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Severe acute respiratory syndrome virus 2 (SARS-CoV-2) is the cause of the current coronavirus disease 19 (COVID-19) pandemic. Protease inhibitors are under consideration as virus entry inhibitors that prevent the cleavage of the coronavirus spike (S) protein by cellular proteases. Herein, we showed that the protease inhibitor aprotinin (but not the protease inhibitor SERPINA1/alpha-1 antitrypsin) inhibited SARS-CoV-2 replication in therapeutically achievable concentrations. An analysis of proteomics and translatome data indicated that SARS-CoV-2 replication is associated with a downregulation of host cell protease inhibitors. Hence, aprotinin may compensate for downregulated host cell proteases during later virus replication cycles. Aprotinin displayed anti-SARS-CoV-2 activity in different cell types (Caco2, Calu-3, and primary bronchial epithelial cell air-liquid interface cultures) and against four virus isolates. In conclusion, therapeutic aprotinin concentrations exert anti-SARS-CoV-2 activity. An approved aprotinin aerosol may have potential for the early local control of SARS-CoV-2 replication and the prevention of COVID-19 progression to a severe, systemic disease.

From a panel of 22 flavonoids, we identified six compounds (apigenin, baicalein, biochanin A, kaempferol, luteolin, naringenin) that inhibited influenza A nucleoprotein production in human lung epithelial (A549) cells infected with the highly pathogenic avian influenza H5N1 virus strain A/Thailand/Kan-1/04 in non-toxic concentrations. Baicalein (IC(50): 18.79±1.17μM, selectivity index 5.82) and biochanin A (IC(50) 8.92±1.87μM, selectivity index 5.60) were selected for further experiments. Both compounds reduced H5N1 infectious titres (baicalein 40μM: 29-fold reduction, biochanin A 40μM: 55-fold reduction after infection at MOI 0.01), virus-induced caspase 3 cleavage, nuclear export of viral RNP complexes, and enhanced the effects of the neuraminidase inhibitor zanamivir. Biochanin A and baicalein also inhibited the replication of the H5N1 strain A/Vietnam/1203/04. Time of addition experiments indicated that both compounds interfere with H5N1 replication after the adsorption period. Further mechanistic investigations revealed clear differences between these two flavonoids. Only baicalein interfered with the viral neuraminidase activity (39±7% inhibition at 100μM, the maximum concentration tested). In contrast to baicalein, biochanin A affected cellular signalling pathways resulting in reduced virus-induced activation of AKT, ERK 1/2, and NF-kB. Moreover, biochanin A inhibited the virus-induced production of IL-6, IL-8, and IP-10 while baicalein inhibited IL-6 and IL-8 production without affecting IP-10 levels. In primary human monocyte-derived macrophages, only baicalein but not biochanin A impaired H5N1 virus replication. Both flavonoids interfered with the H5N1-induced production of IL-6, IP-10, and TNF-α but not of IL-8 in macrophages. These findings indicate that closely related flavonoids can exert anti-H5N1 effects by different molecular mechanisms., (Copyright © 2012 Elsevier B.V. All rights reserved.)

The Pelargonium sidoides extract EPs® 7630 is an approved drug for the treatment of acute bronchitis in Germany. The postulated mechanisms underlying beneficial effects of EPs® 7630 in bronchitis patients include immunomodulatory and cytoprotective effects, inhibition of interaction between bacteria and host cells, and increase of cilliary beat frequency on respiratory cells. Here, we investigated the influence of EPs® 7630 on replication of a panel of respiratory viruses. Determination of virus-induced cytopathogenic effects and virus titres revealed that EPs® 7630 at concentrations up to 100 μg/ml interfered with replication of seasonal influenza A virus strains (H1N1, H3N2), respiratory syncytial virus, human coronavirus, parainfluenza virus, and coxsackie virus but did not affect replication of highly pathogenic avian influenza A virus (H5N1), adenovirus, or rhinovirus. Therefore, antiviral effects may contribute to the beneficial effects exerted by EPs® 7630 in acute bronchitis patients., (Copyright © 2010 Elsevier GmbH. All rights reserved.)

Human cytomegalovirus (HCMV) is a major pathogen in immunocompromised individuals. Here, non-toxic concentrations of the anti-cancer kinase inhibitor sorafenib were shown to inhibit replication of different HCMV strains (including a ganciclovir-resistant strain) in different cell types. In contrast to established anti-HCMV drugs, sorafenib inhibited HCMV major immediate early promoter activity and HCMV immediate early antigen (IEA) expression. Sorafenib is known to inhibit Raf. Comparison of sorafenib with the MEK inhibitor U0126 suggested that sorafenib inhibits HCMV IEA expression through inhibition of Raf but independently of signaling through the Raf downstream kinase MEK 1/2. In concordance, siRNA-mediated depletion of Raf but not of MEK-reduced IEA expression. In conclusion, sorafenib diminished HCMV replication in clinically relevant concentrations and inhibited HCMV IEA expression, a pathophysiologically relevant event that is not affected by established anti-HCMV drugs. Moreover, we demonstrated for the first time that Raf activation is involved in HCMV IEA expression.

Glycyrrhizin is known to exert antiviral and anti-inflammatory effects. Here, the effects of an approved parenteral glycyrrhizin preparation (Stronger Neo-Minophafen C) were investigated on highly pathogenic influenza A H5N1 virus replication, H5N1-induced apoptosis, and H5N1-induced pro-inflammatory responses in lung epithelial (A549) cells. Therapeutic glycyrrhizin concentrations substantially inhibited H5N1-induced expression of the pro-inflammatory molecules CXCL10, interleukin 6, CCL2, and CCL5 (effective glycyrrhizin concentrations 25 to 50 µg/ml) but interfered with H5N1 replication and H5N1-induced apoptosis to a lesser extent (effective glycyrrhizin concentrations 100 µg/ml or higher). Glycyrrhizin also diminished monocyte migration towards supernatants of H5N1-infected A549 cells. The mechanism by which glycyrrhizin interferes with H5N1 replication and H5N1-induced pro-inflammatory gene expression includes inhibition of H5N1-induced formation of reactive oxygen species and (in turn) reduced activation of NFκB, JNK, and p38, redox-sensitive signalling events known to be relevant for influenza A virus replication. Therefore, glycyrrhizin may complement the arsenal of potential drugs for the treatment of H5N1 disease.

The antioxidant N-acetyl-L-cysteine (NAC) had been shown to inhibit replication of seasonal human influenza A viruses. Here, the effects of NAC on virus replication, virus-induced pro-inflammatory responses and virus-induced apoptosis were investigated in H5N1-infected lung epithelial (A549) cells. NAC at concentrations ranging from 5 to 15 mM reduced H5N1-induced cytopathogenic effects (CPEs), virus-induced apoptosis and infectious viral yields 24 h post-infection. NAC also decreased the production of pro-inflammatory molecules (CXCL8, CXCL10, CCL5 and interleukin-6 (IL-6)) in H5N1-infected A549 cells and reduced monocyte migration towards supernatants of H5N1-infected A549 cells. The antiviral and anti-inflammatory mechanisms of NAC included inhibition of activation of oxidant sensitive pathways including transcription factor NF-kappaB and mitogen activated protein kinase p38. Pharmacological inhibitors of NF-kappaB (BAY 11-7085) or p38 (SB203580) exerted similar effects like those determined for NAC in H5N1-infected cells. The combination of BAY 11-7085 and SB203580 resulted in increased inhibitory effects on virus replication and production of pro-inflammatory molecules relative to either single treatment. NAC inhibits H5N1 replication and H5N1-induced production of pro-inflammatory molecules. Therefore, antioxidants like NAC represent a potential additional treatment option that could be considered in the case of an influenza A virus pandemic.

Purpose: Ocular involvement in influenza A virus diseases is common but usually limited to mild conjunctivitis. Rarely, inflammation of the choriocapillaris may result in atrophia of the retinal pigment epithelium (RPE). Primary human retinal pigment epithelial (RPE) cells were infected with seasonal (H1N1 A/New Caledonia/20/99, H3N2 A/California/7/2004) or highly pathogenic avian H5N1 (A/Thailand/1(Kan-1)/04, A/Vietnam/1203/04, A/Vietnam/1194/04) influenza strains., Methods: Influenza A virus replication was studied by investigation of cytopathogenic effects, immune staining for influenza A virus nucleoprotein, determination of virus titers, and electron microscopy. Apoptosis induction was examined by immune staining for activated caspase 3 and cleaved PARP. Proinflammatory gene expression was investigated by quantitative PCR., Results: H5N1 but not seasonal influenza strains replicated to high titers (>10(8) TCID(50)/mL; 50% tissue culture infectious dose/milliliter) in RPE cells. H5N1 infection resulted in RPE cell apoptosis that was abolished by the antiviral drug ribavirin. Pretreatment with type I interferons (interferon-alpha and -beta) or the type II interferon, (interferon-gamma), inhibited H5N1 replication. Moreover, H5N1 infection induced expression of proinflammatory genes (tumor necrosis factor-alpha, CXCL8, CXCL10, CXCL11, and interleukin-6), which was inhibited by ribavirin in a concentration-dependent manner., Conclusions: A novel cell type derived from the central nervous system was permissive to H5N1 influenza virus replication. This findings supports those suggesting H5N1 influenza strains to own a greater potential to spread to nonrespiratory tissues than seasonal human influenza viruses. Moreover, the data warrant the further study of the role of influenza A virus replication in retinal diseases associated with influenza A virus infections.

Aim of the Study: Severe acute respiratory syndrome (SARS) is a life-threatening disease caused by the SARS coronavirus (SARS-CoV). The development of new antiviral agents for SARS-CoV is an important issue. We tried to find potential resource from Traditional Chinese medicine (TCM) for development of new drugs against SARS-CoV., Materials and Methods: Our team recruited the potential TCM formulae (also known as Kampo) from two TCM books, Shang-Han Lun (Discussion of Cold-Induced Disorders) and Wen-Bing Tiau-Bein (Differential Management of Febrile Diseases). Several herbs, which were believed to be beneficial for SARS by experienced TCM doctors were also recruited. In addition, a vegetable polular in Taiwan, China and Malaysia, the tender leaf of Toona sinensis Roem (also known as Cedrela sinensis, belongs to the family Meliacceae) was also recruited under the suggestion of botanic experts. These TCM products and plant extrats were then tested for the effectiveness against SARS-CoV in vitro., Results: Only TSL-1, the extract from tender leaf of Toona sinensis Roem was found to have an evident effect against SARS-CoV with selectivity index 12 approximately 17., Conclusion: This paper reports for the first time that extract from a vegetable, the tender leaf of Toona sinensis Roem, can inhibit SARS-CoV in vitro. Thererfore, the tender leaf of Toona sinensis Roem may be an important resource agninst SARS-CoV.

Here we describe for the first time the productive in vitro infection of human retinal pigment epithelial cells by varicella-zoster virus (VZV), resulting in a typical cytopathic effect (CPE) that is characterized by enlarged cells with increased granularity. Depending on the CPE dissemination, high titers of up to 1.6 x 10(6) PFU of cell-free and cryostable VZV/ml can be recovered.

Cedrus libani are widely used as traditional medicine in Lebanon for treatment of different infection diseases. In the present study we reported the phytochemical composition analyzed by GC-MS of wood essential oil and cones and leaves ethanol extracts. The main components of wood essential oil were himachalol (22.50%), beta-himachalene (21.90%), and alpha-himachalene (10.50%). Leaves ethanol extract was characterized by a high content of germacrene d (29.40%). The same extract obtained from cones essentially contained alpha-pinene (51.0%) and beta-myrcene (13.0%). Moreover, we investigated extracts, essential oil, and identified compounds for their in vitro antiviral activities against herpes simplex virus type 1 (HSV-1). Cytotoxicity was evaluated by MTT assay in Vero cells. Cones and leaves ethanol extracts exhibited an interesting activity with IC50 of 0.50 and 0.66 mg/ml, respectively, at non-cytotoxic concentration. A comparable activity was found when essential oil was tested (IC50 of 0.44 mg/ml).

Objectives: West Nile virus (WNV) infection causes severe meningitis and encephalitis in a subset of patients. WNV-induced apoptosis has been suggested to contribute to WNV pathogenesis. Tetracyclines exert antiviral effects against HIV and inhibit apoptosis in different models of neuronal disease. Here, the effects of the tetracyclines minocycline, demeclocycline and chlortetracycline were observed on WNV replication and WNV-induced apoptosis in different human CNS-derived cell types (primary human brain neurons, primary human retinal pigment epithelial cells and T98G human glioma cell line)., Methods: WNV replication was studied by cytopathic effects and virus yield reduction assay. Cell viability was examined by MTT assay. Apoptosis was investigated by immunostaining for activated caspase 3 and cleaved poly(ADP-ribose) polymerase. Expression and phosphorylation of cellular proteins were examined by western blot., Results: Minocycline exerted the strongest anti-WNV activity. Non-toxic minocycline concentrations that can be achieved in human tissues significantly reduced WNV titres in all cell types tested. Minocycline inhibited WNV-induced apoptosis and suppressed virus-induced activation of c-Jun N-terminal kinase (JNK) and its target c-jun. The JNK inhibitor L-JNKi exerted similar effects to minocycline., Conclusions: These data suggest that minocycline-induced inhibition of JNK activation contributes to minocycline-induced inhibition of WNV replication and WNV-induced apoptosis. Minocycline is a clinically available, inexpensive and generally very well-tolerated drug. It could be readily evaluated for the treatment of humans with serious WNV infection.

Purpose: Human cytomegalovirus (HCMV) replication depends on different cellular pathways, including histone acetylation and extracellular-signal regulated kinases 1 and 2 (Erk 1/2). In the present study, the influence of therapeutic valproic acid (VPA) concentrations was investigated on HCMV replication in retinal pigment epithelial (RPE) cells., Methods: HCMV antigen expression and replication were detected by immunostaining, real-time RT-PCR, and determination of virus titers. Histone acetylation and Erk 1/2 phosphorylation were detected by Western blot., Results: Pretreatment with VPA < or =1 mM enhanced HCMV antigen expression and replication by up to ninefold. In addition to histone deacetylase (HDAC) inhibition, VPA stimulated Erk 1/2 phosphorylation in RPE cells. Investigation of six VPA derivatives revealed that S-2-pentyl-4-pentynoic acid was the only derivative that induced histone hyperacetylation, indicating HDAC inhibition, in the observed concentrations < or =1 mM and that increased HCMV antigen expression. Other derivatives did not enhance HCMV replication in the tested concentrations, although some were found to induce Erk 1/2 phosphorylation. The mitogen-activated protein kinase kinase (MEK) inhibitor PD98059 inhibited VPA-induced Erk 1/2 phosphorylation but did not affect VPA-induced increased HCMV replication. In addition, the structurally nonrelated HDACI trichostatin A enhanced HCMV replication but did not affect Erk 1/2 phosphorylation in RPE cells., Conclusions: The data demonstrate that VPA stimulates HCMV replication by HDAC inhibition independent of Erk 1/2 phosphorylation in therapeutic concentrations in RPE cells. Therefore, patients at risk of HCMV retinitis who are treated with VPA or other HDAC inhibitors should be carefully monitored.

Initial in vitro investigations demonstrated type I interferons (IFNs: IFN-alpha, IFN-beta) to inhibit replication of SARS coronavirus (SARS-CoV), but found the nucleoside analogue ribavirin ineffective in Vero cells. In this report, ribavirin was shown to inhibit SARS-CoV replication in five different cell types of animal or human origin at therapeutically achievable concentrations. Since clinical anti-SARS-CoV activity of type I interferons or ribavirin is limited, we investigated the combination of IFN-beta and ribavirin. Determination of the virus yield indicated highly synergistic anti-SARS-CoV action of the combination suggesting the consideration of ribavirin plus IFN-beta for the treatment of SARS.

Valproic acid (2-propylpentanoic acid, VPA), an effective inhibitor of histone deacetylases (HDAC) is used for the treatment of epilepsia. In this study, structure-activity relationships for the action of structurally modified VPA derivatives on human cytomegalovirus (HCMV) replication and HDAC inhibition were defined. Pretreatment of human foreskin fibroblasts with VPA (0.125-1mM) caused a concentration-dependent increase of HCMV immediate early and antigen late antigen expression. Structure-activity relationships of VPA derivatives for HCMV stimulation were compared to those for teratogenic action and those for HDAC inhibition. Side chain elongation and introduction of a triple bond in 4-position of the other chain caused teratogenicity, stimulated HCMV replication, and increased HDAC inhibition, as demonstrated by enhanced levels of acetylated histones. Teratogenic VPA derivatives with a branched chain in 3-position as well as a non-teratogenic anticonvulsive active VPA derivative did not stimulate HCMV or accumulation of acetylated histones. This demonstrates a strict correlation between inhibition of HDAC and increased HCMV replication.

A novel coronavirus has been identified as an etiological agent of severe acute respiratory syndrome (SARS). To rapidly identify anti-SARS drugs available for clinical use, we screened a set of compounds that included antiviral drugs already in wide use. Here we report that the HIV-1 protease inhibitor, nelfinavir, strongly inhibited replication of the SARS coronavirus (SARS-CoV). Nelfinavir inhibited the cytopathic effect induced by SARS-CoV infection. Expression of viral antigens was much lower in infected cells treated with nelfinavir than in untreated infected cells. Quantitative RT-PCR analysis showed that nelfinavir could decrease the production of virions from Vero cells. Experiments with various timings of drug addition revealed that nelfinavir exerted its effect not at the entry step, but at the post-entry step of SARS-CoV infection. Our results suggest that nelfinavir should be examined clinically for the treatment of SARS and has potential as a good lead compound for designing anti-SARS drugs.

The outbreak of SARS warrants the search for antiviral compounds to treat the disease. At present, no specific treatment has been identified for SARS-associated coronavirus infection. We assessed the antiviral potential of ribavirin, 6-azauridine, pyrazofurin, mycophenolic acid, and glycyrrhizin against two clinical isolates of coronavirus (FFM-1 and FFM-2) from patients with SARS admitted to the clinical centre of Frankfurt University, Germany. Of all the compounds, glycyrrhizin was the most active in inhibiting replication of the SARS-associated virus. Our findings suggest that glycyrrhizin should be assessed for treatment of SARS.

The biological evaluation of mononucleotide prodrugs (pronucleotides) of various nucleoside reverse transcriptase inhibitors (NRTIs) such as zidovudine (AZT), zalcitabine (ddC) and lamivudine (3TC) was reported in human T-lymphoid MOLT-4/8 cells which were grown continuously for more than 1 year in a medium containing cytarabine (Ara-C). In this cell line, expression of deoxycytidine kinase (dCK) and thymidine kinase 1 (TK1) was decreased in comparison to parental cells (3.8 and 2.9-fold, respectively). The lower mRNA level of TK1 correlated significantly with lower enzyme activity, whereas no dCK activity was detectable. In Ara-C-resistant cells, anti-HIV-1 effects of ddC, 3TC and AZT were more than 100-fold lower compared with parental cells. In contrast, the corresponding mononucleoside phosphotriesters bearing S-acyl-2-thioethyl (SATE) groups as biolabile phosphate protection retained anti-HIV-1 activity due to their ability to bypass the first monophosphorylation step catalyzed by dCK or TK1. The results demonstrate that in vitro selection of T-lymphoid cells in the presence of Ara-C results in cross-resistance to deoxycytidine (ddC, 3TC) and thymidine (AZT) analogs and that these cellular resistance mechanisms can be bypassed by the use of bis(SATE) pronucleotides.

Cytomegalovirus (CMV) infection is a major problem in the immunosuppressed patient. It is thought that besides direct CMV induced cell lysis, immunological damage is part of CMV pathogenesis. New antiviral drugs, which combine immunomodulating and antiviral qualities, could be beneficial. Recently, it has been described that desferrioxamine (DFO) and calcium trinatrium diethylenetriaminepentaacetic acid (DTPA) exhibit both properties. In this report the antiviral effects of both compounds against rat CMV (RCMV) are described in vitro and in vivo using a generalised and local infection model. In vitro, both compounds exhibited a significant antiviral effect, DTPA being more potent than DFO. However, in the generalised infection model no effect was seen on mortality, morbidity or presence of virus in internal organs. In rats infected subcutaneously in the hind paw, no effect was seen locally on paw thickness, presence of viral antigens and inflammatory response. In addition, these rats suffered from a generalised infection of low magnitude at 15 days post infection, although both DFO and DTPA were able to lower the level of viral replication. In conclusion, our data indicate that despite in vitro activity, in vivo usage of DFO or DTPA for acute CMV infection is not warranted.

Transplantation-related pathogenic factors such as ischemia or allograft-directed inflammation are associated with oxidative changes that might lead to cellular oxidative stress. The aim of this study was to investigate the impact of oxidative stress on: (1) CMV replication in cultured human endothelial cells and (2) the stimulation of endothelial cells by proinfiammatory cytokines. Both pathomechanisms are known to contribute to graft rejection crises in vivo. Oxidative stress was induced in endothelial cell cultures with 10-200 microM buthionine sulfoximine. Western blotting showed a significant increase in the production of CMV-specific immediate early and late proteins in buthionine sulfoximine-treated cultures. Immunocytochemical staining suggested that this effect was caused by increased numbers of CMV antigen expressing cells (66% immediate early; 78%, late). Quantitative polymerase chain reaction for CMV-specific DNA and virus titration revealed that enhanced viral replication levels correlated with increased virion production. As a measure for the endothelial cell activation status, the surface expression of HLA-ABC and HLA-DR and adhesion molecules (ICAM-1, ELAM-1, VCAM-1) was quantified by fluorometric methods. Whereas oxidative stress alone did not modulate any surface molecule expression, the IFN-gamma-mediated expression of HLA-ABC and HLA-DR and the IL-1-mediated expression of ICAM-1, but not of ELAM-1 and VCAM-1 (IL-1 + TNF-alpha), was amplified. Interestingly, the amplification of HLA molecule expression was even higher in CMV-infected endothelial cells. This study provides evidence that oxidative stress contributes to the regulation of CMV replication, virus shedding, and the activation of endothelial cells by proinflammatory cytokines as it is observed in transplant recipients.

Unlabelled: In AIDS patients cytomegalovirus (HCMV) retinitis is one of the most frequent opportunistic infections. Antiviral therapy aims at preserving vision as long as possible, but more and more HCMV isolates are proving to be resistant to ganciclovir (GCV) and foscarnet (PFA) in vitro. We tested whether desferrioxamine (DFO), an iron chelator with antiherpetic activity, can inhibit clinical virus isolates and laboratory strains. Clinical isolates were obtained from urine samples of AIDS patients with HCMV retinitis. The concentrations of DFO required for 50% and 90% reduction of the production of HCMV in several HCMV strains ranged from 3.1 to 4.9 microns and from 14.2 to 17.3 microns, respectively, Inhibitory effects of DFO on HCMV replication were completely prevented by co-incubation with stoichiometric amounts of FE3+., Case Reports: DFO was administered by daily dose of 1 g i.v. to a patient with AIDS in whom HCMV retinitis continued to progress despite combination therapy with GCV and PFA. The addition of DFO to the combination inhibited progression of the disease. No relapse of HCMV retinitis was seen with 3 months of DFO therapy. The treatment was free of side effects.

The human T-cell line JJHan, commonly used as a substrate for human herpesvirus type 6 (HHV-6) replication, was established in a protein-free 1:1 mixture of Ham's F12 and Iscove's modified Dulbecco's medium. After 250 passages (4 years) in protein-free medium, the cells, designated JJHan-PF, were cocultured with JJHan cells chronically infected with strain AJ of HHV-6 conventionally grown in serum-supplemented medium. The production of viral antigen was monitored by indirect immunofluorescent antibody staining. The infectivity of the virus in JJHan-PF cells was similar to that in JJHan cultures as shown by HHV-6 antigen detection using a convalescent phase serum. The results show that JJHan-PF cells provide a sensitive substrate for HHV-6 replication without the interference of serum or other proteins usually added to the culture medium.

The replication of human cytomegalovirus (HCMV) was investigated in a new human rhabdomyosarcoma cell line (KFR) with morphological and biochemical characteristics of fetal striated muscle precursors (rhabdomyoblasts). KFR cells exhibited the unique property for spontaneous morphological transformation from a poorly-differentiated state into well-differentiated (myotube-like) rhabdomyoblasts. The poorly-differentiated rhabdomyoblasts promoted both complete viral gene expression and the production of infectious virus. In contrast, in well-differentiated rhabdomyoblasts HCMV infection was abortive. The results showed that replication of HCMV in this human rhabdomyosarcoma cell line depended on the state of cellular differentiation.

A protein-free chemically defined medium designated PFEK-1 was developed for culture of VERO cells on polyvinyl formal (PVF) culture surface without serum or other macromolecular supplements. VERO cells proliferated in PFEK-1 medium on PVF surface to a similar extent as cells in serum-supplemented medium without previous adaptation from serum-containing conditions. The protein-free culture infected with coxsackievirus B4, herpes simplex virus types 1 and 2, measles virus and poliovirus types 1, 2 and 3 developed viral titers comparable to those found in conventionally grown cells. The results demonstrated that VERO cells in protein-free culture provide a sensitive substrate for the production of human pathogenic viruses which are not contaminated by serum or other protein factors usually added to a culture medium.

Human carcinoma of the cervix cell line HeLa, adapted to continuous growth in a protein-free chemically defined medium, was used as substrate for the replication of several human pathogenic viruses. Growth characteristics of the cells designated as HeLa-PF in protein-free 1:1 nutrient mixture of Dulbecco's modified MEM and Ham's F-12 supplemented with L-ascorbic acid 2-phosphate were similar to those of the cells grown in a serum-supplemented medium. After 30 months (135 subcultures) in the protein-free medium, HeLa-PF cells were infected with poliovirus types 1, 2 and 3; adenovirus types 2 and 5 and herpes simplex virus type 1. Both adenoviruses and polioviruses developed in HeLa-PF cells titers and showed cytopathic effects comparable to those obtained in conventionally grown and maintained cells; in contrast, significantly lower herpes simplex virus type 1 titers and changed characteristics of the cytopathic effects were observed in HeLa-PF cells. The results show that HeLa-PF cells grown continuously in protein-free medium provide a unique system for the study of virus replication.