two carboxylic acid interferon inducers of resistance stimulated in mice by foot-and-mouth disease vaccine.

Simultaneous injection of divinyl ether-maleic amhydride (DVE/MA) or itaconic-acrylic acid and foot-and-mouth disease virus vaccine enhanced the survival of infant mice to subsequent injections of virus. This enhanced resistance was obtained even with doses of interferon inducers which, when administered alone, failed to protect the mice. There was some increase in serum-neutralizing antibody in mice 7 days after injection of DVE/MA and vaccine, as compared with mice given vaccine alone, but there was no clear connection between antibody level and amount of DVE/MA administered.

Mouse Resistance Against Foot-and-Mouth Disease Virus Induced by Injections of Pyran.

Mouse resistance to foot-and-mouth disease virus (FMDV) was induced by intraperitoneal injections of pyran copolymer. A biphasic pattern of protection occurred with greatest resistance 4 and 48 hr after injection of this polyanion. Viremia was not detectable in pretreated mice challenge-exposed with FMDV. Incubation of virus with pyran did not alter viral infectivity in mice or tissue culture. Serum interferon was demonstrated 1 and 2 days after pyran administration.