PD 404,182 is a virocidal small molecule that disrupts hepatitis C virus and human immunodeficiency virus

We describe a virucidal small molecule, PD 404,182, that is effective against hepatitis C virus (HCV) and human immunodeficiency virus (HIV). The median 50% inhibitory concentrations (IC(50)s) for the antiviral effect of PD 404,182 against HCV and HIV in cell culture are 11 and 1 μM, respectively. The antiviral activity of PD 404,182 is due to the physical disruption of virions that is accompanied to various degrees (depending on the virus and exposure temperature/time) by the release of viral nucleic acids into the surrounding medium. PD 404,182 does not directly lyse liposomal membranes even after extended exposure, and it shows no attenuation in antiviral activity when preincubated with liposomes of various lipid compositions, suggesting that the compound inactivates viruses through interaction with a nonlipid structural component of the virus. The virucidal activity of PD 404,182 appears to be virus specific, as little to no viral inactivation was detected with the enveloped Dengue and Sindbis viruses. PD 404,182 effectively inactivates a broad range of primary isolates of HIV-1 as well as HIV-2 and simian immunodeficiency virus (SIV), and it does not exhibit significant cytotoxicity with multiple human cell lines in vitro (50% cytotoxic concentration, >300 μM). The compound is fully active in cervical fluids, although it exhibits decreased potency in the presence of human serum, retains its full antiviral potency for 8 h when in contact with cells, and is effective against both cell-free and cell-associated HIV. These qualities make PD 404,182 an attractive candidate anti-HIV microbicide for the prevention of HIV transmission through sexual intercourse.     

Robustness of solvent/detergent treatment of plasma derivatives: a data collection from Plasma Protein Therapeutics Association member companies

BACKGROUND: Solvent/detergent (S/D) treatment is an established virus inactivation technology that has been applied in the manufacture of medicinal products derived from human plasma for more than 20 years. Data on the inactivation of enveloped viruses by S/D treatment collected from seven Plasma Protein Therapeutics Association member companies demonstrate the robustness, reliability, and efficacy of this virus inactivation method.
STUDY DESIGN AND METHODS: The results from 308 studies reflecting production conditions as well as technical variables significantly beyond the product release specification were evaluated for virus inactivation, comprising different combinations of solvent and detergent (tri( n -butyl) phosphate [TNBP]/Tween 80, TNBP/Triton X-100, TNBP/Na-cholate) and different products (Factor [F]VIII, F IX, and intravenous and intramuscular immunoglobulins).
RESULTS: Neither product class, process temperature, protein concentration, nor pH value has a significant impact on virus inactivation. A variable that did appear to be critical was the concentration of solvent and detergent.
CONCLUSION: The data presented here demonstrate the robustness of virus inactivation by S/D treatment for a broad spectrum of enveloped test viruses and process variables. Our data substantiate the fact that no transmission of viruses such as human immunodeficiency virus, hepatitis B virus, hepatitis C virus, or of other enveloped viruses was reported for licensed plasma derivatives since the introduction of S/D treatment.     

A throat lozenge containing amyl meta cresol and dichlorobenzyl alcohol has a direct virucidal effect on respiratory syncytial virus, influenza A and SARS-CoV

A potent virucidal mixture containing amyl metacresol and dichlorobenzyl alcohol at low pH inactivated enveloped respiratory viruses influenza A, respiratory synctial virus (RSV) and severe acute respiratory syndrome coronavirus (SARS-CoV) but not viruses with icosahedral symmetry, such as adenoviruses or rhinoviruses. A titre of approximately 3.5 log10 TCID50 was reduced to below the level of detection within two minutes. Electron microscopy of purified influenza A virus showed extensive clumping and morphological changes in spike configuration after contact with the virucidal mixture, but no overt destruction of the viral membrane. We conclude that, formulated as a lozenge, the mixture could have significant effects in reducing the infectivity of certain infectious viruses in the throat and presumably in cough droplets, thus reducing, theoretically, opportunities for person-to-person transmission.     

Interaction of polyunsaturated fatty acids with animal cells and enveloped viruses.

Essential unsaturated fatty acids such as oleic, linoleic, or arachidonic were incorporated into the phospholipids of animal cells and induced in them a change in the fluidity of their membranes. Exposure of enveloped viruses such as arbo-, myxo-, paramyxo-, or herpesviruses to micromolar concentrations of these fatty acids (which are not toxic to animal cells) caused rapid loss of infectivity of these viruses. Naked viruses such as encephalomyocarditis virus, polio virus or simian virus 40 were not affected by incubation with linoleic acid. The loss of infectivity was attributed to a disruption of the lipoprotein envelope of these virions, as observed in an electron microscope.     

Inactivation of enveloped viruses by anthraquinones extracted from plants.

To determine the extent of antiviral activity present in a number of plant extracts, hot glycerin extracts were prepared from Rheum officinale, Aloe barbadensis, Rhamnus frangula, Rhamnus purshianus, and Cassia angustifolia and their virucidal effects were tested against herpes simplex virus type 1. All the plant extracts inactivated the virus. The active components in these plants were separated by thin-layer chromatography and identified as anthraquinones. A purified sample of aloe emodin was prepared from aloin, and its effects on the infectivity of herpes simplex virus type 1 and type 2, varicella-zoster virus, pseudorabies virus, influenza virus, adenovirus, and rhinovirus were tested by mixing virus with dilutions of aloe emodin for 15 min at 37 degrees C, immediately diluting the sample, and assaying the amount of infectious virus remaining in the sample. The results showed that aloe emodin inactivated all of the viruses tested except adenovirus and rhinovirus. Electron microscopic examination of anthraquinone-treated herpes simplex virus demonstrated that the envelopes were partially disrupted. These results show that anthraquinones extracted from a variety of plants are directly virucidal to enveloped viruses.     

Inactivation of parvovirus B19 in coagulation factor concentrates by UVC radiation: assessment by an in vitro infectivity assay using CFU-E derived from peripheral blood CD34+ cells

BACKGROUND: Nonenveloped and thermostable viruses such as parvovirus B19 (B19) can be transmitted to patients who are receiving plasma-derived coagulation factor concentrates treated by the S/D method for inactivating enveloped viruses. Therefore, it is important to develop and validate new methods for the inactivation of nonenveloped viruses. STUDY DESIGN AND METHODS: Suspensions of B19 in coagulation factor concentrates (FVIII) were irradiated with UVC light. B19 infectivity was determined by an indirect immunofluorescence assay using CFU-E, as a host cell, derived from peripheral blood CD34+ cells. The effects of catechins on B19 infectivity and on FVIII activity after UVC illumination were also examined. RESULTS: The indirect immunofluorescence assay estimated the B19 infectivity of samples containing virus copies of 10(5) to 10(11) per 10 microL to be a median tissue culture-infectious dose of 10(0.3) to 10(5.4) per 10 microL. B19 was inactivated by 3 log at 750 J per m(2) of UVC radiation and was undetectable after 1000 or 2000 J per m(2) of irradiation. However, FVIII activity decreased to 55 to 60 percent of pretreatment activity after 2000 J per m(2) of UVC radiation. This was inhibited in the presence of rutin or catechins. Epigallocatechin gallate could maintain FVIII activity at almost 100 percent of pretreatment activity after 2000 J per m(2) of UVC radiation, while B19 infectivity was decreased to undetectable levels, which resulted in >3.9 log inactivation. CONCLUSION: UVC radiation in the presence of catechins, especially epigallocatechin gallate, appears to be an effective method of increasing the viral safety of FVIII concentrates without the loss of coagulation activity.     

Distribution of HIV type 1 (HIV-1) in blood components: detection and significance of high levels of HIV-1 associated with platelets

BACKGROUND: Although inactivation of enveloped viruses transmitted by plasma derivatives has been successful, no methods for virus inactivation or removal have been established for platelet concentrates or red cell (RBC) components. Relatively little is known regarding the extent or significance of virus interactions with the cellular constituents in these components. STUDY DESIGN AND METHODS: Units of whole blood were collected from six HIV type 1 (HIV-1)-positive, asymptomatic individuals and separated into peripheral blood mononuclear cells (PBMNCs), cell-free plasma, white cell-reduced platelet concentrate, and white cell-reduced RBCs. DNA and RNA polymerase chain reaction and virus culture methods were used to study the compartmentalization of HIV-1 immediately after component preparation and after storage. RESULTS: As expected, HIV DNA and infectious virus were detected in fresh blood and in PBMNCs, and virion- associated RNA was detected in fresh plasma from all six donors. The levels of viral nucleic acids in these preparations remained relatively stable with 4 degrees C storage, whereas infectivity of PBMNCs was rapidly lost. Washed RBCs tested negative for HIV in all assays at all time points. Platelets retained high levels of HIV RNA (but not infectivity) after extensive washing, as well as after storage at 4 and 22 degrees C. High-level platelet-associated HIV-1 was also demonstrated in samples collected during early seroconversion. Periseroconversion and postseroconversion levels of platelet-associated HIV-1 correlated with the level of plasma viremia and with the rate of progression to AIDS. Cell-free virus from donor plasma and tissue culture fluid rapidly and firmly attached to platelets from noninfected donors. Infectivity of tissue culture virus bound to platelets was demonstrated in vitro. CONCLUSION: Significant levels of HIV-1 are associated with platelets during all stages of infection. Platelet- associated HIV could either mediate virus clearance or facilitate virus dissemination and expanded tropism. Finally, virus inactivation research must address virus associations with platelets.     

Resistance of porcine circovirus and chicken anemia virus to virus inactivation procedures used for blood products

BACKGROUND: Virus inactivation procedures are used to prevent contamination of plasma-derived blood products with viruses. Pasteurization or prolonged dry heat has proven effective against several enveloped and nonenveloped viruses and provides an additional layer of safety for plasma products. STUDY DESIGN AND METHODS: The resistance of porcine circovirus 2 (PCV2) and chicken anemia virus (CAV), two small, nonenveloped viruses, to standard (pasteurization, 10 hr at 60 degrees C; dry heating, 80 degrees C for 72 hr) and more extreme heat inactivation procedures (temperatures up to 120 degrees C) was determined. The ability of these procedures to inactivate PCV2 and CAV was measured by comparison of in vitro infectivity before and after treatment. RESULTS: Infectivity of PCV2 and CAV was reduced by approximately 1.6 and 1.4 log by pasteurization and by 0.75 and 1.25 log by dry-heat treatment, both substantially more resistant than other viruses previously investigated. PCV2 and CAV were additionally almost completely resistant to dry-heat treatment up to 120 degrees C for 30 minutes (mean log infectivity reductions, 1.25 and 0.6), although both were more effectively inactivated when the temperature of wet-heat treatment was increased to 80 degrees C (>3.2 and >3.6 log infectivity reduction). CONCLUSION: Although neither PCV2 nor CAV are known to infect humans, their inactivation properties may represent those of other small DNA viruses known to be present (e.g., TT virus, small anellovirus) or potentially present in human plasma. Findings of extreme thermal resistance demonstrate that recipients of plasma-derived therapeutics may potentially still be exposed to small DNA viruses, despite the implementation of viral inactivation steps.     

Étude de l’activité virucide in vitro des Gouttes aux Essences® sur le virus para-influenzae de type 3

Respiratory tract infection treatment with essential oils limits the use of antibiotics (Phytothérapie 6 (2004) 175–9). Several different viruses cause those respiratory tract infections: influenza viruses, respiratory syncytial virus, para-influenza viruses, adenoviruses and rhinoviruses... The aim of this study was to show the virucidal action of Gouttes aux Essences®, a complex of five essential oils, against para-influenza type 3 virus, an enveloped RNA virus belonging to the genus respirovirus. The assessment of the virucidal activity of essential oils was performed according to the NF EN 14476 + A1 norm (janvier 2007, phase 2, étape 1) Essential oils, at 20 and 80% dilution, in contact with parainfluenza type 3 virus during 60 minutes at 20 °C, have a significant virucidal activity. This work confirms the interest and the relevance of the respiratory tract infections in vivo treatment with essential oils, which could complete the therapeutic choice and decrease antibiotic unnecessary use.     

Inactivation by phthalocyanine photosensitization of multiple forms of human immunodeficiency virus in red cell concentrates

BACKGROUND: The use of phthalocyanines in conjunction with red light has been shown to inactivate model lipid-enveloped viruses in red cell concentrates. The ability of this treatment to inactivate multiple forms of human immunodeficiency virus (HIV) was evaluated in this study. STUDY DESIGN AND METHODS: The phthalocyanines used were aluminum phthalocyanine tetrasulfonate (AIPcS4) and the silicon phthalocyanines HOSiPcOSi(CH3)2(CH2)3 N(CH3)2 (Pc 4), and HOSiPcOSi(CH3)2 (CH2)3N+(CH3)3I-(Pc 5). HIV was studied in a cell-free form, in an actively replicating form, in latently infected cells, and in blood from HIV-positive patients. RESULTS: All three phthalocyanines inactivate > or = 10(5) infectious doses of cell-free HIV. However, only Pc 4 effectively inactivated actively replicating HIV and latently infected cells. The latter was about four times as sensitive to inactivation as was actively replicating HIV. Increasing the hematocrit of red cells during treatment decreased the rate of inactivation, especially at lower light doses. Under treatment conditions that completely inactivated the laboratory isolates of HIV, cell-associated HIV in blood from HIV-positive patients was also completely inactivated. The polymerase chain reaction signal from the gag gene of HIV was not affected on treatment of cell-free virus, but it was reduced after treatment of cell-associated HIV, particularly in some latently infected cell lines. CONCLUSION: Pc 4 and red light are effective in eliminating the infectivity of HIV in red cell concentrates. The usefulness of this approach for blood banking depends on future demonstration of the preservation of red cell circulatory survival and function in vivo.     

In vitro inactivation of Chlamydia trachomatis and of a panel of DNA (HSV-2, CMV, adenovirus, BK virus) and RNA (RSV, enterovirus) viruses by the spermicide benzalkonium chloride

Kinetics of inactivation by the detergent spermicide benzalkonium chloride (BZK) of Chlamydia trachomatis and of a panel of DNA viruses [herpes simplex virus hominis type 2 (HSV-2), cytomegalovirus (CMV), adenovirus (ADV) and BK virus (BKV)] and RNA [respiratory syncytial virus (RSV) and enterovirus (ENV)] were established in accordance with a standardized in vitro protocol. After a 5 min incubation, inactivation of >95% of HSV-2 and CMV was obtained at a concentration of 0.0025% (w/v) (25 Ig/L); concentrations as low as 0.0005%, 0.0050% and 0.0125%, induced a 3.0 log10 reduction in infectivity of HSV-2 and CMV, RSV and ADV, respectively. After a 60 min incubation, concentrations of 0.0125% and 0.050% provided a 3.0 log10 reduction in infectivity of ENV and BKV, respectively. These features indicate that sensitivity to BZK was very high (HSV-2 and CMV) or high (RSV) for enveloped viruses, intermediate (ADV) or low (ENV and BKV) for non-enveloped viruses. Furthermore, BZK had marked antichlamydial activity, showing >99% killing after only a 1 min incubation at a concentration of 0.00125%. BZK demonstrates potent in vitro activity against the majority of microorganisms causing sexually transmitted infectious diseases, including those acting as major genital cofactors of human immunodeficiency virus transmission. These attributes qualify BZK as a particularly attractive candidate for microbicide development.     

Pharmacokinetics, safety, and antiviral effects of hypericin, a derivative of St. John's wort plant, in patients with chronic hepatitis C virus infection

Hypericin is a natural derivative of the common St. Johns wort plant, Hypericum perforatum. It has in vitro activity against several viruses, including bovine diarrhea virus, a pestivirus with structural similarities to hepatitis C virus (HCV). We conducted a phase I dose escalation study to determine the safety and antiviral activity of hypericin in patients with chronic HCV infection. The first 12 patients received an 8-week course of 0.05 mg of hypericin per kg of body weight orally once a day; 7 patients received an 8-week course of 0.10 mg/kg orally once a day. At the end of the 8-week period of treatment, no subject had a change of plasma HCV RNA level of more than 1.0 log(10). Five of 12 subjects receiving the 0.05-mg/kg/day dosing schedule and 6 of 7 subjects receiving the 0.10-mg/kg/day dosing schedule developed phototoxic reactions. No other serious adverse events associated with hypericin use occurred. The pharmacokinetic data revealed a long elimination half-life (mean values of 36.1 and 33.8 h, respectively, for the doses of 0.05 and 0.1 mg/kg) and mean area under the curve determinations of 1.5 and 3.1 microg/ml x hr, respectively. In sum, hypericin given orally in doses of 0.05 and 0.10 mg/kg/d caused considerable phototoxicity and had no detectable anti-HCV activity in patients with chronic HCV infection.     

亚甲蓝光化学法灭活血制品中巨细胞病毒的研究

目的探讨亚甲蓝光化学法(MBP)灭活血制品中巨细胞病毒(HCMV)的效果。方法将HCMVAD16910%分别加入血浆、红细胞悬液中并进行MBP病毒灭活处理(MB浓度5μmol/L,光照时间1h,光照强度38000lux),分别加至人胚肺成纤维细胞(HELF)单层中培养,与对照细胞比较观察细胞病变情况(CPE)。结果经MBP病毒灭活处理后,含HCMV血浆及红细胞的组织培养半数感染量(TCID50)下降4～6。结论MBP能灭活血液中的HCMV。     

Differential sensitivities of viruses in red cell suspensions to methylene blue photosensitization

Background : Previous studies explored the feasibility of using the photosensitizer methylene blue (MB) as a virucidal agent in red cell suspensions. Under treatment conditions (5 μM [5 μmol/L] MB, 3.4 × 10(4) J/m²) that resulted in more than 6 log₁₀ inactivation of vesicular stomatitis virus (VSV) or of the enveloped bacteriophage φ6, red cell membrane alterations were observed. Increased red cell ion permeability and the binding of plasma proteins to the red cell surface were the most sensitive indicators, which varied in a dose-dependent fashion. Study Design and Methods : Inactivation of three additional extracellular viruses and intracellular human immunodeficiency virus type 1 (HIV-1) was assessed after MB phototreatment of red cell suspensions. Potassium leakage and IgG binding also were characterized in MB-treated red cell suspensions that were exposed to low-fluence light (6 × 10³ J/m²). Results : Different viruses exhibit a wide range of sensitivities to MB photoinactivation. For example, phototreatment conditions (5 μM [5 μmol/L] MB, 3.4 × 10⁴ J/m²) that inactivated more than 6 log₁₀ of VSV did not inactivate the nonenveloped picornavirus, encephalomyocarditis virus. In contrast, lower fluences (6 × 10³ J/m²) inactivated approximately 5 log₁₀ or more of Sindbis virus and approximately 4log₁₀ of extracellular HIV-1. These less stringent phototreatment conditions (5 μM [5 μmol/L] 6 × 10³ J/m²) caused lower rates of red cell potassium leakage (reduction by 6- fold) and little or no binding of plasma proteins to the red cell surface, compared to values observed previously with higher fluences. However, neither 6 × 10³ nor 4.1 × 10⁴ J per m² fluences resulted in any inactivation of intracellular HIV as represented by changes in the amount of p24 antigen produced during co-culture of actively infected H9 cells. Conclusion : MB-based protocols would require the use of high-efficiency (> 6log₁₀) white cell-reduction filters or additional inactivation steps to deplete or inactivate intracellular virus.     

Virucidal effect of myeloperoxidase on human immunodeficiency virus type 1-infected T cells.

Myeloperoxidase is virucidal to human immunodeficiency virus type 1 (HIV-1) in the persistently infected CEM human T-cell line or in acutely infected human peripheral blood mononuclear cells, as judged by viral infectivity and P24 radioimmunoassay. HIV-1 was specifically inactivated by low doses of the human myeloperoxidase (1.4 to 14.3 mU/ml) and the cells were spared. A higher enzyme concentration (143 mU/m) was cytotoxic, but uninfected CEM cells and normal lymphocytes were resistant to > or = 143 mU of myeloperoxidase per ml. The enzyme was virucidal with the Cl- present in medium and did not require exogenous H2O2. Catalase, an antioxidant enzyme, partially inhibited the virucidal effect of myeloperoxidase. Hence, the H2O2 probably came from the HIV-infected cells themselves. These in vitro findings indicate that the myeloperoxidase system is capable of inactivating HIV-1 of infected cells.     

Transmission of parvovirus B19 by coagulation factor concentrates exposed to 100 degrees C heat after lyophilization

BACKGROUND: Double inactivation by solvent/detergent treatment plus heating at 100 degrees C for 30 minutes after lyophilization has been adopted to improve viral safety of factor VIII and factor IX concentrates, particularly with respect to non-lipid-enveloped viruses. The aim of this study was to evaluate the safety of concentrates exposed to these virucidal methods. STUDY DESIGN AND METHODS: Twenty- six previously untreated hemophiliacs, 19 with factor VIII deficiency and 7 with factor IX deficiency, were investigated in a prospective multicenter study over a 12-month follow-up period by the use of serologic and virologic markers for lipid- and non-lipid-enveloped viruses (human immunodeficiency virus types 1 and 2; hepatitis A, B, and C viruses; B19 parvovirus antibodies; and B19 DNA). Overall, 270,000 U of factor VIII and 102,000 U of factor IX concentrate were administered during the study period. RESULTS: None of the 26 patients seroconverted for human immunodeficiency virus or hepatitis C virus. Hepatitis B virus markers remained negative in the 10 unvaccinated hemophiliacs. No hepatitis A virus seroconversion occurred among 17 susceptible patients. B19 seroconversion (IgM) and B19 viremia were observed within 2 weeks of the first concentrate infusion in 8 of 15 susceptible patients, 5 of 11 treated with factor VIII and 3 of 4 with factor IX concentrate. CONCLUSION: This prospective study indicates that very high temperatures applied to lyophilized concentrates appear to prevent the transmission of hepatitis A virus to hemophiliacs. However, B19 parvovirus still contaminates concentrates despite the use of this robust virucidal method.     

Process for the preparation of pathogen-inactivated RBC concentrates by using PEN110 chemistry: preclinical studies

BACKGROUND: A pathogen-inactivation process for RBC concentrates is being developed by using PEN110 chemistry (INACTINE, V.I. Technologies). The objective of this study was to characterize the quality of RBCs prepared by using the PEN110 process and to measure the virucidal effect achieved against two viruses. STUDY DESIGN AND METHODS: Virology and RBC studies were conducted with standard RBC units treated with 0.1-percent (vol/vol) PEN110 at 22 degrees C for 6 hours. The quality of PEN110-treated human RBCs was assessed with biochemical and phenotypic variables. The in vivo viability of PEN110-treated RBCs in baboons was studied with the double-label (51)Cr/(125)I method. RESULTS: Decreases in infectious titer by inactivation of greater than a 5 log 50-percent tissue culture infectious doses per mL of bovine viral diarrhea virus (an enveloped RNA virus) and porcine parvovirus (a nonenveloped DNA virus) was observed. RBC hemolysis was less than 1 percent after 42 days of storage, and no changes in RBC antigens were observed. The in vivo viability of PEN110-treated baboon RBCs was unchanged from control. CONCLUSION: The preparation of RBCs by using the PEN110 process achieved a significant viral reduction of two diverse viruses without causing adverse effects to the RBCs. The process appears to be a promising approach, thus justifying further study.     

Viral membranes: an emerging antiviral target for enveloped viruses?

Evaluation of: Wolf MC, Freiberg AN, Zhang T et al. A broad-spectrum antiviral targeting entry of enveloped viruses. Proc. Natl Acad. Sci. USA 107, 3157–3162 (2010).

The emergence and re-emergence of viruses and the widespread antiviral resistance calls for the development of a broad-spectrum strategy for viral infection. The article under review describes an approach to achieve this goal by developing an antiviral rhodanine derivative effective against enveloped viruses targeting the viral lipid membrane. By intercalating into the viral membrane, the compound irreversibly inactivates the virions with virucidal effects. Potential toxic effects on hosts could be minimized by continuous regeneration of cellular membranes. The present strategy exploits the therapeutic window that exists between static viral membranes and biogenic cellular membranes and provides a useful guideline for future research endeavors towards broad-spectrum antiviral approaches for enveloped viruses. Developing a formulation that ensures efficient delivery and pharmacokinetic properties while minimizing systemic toxicity on cell membranes remains a challenge. The advantages and disadvantages of a viral membrane-targeting approach for the control of emerging and re-emerging viruses will be discussed.     

Virus inactivation during production of intravenous immunoglobulin

The effect of pepsin treatment at pH 4 on the infectivity of several enveloped viruses was assessed under the conditions used during the production of intravenous immunoglobulins. It was shown that the prototypes of four virus families–human immunodeficiency virus (Lentivirinae), herpes simplex virus type 1 and human cytomegalovirus (Herpesviridae), Semliki Forest virus (Togaviridae), and vesicular stomatitis virus (Rhabdoviridae)–were inactivated by this procedure. With vesicular stomatitis virus as a model, the contributions of both low pH and pepsin were demonstrated, and pepsin had a synergistic or additive action.