Potentially Infectious Agents Associated with Shearling Bedpads: Effect of Laundering with Detergent-Disinfectant Combinations on Staphylococcus aureus and Pseudomonas aeruginosa

Glutaraldehyde-tanned woolskins which are used as bedpads to prevent decubitus ulcers were contaminated with Staphylococcus aureus (ATCC 6538) and Pseudomonas aeruginosa (ATCC 15442). Two methods of exposure, direct contact and aerosol, were used in separate experiments. Attempts were made to decrease the bacterial population placed on the woolskins by laundering them in a quaternary ammonium disinfectant, a phenolic disinfectant, or alkalinized glutaraldehyde, in combination with an anionic or nonionic detergent. The effect of a commercial detergent-sanitizer was also studied. Bacterial populations were significantly reduced in all experiments, but only laundering in glutaraldehyde in combination with either detergent resulted in maximum removal of bacteria. Viable bacteria were usually not detected in the rinse water (<1 viable organism/5 ml of rinse water).     

Quantitative Studies on Fabrics as Disseminators of Viruses: V. Effect of Laundering on Poliovirus-Contaminated Fabrics

The effects of laundering with both anionic and nonionic detergents in cold, warm, and hot water on poliovirus-contaminated cotton sheeting, cotton terry cloth, washable wool shirting, wool blanketing, dull nylon jersey, and dacron/cotton shirting were determined. The fabrics were exposed to virus by aerosolization and direct contact (pipette) in separate studies. Although the results varied with each factor used in the study, virus titers on all the fabrics were generally reduced considerably by the laundering process. When the fabrics were dried for 20 hr after laundering, an additional decline in virus titers was seen, often to below detectable levels. The type of detergent used made little difference in effect on virus titer reduction, but the hot wash water markedly reduced the detectable virus. Fabric type was not a major factor in the majority of the experiments, although virus tended to be eliminated more readily from the nylon jersey, and in warm water the virus persisted longer on wool blanketing material laundered in anionic detergent. Sterile fabrics of each type laundered with similar fabrics which contained virus often became contaminated by the virus during the laundering process. Virus titers ranging from undetectable to 10(3.9) cell culture 50% infectious doses/ml were obtained from samples of the rinse water after warm- and cold-water laundering.     

Quantitative Studies on Fabrics as Disseminators of Viruses: IV. Virus Transmission by Dry Contact of Fabrics

Cotton and woolen fabrics and fabrics of synthetic fibers were exposed by direct contact (pipette) and by aerosolization to poliovirus and to vaccinia virus in separate experiments, allowed to dry for 16 hr at 25 C in 35% relative humidity, and randomly tumbled with sterile swatches of the same fabrics for 30 min. By use of a HEp-2 cell assay system, up to 10^3.5 CCID₅₀ of poliovirus per ml and 10^4.4 CCID₅₀ of vaccinia virus per ml were recovered from the originally sterile fabrics as early as 1 to 10 min after contact. Maximum transfer of both viruses was achieved with wool blanket material, although high titers of vaccinia virus were recovered from all fabrics tested. Poliovirus placed on the fabrics in an aerosol tended to be transferred to the sterile fabrics at a greater rate than when it was placed on the fabrics by direct contact. The method of exposure had essentially no effect on the rate of transfer of vaccinia virus.     

Procedure for the Evaluation of the Virucidal Effectiveness of an Ethylene Oxide Gas Sterilizer

A quantitative, reproducible method was developed for the evaluation of the virucidal activity of test gases. Using this method, we determined the virucidal effectiveness of a Steri-Vac ethylene oxide gas sterilizer. Wool gabardine material was exposed to high concentrations of herpes simplex, vaccinia, parainfluenza, or polio viruses and was processed through the sterilizer. Two time-temperature cycles of the machine, 29 C for 180 min and 60 C for 48 min, were used in separate experiments. The viruses were exposed to the gas when freshly pipetted onto the fabric or when pipetted on the material and allowed to dry 16 to 24 hr. In two experiments carried out under each condition, the virus titers were reduced by the sterilization process to less than detectable limits. These titer reductions were for the herpes virus >/= 2.7 to 5.0 log, for vaccinia virus >/= 4.0 to 6.1 log, for parainfluenza virus >/= 1.8 to 4.9 log, and for poliovirus >/= 4.9 to 7.7 log. The observed reductions in virus titers were the same whether the virus-contaminated fabrics were sealed in polyethylene packages or held in open petri dishes during exposure to ethylene oxide.     

Quantitative Studies on Fabrics as Disseminators of Viruses: II. Persistence of Poliomyelitis Virus on Cotton and Wool Fabrics

The length of time that poliovirus could be recovered from wool gabardine and blanket, and from cotton sheeting, terry cloth, and knit jersey fabrics was determined under conditions of controlled temperature and humidity (25 C in 35 and 78% relative humidities). Three types of exposure of the fabrics to viruses were used: direct contact, aerosol, and virus-containing household dust having a high content of textile fibers. When held in 35% relative humidity, virus persisted for 20 weeks on wool fabrics, but only 1 to 4 weeks on cotton fabrics. At this relative humidity, virus titers on wool fabrics decreased rapidly to low but detectable levels which persisted for long periods of time, whereas in 78% relative humidity the decrease in virus titer was less rapid, but the period of viral persistence was shorter. Generally, virus titers on cotton fabrics held in both relative humidities decreased exponentially to an undetectable level. The method of exposure to virus had a definite effect on the duration of viral persistence on a given fabric. Virus contained in household dust was least stable.     

Quantitative Studies on Fabrics as Disseminators of Viruses: III. Persistence of Vaccinia Virus on Fabrics Impregnated with a Virucidal Agent

Eight compounds were tested in vitro for virucidal and antiviral activity against poliovirus and vaccinia virus. These compounds included five quaternary ammonium salts, two bromosalicylanilides, and neomycin sulfate, an antibiotic. None of the compounds was active against poliovirus, but virucidal activity was demonstrated against vaccinia virus with three of the quarternary ammonium compounds: n-alkyl (C14, C12, C16) dimethyl benzyl ammonium chloride, di-isobutyl cresoxy ethoxy ethyl dimethyl benzyl ammonium chloride monohydrate, and n-alkyl (60% C14, 30% C16, 5% C12, 5% C18) dimethyl benzyl ammonium chlorides plus n-alkyl (50% C12, 30% C14, 17% C16, 3% C18) dimethyl ethylbenzyl ammonium chlorides. Wool blanketing, wool gabardine, and cotton sheeting materials were impregnated with the first of the above virucidal compounds, and the persistence of vaccinia virus on these fabrics was compared with the persistence of the agent on nonimpregnated fabrics of the same type held at 25 C in 35 and 78% relative humidity. No virus could be recovered from the chemically treated fabrics at any time after virus exposure, whereas the virus persisted as long as 4 weeks on nonimpregnated materials. Viable vaccinia virus was also found to persist less than 1 day on a cotton fabric finished with a wash-and-wear modified triazone resin. Poliovirus persisted less than 5 days on this wash-and-wear fabric.     

Residual Disinfection of Wool Blankets Treated with Formaldehyde

SUMMARY: Treatment with formaldehyde during laundering conferred a persistent residual disinfectant action on wool blankets but not on cotton ones. The former was shown by a slower rate of bacterial contamination of blankets during use or by the more rapid disappearance of nonsporing bacteria from contaminated blankets during storage. The formaldehyde treated blankets had no perceptible odour during use, and did not cause irritation of the skin or mucous membranes. It is suggested that the formaldehyde treatment of wool blankets might be worthy of trial as a means of reducing bacterial contamination in rooms occupied by patients with a high risk of infection.     

Virucidal activity of the new disinfectant monopercitric acid

AIMS: The virucidal efficacy of monopercitric acid (MPCA) was evaluated against the enveloped vaccinia virus as well as the nonenveloped adenovirus type 2 and poliovirus type 1. The results were compared with that obtained with peracetic acid (PAA). METHODS AND RESULTS: In the virucidal suspension test without and with protein burden, all viruses were inactivated by 0.5% MPCA within 0.5 min or by 0.1% MPCA within 5 min as measured by a >10(4)-fold reduction in virus titres. For MPCA, there was a better virucidal efficacy than for PAA which inactivated all viruses included in the test within 15-30 min at a concentration of 0.2%. SIGNIFICANCE AND IMPACT OF THE STUDY: The high virucidal activity, short exposure times, and nontoxic by-products seem to make MPCA suitable as disinfectant for medical use and should warrant further investigation.     

Limited expression of poliovirus by vaccinia virus recombinants due to inhibition of the vector by proteinase 2A.

A recombinant vaccinia virus was constructed that expressed poliovirus coat precursor protein P1 fused to about two-thirds of the 2A proteinase. The truncated 2A segment could be cleaved away from the P1 region by coinfecting with poliovirus type 1, 2, or 3 or with human rhinovirus 14 but not with encephalomyocarditis virus. Further cleavage of the vector-derived P1 to yield mature poliovirus capsid proteins was not observed. Attempts to isolate vaccinia virus recombinants containing portions of the poliovirus genome that encompassed the complete gene for proteinase 2A were unsuccessful, unless expression of functional 2A was abolished by insertion of a frameshift mutation. We conclude that an activity of the 2A proteinase, probably its role in translational inhibition, prevented isolation of vaccinia virus recombinants that expressed 2A.     

Therapy and long-term prophylaxis of vaccinia virus respiratory infections in mice with an adenovirus-vectored interferon alpha (mDEF201)

An adenovirus 5 vector encoding for mouse interferon alpha, subtype 5 (mDEF201) was evaluated for efficacy against lethal vaccinia virus (WR strain) respiratory infections in mice. mDEF201 was administered as a single intranasal treatment either prophylactically or therapeutically at doses of 10(6) to 10(8) plaque forming units/mouse. When the prophylactic treatment was given at 56 days prior to infection, it protected 90% of animals from death (100% protection for treatments given between 1-49 days pre-infection), with minimal weight loss occurring during infection. Surviving animals re-challenged with virus 22 days after the primary infection were protected from death, indicating that mDEF201 did not compromise the immune response against the initial infection. Post-exposure therapy was given between 6-24 h after vaccinia virus exposure and protection was afforded by a 10(8) dose of mDEF201 given at 24 h, whereas a 10(7) dose was effective up to 12 h. Comparisons were made of the ability of mDEF201, given either 28 or 1 day prior to infection, to inhibit tissue virus titers and lung infection parameters. Lung, liver, and spleen virus titers were inhibited to nearly the same extent by either treatment, as were lung weights and lung hemorrhage scores (indicators of pneumonitis). Lung virus titers were significantly (>100-fold) lower than in the placebo group, and the other infection parameters in mDEF201 treated mice were nearly at baseline. In contrast, viral titers and lung infection parameters were high in the placebo group on day 5 of the infection. These results demonstrate the long-acting prophylactic and treatment capacity of mDEF201 to combat vaccinia virus infections.     

Use of Disposable Micro Tissue Culture Plates for Antiviral and Interferon Induction Studies

A reproducible test system requiring small amounts of test compound was developed for evaluating antiviral and interferon-inducing activity. In the antiviral experiments, KB cells were grown in disposable polystyrene microplates covered with a standard domestic plastic wrap. Viruses used in the system were types 1 and 2 herpes simplex virus, vaccinia virus, type 3 adenovirus, myxoma virus, pseudorabies virus, type 3 parainfluenza virus, types 1A and 13 rhinovirus, vesicular stomatitis virus, coxsackievirus B, and type 2 poliovirus. Inhibition of viral cytopathogenic effect was the primary criterion of evaluation of antiviral activity. Reduction in cell and supernatant fluid virus titers was used as a secondary means of evaluation. The microplate system was adaptable for determining prophylactic, therapeutic, and inactivating effects against viruses. Mouse L-929 cells were used for the interferon induction studies, with vesicular stomatitis virus utilized as the indicator of interferon activity. Known active compounds evaluated in this microplate system had activity similar to that seen in macro in vitro systems.     

The combined effects of various disinfectants against poliovirus 1 in a municipal wastewater effluent

Abstract The effects of the combination of any two of the following disinfectants: chlorine, chlorine dioxide, ozone and peracetic acid were investigated using poliovirus 1 as a model virus in a municipal sewage effluent. It was noted that the efficacy of chlorine was enhanced in the presence of either chlorine dioxide, ozone or peracetic acid. Maximum enhancement was achieved when peracetic acid was present with either chlorine or ozone and less enhancement was noted when the peracetic acid was added before chlorine dioxide. Similar results were noted when hydrochloric acid was used instead of peracetic acid. It may be concluded that the application of any two of the disinfectants studied were rarely synergistic but merely additive or complementary (i.e., one disinfectant provides the other with a medium in which it functions better).     

Fluorescent-Antibody Studies with Antisera Against Heated and Unheated Poliovirus Type 1

Fluorescent-antibody (FA) reagents were prepared from sera of guinea pigs immunized with either native infectious poliovirus type 1 or poliovirus type 1 which had been heated at 56 C for 30 min. Conjugates made from sera of animals immunized with heated virus gave higher direct FA staining titers on air-dried, acetone-fixed, infected cells than conjugates made from sera of animals immunized with native infectious virus. Evidence was obtained that complement-fixing antibody reactive with heated antigen was responsible for the FA staining. Two conjugates prepared from sera of guinea pigs immunized with heated poliovirus type 1 were successfully used to identify 21 type 1 viruses isolated from a group of 44 stool suspensions studied as unknowns. These conjugates did not stain any of 23 heterologous enteroviruses present in the remainder of the stools and gave minimal non-specific staining.     

Protection of mice and swine from pseudorabies virus conferred by vaccinia virus-based recombinants.

Glycoproteins gp50, gII, and gIII of pseudorabies virus (PRV) were expressed either individually or in combination by vaccinia virus recombinants. In vitro analysis by immunoprecipitation and immunofluorescence demonstrated the expression of a gII protein of approximately 120 kDa that was proteolytically processed to the gIIb (67- to 74-kDa) and gIIc (58-kDa) mature protein species similar to those observed in PRV-infected cells. Additionally, the proper expression of the 90-kDa gIII and 50-kDa gp50 was observed. All three of these PRV-derived glycoproteins were detectable on the surface of vaccinia virus-PRV recombinant-infected cells. In vivo, mice were protected against a virulent PRV challenge after immunization with the PRV glycoprotein-expressing vaccinia virus recombinants. The coexpression of gII and gIII by a single vaccinia virus recombinant resulted in a significantly reduced vaccination dose required to protect mice against PRV challenge. Inoculation of piglets with the various vaccinia virus-PRV glycoprotein recombinants also resulted in protection against virulent PRV challenge as measured by weight gain. The simultaneous expression of gII and gp50 in swine resulted in a significantly enhanced level of protection as evaluated by weight evolution following challenge with live PRV.     

Modified vaccinia virus Ankara immunization protects against lethal challenge with recombinant vaccinia virus expressing murine interleukin-4

Recent events have raised concern over the use of pathogens, including variola virus, as biological weapons. Vaccination with Dryvax is associated with serious side effects and is contraindicated for many people, and the development of a safer effective smallpox vaccine is necessary. We evaluated an attenuated vaccinia virus, modified vaccinia virus Ankara (MVA), by use of a murine model to determine its efficacy against an intradermal (i.d.) or intranasal (i.n.) challenge with vaccinia virus (vSC8) or a recombinant vaccinia virus expressing murine interleukin-4 that exhibits enhanced virulence (vSC8-mIL4). After an i.d. challenge, 15 of 16 mice who were inoculated with phosphate-buffered saline developed lesions, one dose of intramuscularly administered MVA was partially protective (3 of 16 mice developed lesions), and the administration of two or three doses of MVA was completely protective (0 of 16 mice developed lesions). In unimmunized mice, an i.n. challenge with vSC8 caused a significant but self-limited illness, while vSC8-mIL4 resulted in lethal infections. Immunization with one or two doses of MVA prevented illness and reduced virus titers in mice who were challenged with either vSC8 or vSC8-mIL4. MVA induced a dose-related neutralizing antibody and vaccinia virus-specific CD8+-T-cell response. Mice immunized with MVA were fully protected from a low-dose vSC8-mIL4 challenge despite a depletion of CD4+ cells, CD8+ cells, or both T-cell subsets or an antibody deficiency. CD4+- or CD8+-T-cell depletion reduced the protection against a high-dose vSC8-mIL4 challenge, and the depletion of both T-cell subsets was associated with severe illness and higher vaccinia virus titers. Thus, MVA induces broad humoral and cellular immune responses that can independently protect against a molecularly modified lethal poxvirus challenge in mice. These data support the continued development of MVA as an alternative candidate vaccine for smallpox.     

Sickness and recovery of dogs challenged with a street rabies virus after vaccination with a vaccinia virus recombinant expressing rabies virus N protein.

Dogs were vaccinated intradermally with vaccinia virus recombinants expressing the rabies virus glycoprotein (G protein) or nucleoprotein (N protein) or a combination of both proteins. The dogs vaccinated with either the G or G plus N proteins developed virus-neutralizing antibody titers, whereas those vaccinated with only the N protein did not. All dogs were then challenged with a lethal dose of a street rabies virus, which killed all control dogs. Dogs vaccinated with the G or G plus N proteins were protected. Five (71%) of seven dogs vaccinated with the N protein sickened, with incubation periods 3 to 7 days shorter than that of the control dogs; however, three (60%) of the five rabid dogs recovered without supportive treatment. Thus, five (71%) of seven vaccinated with the rabies N protein were protected against a street rabies challenge. Our data indicate that rabies virus N protein may be involved in reducing the incubation period in dogs primed with rabies virus N protein and then challenged with a street rabies virus and, of more importance, in subsequent sickness and recovery.     

Coinfection with recombinant vaccinia viruses expressing poliovirus P1 and P3 proteins results in polyprotein processing and formation of empty capsid structures.

The assembly process of poliovirus occurs via an ordered proteolytic processing of the capsid precursor protein, P1, by the virus-encoded proteinase 3CD. To further delineate this process, we have isolated a recombinant vaccinia virus which expresses, upon infection, the poliovirus P1 capsid precursor polyprotein with an authentic carboxy terminus. Coinfection of HeLa cells with the P1-expressing vaccinia virus and with a second recombinant vaccinia virus which expresses the poliovirus proteinase 3CD resulted in the correct processing of P1 to yield the three individual capsid proteins VP0, VP3, and VP1. When extracts from coinfected cells were fractionated on sucrose density gradients, the VP0, VP3, and VP1 capsid proteins were immunoprecipitated with type 1 poliovirus antisera from fractions corresponding to a sedimentation consistent for poliovirus 75S procapsids. Examination of these fractions by electron microscopy revealed structures which lacked electron-dense cores and which corresponded in size and shape to those expected for poliovirus empty capsids. We conclude that the expression of the two poliovirus proteins P1 and 3CD in coinfected cells is sufficient for the correct processing of the capsid precursor to VP0, VP3, and VP1 as well as for the assembly of poliovirus empty capsid-like structures.     

Effect of the Deletion of Genes Encoding Proteins of the Extracellular Virion Form of Vaccinia Virus on Vaccine Immunogenicity and Protective Effectiveness in the Mouse Model

Antibodies to both infectious forms of vaccinia virus, the mature virion (MV) and the enveloped virion (EV), as well as cell-mediated immune response appear to be important for protection against smallpox. EV virus particles, although more labile and less numerous than MV, are important for dissemination and spread of virus in infected hosts and thus important in virus pathogenesis. The importance of the EV A33 and B5 proteins for vaccine induced immunity and protection in a murine intranasal challenge model was evaluated by deletion of both the A33R and B5R genes in a vaccine-derived strain of vaccinia virus. Deletion of either A33R or B5R resulted in viruses with a small plaque phenotype and reduced virus yields, as reported previously, whereas deletion of both EV protein-encoding genes resulted in a virus that formed small infection foci that were detectable and quantifiable only by immunostaining and an even more dramatic decrease in total virus yield in cell culture. Deletion of B5R, either as a single gene knockout or in the double EV gene knockout virus, resulted in a loss of EV neutralizing activity, but all EV gene knockout viruses still induced a robust neutralizing activity against the vaccinia MV form of the virus. The effect of elimination of A33 and/or B5 on the protection afforded by vaccination was evaluated by intranasal challenge with a lethal dose of either vaccinia virus WR or IHD-J, a strain of vaccinia virus that produces relatively higher amounts of EV virus. The results from multiple experiments, using a range of vaccination doses and virus challenge doses, and using mortality, morbidity, and virus dissemination as endpoints, indicate that the absence of A33 and B5 have little effect on the ability of a vaccinia vaccine virus to provide protection against a lethal intranasal challenge in a mouse model.     

Immunization with a vaccinia virus recombinant expressing herpes simplex virus type 1 glycoprotein D: long-term protection and effect of revaccination.

Previously we showed that mice immunized with a vaccinia virus vector expressing the herpes simplex virus type 1 (HSV-1) glycoprotein D (gD) gene (vaccinia/gD) were protected against both lethal and latent infections with HSV-1 for at least 6 weeks after immunization (K. J. Cremer, M. Mackett, C. Wohlenberg, A. L. Notkins, and B. Moss, Science 228:737-740, 1985). In the experiments described here, we examined long-term immunity to HSV following vaccinia/gD vaccination, the effect of revaccination with vaccinia/gD, and the impact of previous immunity to vaccinia virus on immunization with the gD recombinant. Mice immunized with vaccinia/gD showed 100, 100, and 80% protection against lethal infection with HSV-1 at 18, 44, and 60 weeks postimmunization, respectively. Protection against latent trigeminal ganglionic infection was 70, 50, and 31% at 6, 41, and 60 weeks postvaccination, respectively. To study the effect of reimmunization on antibody levels, mice vaccinated with vaccinia/gD were given a second immunization (booster dose) 3 months after the first. These mice developed a 10-fold increase in neutralizing-antibody titer (221 to 2,934) and demonstrated a significant increase in protection against lethal HSV-1 challenge compared with animals that received only one dose of vaccinia/gD. To determine whether preexisting immunity to vaccinia virus inhibited the response to vaccination with vaccinia/gD virus, mice were immunized with a recombinant vaccinia virus vector expressing antigens from either influenza A or hepatitis B virus and were then immunized (2 to 3 months later) with vaccinia/gD. These mice showed reduced titers of neutralizing antibody to HSV-1 and decreased protection against both lethal and latent infections with HSV-1 compared with animals vaccinated only with vaccinia/gD. We conclude that vaccination with vaccinia/gD produces immunity against HSV-1 that lasts over 1 year and that this immunity can be increased by a booster but that prior immunization with a vaccinia recombinant virus expressing a non-HSV gene reduces the levels of neutralizing antibody and protective immunity against HSV-1 challenge.