Strong alkaline electrolyzed water efficiently inactivates SARS-CoV-2, other viruses, and Gram-negative bacteria.

Air spaces and material surfaces in a pathogen-contaminated environment can often be a source of infection to humans, and disinfection has become a common intervention focused on reducing the contamination levels. In this study, we examined the efficacy of SAIW, a unique electrolyzed water with chlorine-free, high pH, high concentration of dissolved hydrogen, and low oxygen reduction potential, for the inactivation of several viruses and bacteria. Infectivity assays revealed that initial viral titers of enveloped and non-enveloped viruses, including severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), influenza A virus, herpes simplex virus type 1, human coronavirus, feline calicivirus, and canine parvovirus, were reduced by 2.9- to 5.5-log10 within 30 s of SAIW exposure. Similarly, the culturability of three Gram-negative bacteria (Escherichia coli, Salmonella, and Legionella) dropped down by 1.9- to 4.9-log10 within 30 s of SAIW treatment. Mechanistically, treatment with SAIW was found to significantly decrease the binding and subsequent entry efficiencies of SARS-CoV-2 on Vero cells. Finally, we showed that this chlorine-free electrolytic ion water had no acute inhalation toxicity in mice, demonstrating that SAIW holds promise for a safer antiviral and antibacterial disinfectant.

Inhibitory Effects of Antiviral Drug Candidates on Canine Parvovirus in F81 cells.

Canine parvovirus (CPV) is a common etiological agent of acute enteritis, which occurs globally in domestic and wild carnivores. Despite the widespread use of inactivated or live attenuated vaccines, the emergence of antigenic variants and the influence of maternal antibodies have raised some concerns regarding the efficacy of commercial vaccines. While no specific antiviral therapy for CPV infection exists, the only treatment option for the infection is supportive therapy based on symptoms. Thus, there is an urgent medical need to develop antiviral therapeutic options to reduce the burden of CPV-related disease. In this study, a cytopathic effect (CPE)-based high-throughput screening assay was used to screen CPV inhibitors from a Food and Drug Administration (FDA)-approved drug library. After two rounds of screening, seven out of 1430 screened drugs were found to have >50% CPE inhibition. Three drugs-Nitazoxanide, Closantel Sodium, and Closantel-with higher anti-CPV effects were further evaluated in F81 cells by absolute PCR quantification and indirect immunofluorescence assay (IFA). The inhibitory effects of all three drugs were dose-dependent. Time of addition assay indicated that the drugs inhibited the early processes of the CPV replication cycle, and the inhibition effects were relatively high within 2 h postinfection. Western blot assay also showed that the three drugs had broad-spectrum antiviral activity against different subspecies of three CPV variants. In addition, antiapoptotic effects were observed within 12 h in Nitazoxanide-treated F81 cells regardless of CPV infection, while Closantel Sodium- or Closantel-treated cells had no pro- or antiapoptotic effects. In conclusion, Nitazoxanide, Closantel Sodium, and Closantel can effectively inhibit different subspecies of CPV. Since the safety profiles of FDA-approved drugs have already been extensively studied, these three drugs can potentially become specific and effective anti-CPV drugs.

In vitro virucidal activity of sodium hypochlorite against canine parvovirus type 2.

Canine parvovirosis is a very contagious, severe and often lethal infectious disease of dogs caused by canine parvovirus type 2 (CPV-2). Parvoviruses are very resistant to several disinfectants while are sensitive to halogens such as sodium hypochlorite which is often used for decontamination of veterinary clinics and animal housing facilities due to its broad spectrum of activity. If compliance with vaccination programmes and with proper disinfection plans is ensured, there should be no continuous, nor frequent, CPV-2 outbreaks in kennels and veterinary clinics. However, a continuous spread of CPV-2 infections is observed, even in kennels where an appropriate vaccination programme is applied, and this imposes a re-evaluation of disinfection protocols using sodium hypochlorite. The aim of the present study was to determine the effect of concentration, contact time and presence of organic matter on the virucidal activity of sodium hypochlorite against several CPV-2 strains. A sensitive in vitro assay capable of measuring the infectivity of CPV-2 was employed to determine the efficacy of three different concentrations of sodium hypochlorite. The data indicate that using a 0.75% sodium hypochlorite solution for a short contact time (1 min) can reduce significantly the CPV-2 titres and that even lower concentrations, i.e. 0.37%, can efficiently inactivate the viruses provided that the contact time is extended to 15 min. Results also confirm the importance of cleaning before disinfection since the presence of organic matter totally abrogated the virucidal activity of sodium hypochlorite solutions against the three CPV-2 strains.

Serum Derived Transfer Factor Stimulates the Innate Immune System to Improve Survival Traits in High Risk Pathogen Scenarios.

Preclinical Research Transfer Factors (TFs) are low molecular weight (<5,000 daltons) biological response mediators. In the present study, a serum derived TF improved the ability of the recipient animal to survive high-risk infectious challenges (salmonellosis and canine parvoviral enteritis (CPV)) by altering the host's cytokine response profile. Mice mortally challenged with 5,000 colony-forming units of Salmonella experienced a group mortality of 73% while mice treated with a single 5 mg dose of the TF demonstrated a significant decrease in morbidity (7%, p ≤ 0.01). The splenic bacterial load in untreated mice was over 10,000 times higher than that in the TF treated mice. Twenty-four hours post-administration, the treated murine population expressed a rapid temporal increase in serum IL-6 (26-fold) and INF-γ (77-fold) concentrations. IL-6 can act as a critical signal regulating action against bacterial pathogens. A comparative double-blind study performed using dogs confirmed to be undergoing a canine parvovirus challenge showed that when conventional supportive therapy was supplemented with a single 5 mg TF dose there was a reduction (p ≤ 0.01) in group mortality (68% of the TF treated group survived versus 32% of the placebo group), an observation consistent with the observed increase in INF-γ, a cytokine associated with promoting antiviral activity. Drug Dev Res 78 : 189-195, 2017. © 2017 Wiley Periodicals, Inc.

Antiviral activity of phosphorylated Radix Cyathulae officinalis polysaccharide against Canine Parvovirus in vitro.

Phosphorylated Radix Cyathulae officinalis Kuan polysaccharides (pRCPS) was prepared according to three-factors, ratio of STMP (%) and STPP (%), reaction time and reaction temperature, and three level L9(34) orthogonal design. The antiviral activity of nine pRCPS (pRCPS1-9) was systematically evaluated by three methods pre-adding mode, mixed mode, and post-adding mode. Cellular activity was tested by the CCK-8 assay. The results showed that the optimal modification conditions were the ratio of STMP (%) and STPP (%) 1:4, reaction time 2h and reaction temperature 65°C. Six pRCPS (pRCPS1-4, pRCPS7, pRCPS9) exhibited significant anti-viral activity in pre-adding mode (P<0.05). Eight pRCPS (pRCPS1-4, pRCPS5, pRCPS6, pRCPS7, and pRCPS9) showed dramatic anti-viral activity in the mixed mode (P<0.05). Six pRCPS (pRCPS1-4, pRCPS6, pRCPS9) showed antiviral activity in the post-adding mode (P<0.05). Taken together, four pRCPS (pRCPS1-4) demonstrated significant antiviral activity in all the test modes (P<0.05) and their antiviral efficacy were significantly stronger than unmodified RCPS (P<0.05). Those results indicated that four pRCPS (pRCPS1-4) possessed significant antiviral activity and may have potential as a new CPV therapeutic compound, and phosphorylation could significantly enhance the antiviral effect of RCPS. Moreover, phosphorylation modification technique could be valuable as a method to promote the antiviral activity of polysaccharide.

Evaluation of Immunity and Seropositivity of IgG Antibodies to Canine Parvoviruses in Vaccinated and Unvaccinated Dogs in Abeokuta, Nigeria.

Canine Parvovirus (CPV) is a very contagious and virulent viral disease affecting domestic dogs all over the world causing high morbidity and mortality in dogs, especially puppies. This study aimed at determining the seropositivity of IgG antibodies against CPV in vaccinated and unvaccinated dogs and to evaluate the immune status of dogs presented in Abeokuta. Forty-eight dogs were enrolled in this study. These dogs were presented at random for treatment, routine checkup, and vaccination at the State Veterinary Hospital and Veterinary Teaching Hospital all in Abeokuta. All the dogs were fully maintained under domestic setting. Selection for study was done based on thorough examination of the dogs and their medical records. The clients were informed of the nature of the investigation. Blood samples were collected and analyzed for anti-CPV-IgG. In principle, protective immunity correlates with high antibody titers and this was determined using a commercially available immunocomb® test kit for anti-CPV IgG antibody. Of 48 dogs sampled, 38 (79.2%) had high level of anti-CPV antibody titer and 10 (20.8%) had low level of anti-CPV antibody titer. Twenty six (54.2%) were males while 22 (45.8%) were females. Forty-five (93.75%) dogs were exotic breeds while 3 (6.25%) dogs were mongrels. Thirty (62.5%) of the dogs were less than one year old and the age range of all dogs sampled was between 7 weeks and 7 years. There was no significant difference (P > 0.05) between sex and the level of immunity but significant differences (P < 0.05) were observed between ages of dogs, breeds, post-vaccination period, and the level of immunity. In conclusion, this study has further confirmed the presence of IgG antibodies against canine parvovirus among dogs in Abeokuta, Nigeria. Of all variables evaluated, ages of dogs, breeds and post-vaccination period were the main correlates of the level of immunity to CPV. This study also showed agreement with previous studies in the diagnostic value of using the immunoblot ELISA assay for the rapid detection of anti-CPV IgG antibody.

Antiviral effect of lithium chloride on infection of cells by canine parvovirus.

Canine parvovirus type 2 causes significant viral disease in dogs, with high morbidity, high infectivity, and high mortality. Lithium chloride is a potential antiviral drug for viruses. We determined the antiviral effect of Lithium Chloride on canine parvovirus type 2 in feline kidney cells. The viral DNA and proteins of canine parvovirus were suppressed in a dose-dependent manner by lithium chloride. Further investigation verified that viral entry into cells was inhibited in a dose-dependent manner by lithium chloride. These results indicated that lithium chloride could be a potential antiviral drug for curing dogs with canine parvovirus infection. The specific steps of canine parvovirus entry into cells that are affected by lithium chloride and its antiviral effect in vivo should be explored in future studies.

Aptamers targeting rabies virus-infected cells inhibit viral replication both in vitro and in vivo.

Rabies is an acute fatal encephalitis disease that affects many warm-blooded mammals. The causative agent of the disease is Rabies virus (RABV). Currently, no approved therapy is available once the clinical signs have appeared. Aptamers, oligonucleotide ligands capable of binding a variety of molecular targets with high affinity and specificity, have recently emerged as promising therapeutic agents. In this study, sixteen high-affinity single-stranded DNA (ssDNA) aptamers were generated by cell-SELEX. Viral titer assays revealed aptamers could specifically inhibit the replication of RABV in cells but did not inhibit the replication of canine distemper virus or canine parvovirus. In addition, the FO21 and FO24 aptamers, with and without PEGylation, were found to effectively protect mice against lethal RABV challenge. When mice were inoculated with aptamers for 24h prior to inoculation with CVS-11, approximately 87.5% of the mice survived. Here, we report aptamers that could significantly protect the mice from a lethal dose of RABV in vitro and in vivo, as demonstrated by the results for survival rate, weight loss and viral titers. These results indicate that FO21 and FO24 aptamers are a promising agent for specific antiviral against RABV infections.

Inhibition of canine parvovirus replication in cultured cells by small interfering RNAs expressed from plasmid vectors.

Small interfering RNAs (siRNAs) target complementary mRNA for specific degradation, a mechanism many viruses are susceptible too. Thus, siRNA degradation of target RNAs can be exploited as novel therapeutics. In this report, we show that the vector-based siRNAs (psiSTRIKEs) expressed by a human U6 promoter could efficiently inhibit CPV replication in cell culture. A series of PsiSTRIKE vectors expressing siRNA were constructed that target structural protein genes or nonstructural protein genes of CPV genome. These plasmids were transfected into FK81 cells via lipofectin and the stable transfection clones were selected. The immunostaining, plaque assay, and cell proliferation assay of the cells infected by CPV were performed. The results show that siRNAs against nonstructural protein genes effectively inhibited CPV replication. The inhibition efficiencies detected by immunostaining assay of psiSTRIKE/vp1510, psiSTRIKE/NS160, and psiSTRIKE/NS1939 were 66%, 76% and 78%, respectively at 48h, and 69%, 46% and 67%, respectively at 96h. Plaque assay showed that, comprising to the control, the psiSTRIKE/NS160 reduced the virion production by 100-fold, and psiSTRIKE/NS1939 or psiSTRIKE/VP1510 reduced the virion production 13-fold. When compared to control, the viability of cells transfected psiSTRIKE/NS160 increased 78% and 124%, respectively at 72 and 120h. Our study may provide a potential therapy against CPV infection.

Validation of model virus removal and inactivation capacity of an erythropoietin purification process.

Human erythropoietin (hEpo) production requires mammalian cells able to make complex post-translational modifications to guaranty its biological activity. As mammalian cell can be reservoir of pathogenic viruses and several animal origin components are usually used in the cultivation of mammalian cells, hEpo contamination with viruses is something of great concern. As consequence, this study investigated the viral removal and inactivation capacity of a recombinant-hEpo (rec-hEpo) purification process. Canine parvovirus, Human poliovirus type-2, Bovine viral diarrhea virus and Human immunodeficiency virus type-1 were used for measuring process viral removal and inactivation capacities. In conclusion, this study corroborated that the assessed rec-hEpo purification process has enough capacity (5.0-19.4 Logs) for removing and inactivating these model viruses and sodium hydroxide demonstrated to be a robust sanitization solution for chromatography columns (5.0 (PV-2)-6.7 (CPV) Logs).

Virus inactivation in albumin by a combination of alkali conditions and high temperature.

Non-enveloped viruses such as HAV and B19 are of potential concern in plasma products. In the case of albumin, pasteurisation at 60 °C for 10 h is generally used for virus inactivation. However this procedure is only partially effective against some non-enveloped viruses. Using a range of non-enveloped viruses i.e. HAV, SV40, CPV, treatment at a high pH of about 9.5 and a temperature of 60 °C for 10 h was found to be effective for virus inactivation. These extreme conditions caused no increase in aggregate composition of the albumin. In addition the albumin composition was stable over a period of at least 6 months. The ligand binding properties of the albumin, as determined using the dye phenol red, were also not affected by this treatment. This procedure has the potential for increasing the spectrum of viruses inactivated by the 60 °C pasteurisation step.

Evaluation of the antiviral activity of chlorine dioxide and sodium hypochlorite against feline calicivirus, human influenza virus, measles virus, canine distemper virus, human herpesvirus, human adenovirus, canine adenovirus and canine parvovirus.

We evaluated the antiviral activity of a chlorine dioxide gas solution (CD) and sodium hypochlorite (SH) against feline calicivirus, human influenza virus, measles virus, canine distemper virus, human herpesvirus, human adenovirus, canine adenovirus and canine parvovirus. CD at concentrations ranging from 1 to 100 ppm produced potent antiviral activity, inactivating >or= 99.9% of the viruses with a 15 sec treatment for sensitization. The antiviral activity of CD was approximately 10 times higher than that of SH.

Use of oseltamivir in the treatment of canine parvoviral enteritis.

OBJECTIVE: To determine if oseltamivir with standard therapy for canine parvoviral enteritis ameliorates disease morbidity, mortality, or both; to document significant adverse effects associated with its use. DESIGN: Prospective, randomized, blinded, placebo-controlled clinical trial. SETTING: University veterinary teaching hospital. ANIMALS: Thirty-five dogs. INTERVENTIONS: Standard therapy was administered to all dogs. Treatment dogs also received oseltamivir, while control dogs received an equivalent volume of placebo. MEASUREMENTS AND MAIN RESULTS: Dogs were monitored daily according to a clinical scoring system, physical parameters, and diagnostic evaluations. Dogs in the treatment group gained a significant percentage of weight during hospitalization (mean, +2.6%; SD, 7.1%) versus the control dogs (mean, -4.5%; SD, 6.9%) (P=0.006). Treatment dogs did not have any significant changes in their white blood cell (WBC) count, while control dogs experienced a significant drop in their WBC counts during their initial stay. In addition, it did not appear that oseltamivir use was associated with any major adverse clinical effects. CONCLUSIONS: While a clear advantage to the use of oseltamivir was not established, a significant weight loss during hospitalization, as well as a significant decrease in WBC count were documented in the control group. No major adverse effects were identified that could be associated with oseltamivir administration. Based on these results, the true role of oseltamivir in the treatment of parvoviral enteritis remains speculative, although it is believed that further investigation is warranted.

Desipramine induces disorder in cholesterol-rich membranes: implications for viral trafficking.

In this study, the effect of desipramine (DMI) on phospholipid bilayers and parvoviral entry was elucidated. In atomistic molecular dynamics simulations, DMI was found to introduce disorder in cholesterol-rich phospholipid bilayers. This was manifested by a decrease in the deuterium order parameter S(CD) as well as an increase in the membrane area. Disordering of the membrane suggested DMI to destabilize cholesterol-rich membrane domains (rafts) in cellular conditions. To relate the raft disrupting ability of DMI with novel biological relevance, we studied the intracellular effect of DMI using canine parvovirus (CPV), a virus known to interact with endosomal membranes and sphingomyelin, as an intracellular probe. DMI was found to cause retention of the virus in intracellular vesicular structures leading to the inhibition of viral proliferation. This implies that DMI has a deleterious effect on the viral traffic. As recycling endosomes and the internal vesicles of multivesicular bodies are known to contain raft components, the effect of desipramine beyond the plasma membrane step could be caused by raft disruption leading to impaired endosomal function and possibly have direct influence on the penetration of the virus through an endosomal membrane.

Immunological effects of recombinant feline interferon-omega (KT-80) administration in the dog.

The immunological effects of recombinant feline interferon-omega (rFeIFN-omega ; KT-80, Toray) were examined on administration to healthy dogs. The activities of whole blood cells, macrophages, and natural killer cells were enhanced. Moreover, the whole blood activity was examined when KT-80 was administered to dogs which had been diagnosed as having natural canine parvovirus (CPV) infection. Only some cases in which the activity increased until 3 hr post-administration survived. These results suggest that rFeIFN-omega (KT-80) treatment enhanced the cellular immunity of normal dogs, and could exert significant therapeutic effects on only natural CPV infected dogs with induced continuous immunoenhancement.

Viruses adsorbed on musculoskeletal allografts are inactivated by terminal ethylene oxide disinfection.

In 1987 it was anticipated that unsterilized tissues would transmit virus diseases such as hepatitis and HIV-1 from infected donors so a freeze-drying process for musculoskeletal tissue was developed to include terminal ethylene oxide (EO) exposure for 14 h. We found no studies of EO efficacy when viruses were associated with human allografts so we studied the antiviral effect of terminal EO disinfection using all but the final freeze-drying phase of this clinical processing protocol (CPP). Specifically we looked at EO inactivation of HIV-1, a human hepatitis B surrogate and test viruses known to be highly resistant to disinfecting agents, including irradiation. Freeze-drying, ordinarily required after EO disinfection and part of the CPP, was not done. Suspensions of HIV-1, Bovine viral diarrhea, Reovirus type 3, Duck hepatitis B, Poliomyelitis and Canine parvovirus were adsorbed on glass, demineralized bone powder, and preprocessed strips of femoral cortex, iliac wedges, cancellous blocks and patellar bone-tendon-bone preparations and subjected to EO disinfection. Test viruses were inactivated at the end of 7 h of EO disinfection, providing a safety factor in the CPP of at least 100%. Because allografts can transmit viruses, terminal EO disinfection should provide safer musculoskeletal allografts than non-disinfected tissues or those irradiated with a standard irradiation dose. New spontaneously appearing viruses would probably be inactivated with this terminal EO disinfection but they and viral bioweapons will require individual validation to assure viral inactivation.

The band III ligand dipyridamole protects human RBCs during photodynamic treatment while extracellular virus inactivation is not affected.

BACKGROUND: Recently, the potential usefulness of dipyridamole (DIP) in protecting RBCs against the harmful side effects of photodynamic sterilization was demonstrated. In the present study, the use of DIP for selective protection of RBCs was investigated under conditions more relevant for blood bank practice. STUDY DESIGN AND METHODS: WBC-reduced RBC suspensions (30% Hct) were treated with 1,9-dimethylmethylene blue and red light, and the influence of the inclusion of DIP on photohemolysis was assessed as a function of sensitizer concentration, light dose, and storage time. Furthermore, the possible interference of DIP with inactivation of extracellular virus by use of a panel of different viruses (HIV-1, pseudorabies virus [PRV], bovine viral diarrhea virus [BVDV], VSV, encephalomyocarditis, and canine parvovirus) was investigated. RESULTS: In WBC-reduced RBC suspensions (30% Hct), DIP exerted a clear protective effect against photohemolysis. Part of this protection was achieved with concentrations near the dissociation constant for band III binding. Importantly, efficiency of inactivation of extracellular HIV-1, PRV, BVDV, and VSV was not significantly impaired by the inclusion of DIP. Phototreatment conditions, resulting in a 4 to 5 log inactivation of extracellular HIV-1 and PRV, resulted in a high level of hemolysis after 28 days of storage. This long-term hemolysis could be decreased, but not completely prevented, by the inclusion of DIP. CONCLUSION: Photohemolysis in RBC concentrates can be reduced substantially by the application of DIP, while the efficacy of inactivation of HIV-1 and other viruses remains unchanged.

Canine and feline parvoviruses can use human or feline transferrin receptors to bind, enter, and infect cells.

Canine parvovirus (CPV) enters and infects cells by a dynamin-dependent, clathrin-mediated endocytic pathway, and viral capsids colocalize with transferrin in perinuclear vesicles of cells shortly after entry (J. S. L. Parker and C. R. Parrish, J. Virol. 74:1919-1930, 2000). Here we report that CPV and feline panleukopenia virus (FPV), a closely related parvovirus, bind to the human and feline transferrin receptors (TfRs) and use these receptors to enter and infect cells. Capsids did not detectably bind or enter quail QT35 cells or a Chinese hamster ovary (CHO) cell-derived cell line that lacks any TfR (TRVb cells). However, capsids bound and were endocytosed into QT35 cells and CHO-derived TRVb-1 cells that expressed the human TfR. TRVb-1 cells or TRVb cells transiently expressing the feline TfR were susceptible to infection by CPV and FPV, but the parental TRVb cells were not. We screened a panel of feline-mouse hybrid cells for susceptibility to FPV infection and found that only those cells that possessed feline chromosome C2 were susceptible. The feline TfR gene (TRFC) also mapped to feline chromosome C2. These data indicate that cell susceptibility for these viruses is determined by the TfR.

Intracellular route of canine parvovirus entry.

The present study was designed to investigate the endocytic pathway involved in canine parvovirus (CPV) infection. Reduced temperature (18 degrees C) or the microtubule-depolymerizing drug nocodazole was found to inhibit productive infection of canine A72 cells by CPV and caused CPV to be retained in cytoplasmic vesicles as indicated by immunofluorescence microscopy. Consistent with previously published results, these data indicate that CPV enters a host cell via an endocytic route and further suggest that microtubule-dependent delivery of CPV to late endosomes is required for productive infection. Cytoplasmic microinjection of CPV particles was used to circumvent the endocytosis and membrane fusion steps in the entry process. Microinjection experiments showed that CPV particles which were injected directly into the cytoplasm, thus avoiding the endocytic pathway, were unable to initiate progeny virus production. CPV treated at pH 5.0 prior to microinjection was unable to initiate virus production, showing that factors of the endocytic route other than low pH are necessary for the initiation of infection by CPV.

Inactivation kinetics of model and relevant blood-borne viruses by treatment with sodium hydroxide and heat.

To determine the efficacy of a clean-in-place system for the inactivation of viruses present in human plasma, the effect of 0.1 M sodium hydroxide at 60 degrees C on viral infectivity was investigated. Inactivation of the following model and relevant viruses were followed as a function of time: human hepatitis A virus (HAV), canine parvovirus (CPV; a model for human parvovirus B-19) pseudorabies virus (PRV, a model for hepatitis B virus), and bovine viral diarrhoea virus (BVDV, a model for hepatitis C virus and human immunodeficiency virus). Infectivity of CPV was determined by a novel in situ EIA method which will prove useful for studies to validate parvovirus inactivation or removal. Infectivity of BVDV, PRV and CPV were shown to be reproducibly inactivated below the limit of detection by 0.1 M NaOH at 60 degrees C within 30 s. HAV was inactivated to below the limit of detection within 2 min. Treatment with heat alone also resulted in some log reduction for all viruses tested except for CPV which remained unaffected after heating at 60 degrees C for 16 min. Treatment of HAV with hydroxide alone (up to 1.0 m) at 15 degrees C did not lead to rapid inactivation. Collectively, these data suggest that 0.1 M NaOH at 60 degrees C for two min should be sufficient to inactivate viruses present in process residues.