A recombinant virus between the Sabin 1 and Sabin 3 vaccine strains of poliovirus as a possible candidate for a new type 3 poliovirus live vaccine strain.

Biological tests including the monkey neurovirulence test performed on recombinants between the virulent Mahoney and attenuated Sabin 1 strains of type 1 poliovirus indicated that the genome region encoding mainly the viral capsid proteins had little correlation with the neurovirulence or attenuation phenotype of the virus. The results suggested that new vaccine strains of type 2 and type 3 polioviruses may be constructed in vitro by replacing the sequence encoding the antigenic determinants in viral capsid proteins of the Sabin 1 genome by the corresponding sequences of the type 2 and type 3 genome, respectively. Accordingly, we constructed recombinants between the Sabin 1 and Sabin 3 strains of poliovirus in which genome sequences of the Sabin 1 strain encoding most or all capsid proteins were replaced by the corresponding genome sequences of the Sabin 3 strain. One of the recombinant viruses thus constructed was fully viable and showed antigenicity and immunogenicity identical to those of type 3 poliovirus. The monkey neurovirulence tests and in vitro phenotypic marker tests (temperature sensitivity of growth, sodium bicarbonate concentration dependency of growth under agar overlay, and size of plaque) were performed on the recombinant virus. The stability of the virus in regard to the temperature sensitivity phenotype was also tested. The results suggested that the recombinant virus is a possible candidate for a new type 3 poliovirus vaccine strain.     

Long-term survival of hepatitis A virus and poliovirus type 1 in mineral water.

The survival in mineral water of hepatitis A virus (HAV) and poliovirus type 1 was compared, under controlled experimental conditions, at 4 degrees C and room temperature. Viral infectivity titers were determined by cell culture titration, while HAV antigenicity was monitored by radioimmunoassay-endpoint titration. Both viruses persisted longest at 4 degrees C. At this temperature, after 1 year of exposure, the inactivation of either HAV or poliovirus type 1 was not important. At room temperature, poliovirus type 1 was not detected after 300 days, whereas HAV was still infectious. For both temperatures, the computed regression coefficients of best-fit lines for inactivation rates for the two viruses were significantly different. The survival of HAV was also studied at 4 degrees C and room temperature in mineral water with 5- and 50-micrograms/ml protein concentrations (i.e., purity of the virus suspension) for 120 days. As shown by a comparison of the regression coefficients for the inactivation rates, the stability of HAV in mineral water depends on protein concentration and temperature. Radioimmunoassay-endpoint titration results showed inactivation patterns similar to those of cell culture titration, with the most significant reduction in HAV antigenicity at room temperature. At the two temperatures, the infectivity of HAV declined at a faster rate than the antigenicity.     

Comparison of the airborne survival of calf rotavirus and poliovirus type 1 (Sabin) aerosolized as a mixture

A mixture of a cell culture-adapted strain (C-486) of calf rotavirus and poliovirus type 1 (Sabin) was prepared in tryptose phosphate broth containing 0.1% uranine (physical tracer) and antifoam at a final concentration of 0.001%. By using a six-jet Collison nebulizer, the mixture was aerosolized into a 300-liter stainless-steel rotating (4 rpm) drum. The temperature of the air inside the drum was kept at 20 +/- 1 degrees C, and the virus aerosols were held at the following three levels of relative humidity (RH): low (30 +/- 5%), medium (50 +/- 5%), and high (80 +/- 5%). An all-glass impinger, containing 10.0 ml of tryptose phosphate broth with antifoam, was used to collect samples of air from the drum. Both viruses were propagated and quantitated in MA-104 cells. The calf rotavirus was found to survive well at mid-range RH, where 60% of the infectious virus could be detected even after 24 h of virus aerosolization. At the low RH, the half-life of the infectious rotavirus was ca. 14 h. On the other hand, no infectious poliovirus could be recovered from the drum air at the low and medium RH. At the high RH, more than 50% of the infectious rotavirus became undetectable within 90 min of aerosolization. In contrast to this, the half-life of the poliovirus at the high RH was about 10 h. These data, based on the aerosolization of virus mixtures, therefore suggest that there is a pronounced difference in the way RH influences the airborne survival of these two types of viruses held under identical experimental conditions.     

Determinants of attenuation and temperature sensitivity in the type 1 poliovirus Sabin vaccine.

To identify determinants of attenuation in the poliovirus type 1 Sabin vaccine strain, a series of recombinant viruses were constructed by using infectious cDNA clones of the virulent type 1 poliovirus P1/Mahoney and the attenuated type 1 vaccine strain P1/Sabin. Intracerebral inoculation of these viruses into transgenic mice which express the human receptor for poliovirus identified regions of the genome that conferred reduced neurovirulence. Exchange of smaller restriction fragments and site-directed mutagenesis were used to identify the nucleotide changes responsible for attenuation. P1/Sabin mutations at nucleotides 935 of VP4, 2438 of VP3, and 2795 and 2879 of VP1 were all shown to be determinants of attenuation. The recombinant viruses and site-directed mutants were also used to identify the nucleotide changes which are involved in the temperature sensitivity of P1/Sabin. Determinants of this phenotype in HeLa cells were mapped to changes at nucleotides 935 of VP4, 2438 of VP3, and 2741 of VP1. The 3Dpol gene of P1/Sabin, which contains three amino acid differences from its parent P1/Mahoney, also contributes to the temperature sensitivity of P1/Sabin; however, mutants containing individual amino acid changes grew as well as P1/Mahoney at elevated temperatures, suggesting that either some combination or all three changes are required for temperature sensitivity. In addition, the 3'-noncoding region of P1/Sabin augments the temperature-sensitive phenotype conferred by 3Dpol. Although nucleotide 2741, 3Dpol, and the 3'-noncoding region of P1/Sabin contribute to the temperature sensitivity of P1/Sabin, they do not contribute to attenuation in transgenic mice expressing the poliovirus receptor, demonstrating that determinants of attenuation and temperature sensitivity can be genetically separated.     

Comparison of the airborne survival of calf rotavirus and poliovirus type 1 (Sabin) aerosolized as a mixture.

A mixture of a cell culture-adapted strain (C-486) of calf rotavirus and poliovirus type 1 (Sabin) was prepared in tryptose phosphate broth containing 0.1% uranine (physical tracer) and antifoam at a final concentration of 0.001%. By using a six-jet Collison nebulizer, the mixture was aerosolized into a 300-liter stainless-steel rotating (4 rpm) drum. The temperature of the air inside the drum was kept at 20 +/- 1 degrees C, and the virus aerosols were held at the following three levels of relative humidity (RH): low (30 +/- 5%), medium (50 +/- 5%), and high (80 +/- 5%). An all-glass impinger, containing 10.0 ml of tryptose phosphate broth with antifoam, was used to collect samples of air from the drum. Both viruses were propagated and quantitated in MA-104 cells. The calf rotavirus was found to survive well at mid-range RH, where 60% of the infectious virus could be detected even after 24 h of virus aerosolization. At the low RH, the half-life of the infectious rotavirus was ca. 14 h. On the other hand, no infectious poliovirus could be recovered from the drum air at the low and medium RH. At the high RH, more than 50% of the infectious rotavirus became undetectable within 90 min of aerosolization. In contrast to this, the half-life of the poliovirus at the high RH was about 10 h. These data, based on the aerosolization of virus mixtures, therefore suggest that there is a pronounced difference in the way RH influences the airborne survival of these two types of viruses held under identical experimental conditions.     

Comparative sensitivities of Sabin and Mahoney poliovirus type 1 prototype strains and two recent isolates to low concentrations of glutaraldehyde.

Significant intratypic differences in the glutaraldehyde (GTA) sensitivity of echovirus isolates have been shown. While exploring ways to optimize the study of GTA sensitivity of enteroviruses, we also observed intratypic differences in poliovirus type 1 isolates collected in France. A suspension procedure was used for assessing the virucidal effect of GTA at low concentrations (< or = 0.10%) against purified viruses. Two recent isolates of poliovirus type 1 tested were first fully characterized by the PCR restriction fragment length polymorphism (RFLP) test. The RFLP pattern of clinical isolate 5617 was similar to that of poliovirus type 1 LS-c, 2ab (Sabin strain), confirming the vaccine origin of strain 5617. The RFLP pattern of strain 5915 recovered from sewage was different from that of the Mahoney strain, suggesting a genetic variation in this wild isolate. We then analyzed under the same controlled conditions the GTA sensitivities of both isolates and their respective prototype strains. The wild Mahoney and 5915 strains exhibited significantly lower sensitivities to GTA than did the vaccine Sabin and 5617 strains. The inactivation rates of clinical isolates 5617 and 5915 were very similar to those of their corresponding reference Sabin and Mahoney strains. Both the conformational structure of the capsid of each strain and the amino acid constitution of structural polypeptides could be involved in the variations observed. The relevance of our comparative sensitivity studies to standardization of virucidal tests is discussed.     

Comparison of talc-Celite and polyelectrolyte 60 in virus recovery from sewage: development of technique and experiments with poliovirus (type 1, Sabin)-contaminated multilitre samples.

For virus recovery from sewage, a mixture of talc and Celite was tested as a possible inexpensive substitute for polyelectrolyte 60 (PE 60). After adjustment of pH to 6 and the addition of 45-60 plaque forming units (PFU)/ml of poliovirus type I (Sabin) to the sewage sample under test, 100 ml of it was passed through either a PE 60 (400 mg) or a talc (300 mg)-Celite (100 mg) layer; the layer-adsorbed virus was eluted with 10 ml of 10% fetal calf serum (FCS) in saline (pH 7.2). In these experiments, PE 60 layers recovered 73-80% (mean 76%) of the input virus. In comparison, virus recoveries with the talc-Celite layers were 65-70% (mean 68%). Passage of 5 litres of raw sewage (containing 50 to 1.26 X 10(5) PFU/100 ml of the poliovirus) through the talc (15 g)-Celite (5 g) layers and virus elution with 50 ml of 10% FCS in saline gave virus recoveries of 33-63% (mean 49%). Except for pH adjustment and prefiltration through two layers of gauze to remove large solids, no other sample pretreatment was found to be necessary. Application of this technique to recovery of indigenous viruses from field samples of raw sewage and effluents has been highly satisfactory.     

Role of mutations G-480 and C-6203 in the attenuation phenotype of Sabin type 1 poliovirus.

Of the 55 point mutations which distinguish the type 1 poliovirus vaccine strain (Sabin 1) from its neurovirulent progenitor (P1/Mahoney), two have been strongly implicated by previous studies as determinants of the attenuation phenotype. A change of an A to a G at position 480, located within the 5' noncoding region, has been suggested to be the major attenuating mutation, analogous to the mutations at positions 481 and 472 in poliovirus types 2 and 3, respectively. In addition, the change of a U to a C at position 6203, resulting in an amino acid change in the polymerase protein 3D, has also been implicated as a determinant of attenuation, albeit to a lesser extent. To assess the contributions of these mutations to attenuation and temperature sensitivity, reciprocal changes were generated at these positions in infectious cDNA clones of Sabin 1 and P1/Mahoney. Assays in tissue culture and primates indicated that the two mutations make some contribution to the temperature sensitivity of the Sabin 1 strain but that neither is a strong determinant of attenuation.     

Inactivation of hepatitis A HM-175/18f, reovirus T1 Lang and MS2 during alkaline stabilization of human biosolids

AIM: To compare the inactivation rates of male-specific bacteriophage-2 (MS2), hepatitis A HM-175/18f (HM-175) and reovirus T1 Lang (T1 L) during alkaline stabilization of wastewater residues. METHODS AND RESULTS: A bench scale alkaline stabilization model was used to evaluate the inactivation of MS2 seeded into raw sludge simultaneously with HM-175 or T1 L. Stabilization was performed in triplicate at 28 and 4 degrees C for both viral combinations. During stabilization at 28 and 4 degrees C, MS2 and T1 L concentrations were similar at each time point (t = 0.1, 2, 12 and 24 h). MS2 and HM-175 concentrations were also similar at each time point during stabilization at 28 degrees C. At 4 degrees C, MS2 and HM-175 concentrations were not similar at the first two time points (t = 0.1 and 2 h), but were similar at later time points (t = 12 and 24 h). CONCLUSIONS: The inactivation rates of T1 L at 4 degrees C and both T1 L and HM-175 at 28 degrees C were similar to the inactivation rate of MS2 at all time points. At 4 degrees C, MS2 was inactivated at a faster rate during the first two time points (t = 0.1 and 2 h) than HM-175, but was inactivated similarly at later time points (t = 12 and 24 h). SIGNIFICANCE AND IMPACT OF THE STUDY: Phages, such as MS2, would be ideal indicators for the presence of enteric viruses in wastewater residues because of their ubiquity, nonpathogenic nature, low cost and time associated with their detection. The findings of this study suggest that MS2 could serve as an indicator for monitoring the persistence of enteric viruses, such as HM-175 and T1 L, during alkaline stabilization performed at moderate temperatures (28 degrees C), but may not serve as an indicator for HM-175 at reduced temperature (4 degrees C). The utility of MS2 as an indicator of viral persistence during biosolids treatment should be further evaluated, as the increased efficiency and frequency of pathogen monitoring associated with their use may reduce the potential public health risk associated with biosolids, facilitating a greater acceptance for their land application.     

Trypsin sensitivity of the Sabin strain of type 1 poliovirus: cleavage sites in virions and related particles.

Treatment of the Sabin strain of type 1 poliovirus with trypsin produced two stable fragments of capsid protein VP1 which remained associated with the virions. Trypsinized virus was fully infectious and was neutralized by type-specific antisera. The susceptible site in the Sabin 1 strain was between the lysine at position 99 and the asparagine at position 100. A similar tryptic cleavage occurred in the Leon and Sabin strains of type 3 poliovirus, probably at the arginine at position 100, but not in the type 1 Mahoney strain, which lacks a basic residue at either position 99 or position 100. Tryptic treatment of heat-treated virus and 14S assembly intermediates produced unique stable fragments which were different from those produced in virions. The implications of our results for future characterization of the surface structures of these particles and structural rearrangements in the poliovirus capsid are discussed.     

In vivo protocol for testing efficacy of hand-washing agents against viruses and bacteria: experiments with rotavirus and Escherichia coli

Ten antiseptic formulations, an unmedicated liquid soap, and tap water alone were compared for their capacities to eliminate human rotavirus from the finger pads of adult volunteers; three of the antiseptics, the soap, and the tap water alone were also tested against Escherichia coli. A fecal suspension of virus or bacterium was placed on each finger pad and air dried. The contaminated site was exposed to the test product for 10 s, rinsed in tap water, and dried on a paper towel. The residual virus or bacterium was then eluted. Selected agents were also tested by an analogous whole-hand method by which the entire palm surfaces of both hands were contaminated. Alcohols (70%) alone or with Savlon reduced the virus titer by greater than 99%, whereas the reductions by Proviodine, Dettol, and Hibisol ranged from 95 to 97%. Aqueous solutions of chlorhexidine gluconate were significantly less effective for virus removal or inactivation than 70% alcohol solutions. Furthermore, Savlon in water (1:200) was found to be much less effective in eliminating the virus (80.6%) than the bacterium (98.9%). The tap water alone and the soap reduced the virus titers by 83.6 and 72.5% and the bacterial titers by 90 and 68.7%, respectively. The results of the whole-hand method agreed well with those of the finger pad protocol. We conclude that the finger pad method is a suitable model for testing the in vivo efficacy of hand-washing agents and emphasize the need for using appropriate test viruses and bacteria.     

Evolution of the Sabin strain of type 3 poliovirus in an immunodeficient patient during the entire 637-day period of virus excretion

A 20-year-old female hypogammaglobulinemic patient received monotypic Sabin 3 vaccine in 1962. The patient excreted type 3 poliovirus for a period of 637 days without developing any symptoms of poliomyelitis, after which excretion appeared to have ceased spontaneously. The evolution of Sabin 3 throughout the entire period of virus excretion was studied by characterization of seven sequential isolates from the patient. The isolates were analyzed in terms of their antigenic properties, virulence, sensitivity for growth at high temperatures, and differences in nucleotide sequence from the Sabin type 3 vaccine. The isolates followed a main lineage of evolution with a rate of nucleotide substitution that was very similar to that estimated for wild-type poliovirus during person-to-person transmission. There was a delay in the appearance of antigenic variants compared to sequential type 3 isolates from healthy vaccines, which could be one of the possible explanations for the long-term excretion of virus from the patient. The distribution of mutations in the isolates identified regions of the virus possibly involved in adaptation for growth in the human gut and virus persistence. None of the isolates showed a full reversion of the attenuated and temperature-sensitive phenotypes of Sabin 3. Information of this sort will help in the assessment of the risk of spread of virulent polioviruses from long-term excretors and in the design of therapies to stop long-term excretion. This will make an important contribution to the decision-making process on when to stop vaccination once wild poliovirus has been eradicated.     

In vivo efficacy of Ingavirin against pandemic A (H1N1/09)v influenza virus

Ingavirin was shown to be efficient in inhibition of the pandemic influenza virus strains A/California/04/2009 (H1N1)v, A/California/07/2009 (H1N1)v, A/Moscow/225/2009 (H1N1)v and A/Moscow/226/2009 (H1N1)v. as well as the influenza virus strain A/Aichi/2/68 (H3N2) in the lungs of the infected mice. After oral administration of Ingavirin the titers of the influenza virus strains in the lung homogenates lowered.     

In vivo protocol for testing efficacy of hand-washing agents against viruses and bacteria: experiments with rotavirus and Escherichia coli.

Ten antiseptic formulations, an unmedicated liquid soap, and tap water alone were compared for their capacities to eliminate human rotavirus from the finger pads of adult volunteers; three of the antiseptics, the soap, and the tap water alone were also tested against Escherichia coli. A fecal suspension of virus or bacterium was placed on each finger pad and air dried. The contaminated site was exposed to the test product for 10 s, rinsed in tap water, and dried on a paper towel. The residual virus or bacterium was then eluted. Selected agents were also tested by an analogous whole-hand method by which the entire palm surfaces of both hands were contaminated. Alcohols (70%) alone or with Savlon reduced the virus titer by greater than 99%, whereas the reductions by Proviodine, Dettol, and Hibisol ranged from 95 to 97%. Aqueous solutions of chlorhexidine gluconate were significantly less effective for virus removal or inactivation than 70% alcohol solutions. Furthermore, Savlon in water (1:200) was found to be much less effective in eliminating the virus (80.6%) than the bacterium (98.9%). The tap water alone and the soap reduced the virus titers by 83.6 and 72.5% and the bacterial titers by 90 and 68.7%, respectively. The results of the whole-hand method agreed well with those of the finger pad protocol. We conclude that the finger pad method is a suitable model for testing the in vivo efficacy of hand-washing agents and emphasize the need for using appropriate test viruses and bacteria.     

A mutation present in the amino terminus of Sabin 3 poliovirus VP1 protein is attenuating.

The attenuated phenotype of Sabin 3 poliovirus compared with its neurovirulent progenitor strain has been largely accounted for by mutations in the genome at positions 472 and 2034 (G. D. Westrop, K. A. Wareham, D. M. A. Evans, G. Dunn, P. D. Minor, D. I. Magrath, F. Taffs, S. Marsden, M. A. Skinner, G. C. Schild, and J. W. Almond, J. Virol. 63:1338-1344, 1989). By sequencing vaccine virus RNA, we recently identified another Sabin 3-specific mutation at position 2493 (U----C), which predicts an Ile----Thr change at the sixth residue of VP1 (C. Weeks-Levy, J. M. Tatem, S. J. DiMichele, W. Waterfield, A. F. Georgiu, and S. J. Mento, Virology 185:934-937, 1991). Viruses generated by using cDNAs which represent the vaccine sequence (LED3) and a derivative (VR318) possessing a single base change to the wild-type nucleotide (U) at 2493 were used to determine the impact of the 2493 mutation on virus phenotype. The VP1 proteins of LED3 and VR318 viruses were distinguishable by denaturing electrophoretic analysis. LED3 produced smaller plaques in Vero cells than VR318 virus did. Neurovirulence testing of these cDNA-derived viruses in monkeys demonstrated that the 2493 mutation in LED3 virus is attenuating.     

Antiviral activity of Basidiomycete mycelia against influenza type A (serotype H1N1) and herpes simplex virus type 2 in cell culture

In this study, we investigated the in vitro antiviral activity of the mycelia of higher mushrooms against influenza virus type A(serotype H1N1) and herpes simplex virus type 2(HSV-2), strain BH. All 10 investigated mushroom species inhibited the reproduction of influenza virus strain A/FM/1/47(H1N1) in MDCK cells reducing the infectious titer by 2.0–6.0 lg ID50. Four species, Pleurotus ostreatus, Fomes fomentarius, Auriporia aurea, and Trametes versicolor, were also determined to be effective against HSV-2 strain BH in RK-13 cells, with similar levels of inhibition as for influenza. For some of the investigated mushroom species—Pleurotus eryngii, Lyophyllum shimeji, and Flammulina velutipes—this is the first report of an anti-influenza effect. This study also reports the first data on the medicinal properties of A. aurea, including anti-influenza and antiherpetic activities. T. versicolor 353 mycelium was found to have a high therapeutic index(324.67), and may be a promising material for the pharmaceutical industry as an anti-influenza and antiherpetic agent with low toxicity. Mycelia with antiviral activity were obtained in our investigation by bioconversion of agricultural wastes(amaranth flour after CO2 extraction), which would reduce the cost of the final product and solve some ecological problems.     

Antiviral activity of Basidiomycete mycelia against influenza type A(serotype H1N1) and herpes simplex virus type 2 in cell culture

In this study, we investigated the in vitro antiviral activity of the mycelia of higher mushrooms against influenza virus type A(serotype H1N1) and herpes simplex virus type 2(HSV-2), strain BH. All 10 investigated mushroom species inhibited the reproduction of influenza virus strain A/FM/1/47(H1N1) in MDCK cells reducing the infectious titer by 2.0–6.0 lg ID50. Four species, Pleurotus ostreatus, Fomes fomentarius, Auriporia aurea, and Trametes versicolor, were also determined to be effective against HSV-2 strain BH in RK-13 cells, with similar levels of inhibition as for influenza. For some of the investigated mushroom species—Pleurotus eryngii, Lyophyllum shimeji, and Flammulina velutipes—this is the first report of an anti-influenza effect. This study also reports the first data on the medicinal properties of A. aurea, including anti-influenza and antiherpetic activities. T. versicolor 353 mycelium was found to have a high therapeutic index(324.67), and may be a promising material for the pharmaceutical industry as an anti-influenza and antiherpetic agent with low toxicity. Mycelia with antiviral activity were obtained in our investigation by bioconversion of agricultural wastes(amaranth flour after CO2 extraction), which would reduce the cost of the final product and solve some ecological problems.     

Evolution of the Sabin type 1 poliovirus in humans: characterization of strains isolated from patients with vaccine-associated paralytic poliomyelitis.

Attenuated strains of the Sabin oral poliovirus vaccine replicate in the human gut and in rare cases cause vaccine-associated paralytic poliomyelitis (VAPP). Reversion of vaccine strains toward a pathogenic phenotype is probably one of the main causes of VAPP, a disease most frequently associated with type 3 and type 2 strains and more rarely with the type 1 (Sabin 1) strain. To identify the determinants and mechanisms of safety versus pathogenicity of the Sabin 1 strain, we characterized the genetic and phenotypic changes in six Sabin 1-derived viruses isolated from immunocompetent patients with VAPP. The genomes of these strains carried either few or numerous mutations from the original Sabin 1 genome. As assessed in transgenic mice carrying the human poliovirus receptor (PVR-Tg mice), all but one strain had lost the attenuated phenotype. Four strains presented only a moderate neurovirulent phenotype, probably due at least in part to reversions to the wild-type genotype, which were detected in the 5' noncoding region of the genome. The reversions found in most strains at nucleotide position 480, are known to be associated with an increase in neurovirulence. The construction and characterization of Sabin 1 mutants implicated a reversion at position 189, found in one strain, in the phenotypic change. The presence of 71 mutations in one neurovirulent strain suggests that a vaccine-derived strain can survive for a long time in humans. Surprisingly, none of the strains analyzed were as neurovirulent to PVR-Tg mice as was the wild-type parent of Sabin 1 (Mahoney) or a previously identified neurovirulent Sabin 1 mutant selected at a high temperature in cultured cells. Thus, in the human gut, the Sabin 1 strain does not necessarily evolve toward the genetic characteristics and high neuropathogenicity of its wild-type parent.     

Utilization of Chimeras between Human (HM-175) and Simian (AGM-27) Strains of Hepatitis A Virus To Study the Molecular Basis of Virulence

Chimeras between human (HM-175) and simian (AGM-27) strains of hepatitis A virus (HAV) were constructed to evaluate the effect of the 2C gene of AGM-27 on HAV replication in cell culture and virulence in tamarins (Saguinus mystax) and chimpanzees (Pan troglodytes). Kinetic studies and radioimmunofocus assays demonstrated that replacement of the 2C gene of HAV/7, a cell culture-adapted strain of HM-175, with that of AGM-27 drastically reduced the ability of the virus to replicate in cultured cells. Intragenic chimeras containing AGM-27 sequences in either the 5′ or 3′ half of the 2C gene replicated in cell culture at an intermediate level. Whereas HAV/7 is attenuated for tamarins, a chimera containing the simian virus 2C gene in the HAV/7 background was virulent in tamarins, demonstrating that the simian virus 2C gene alone can confer the phenotype of virulence to an otherwise attenuated virus. Clusters of AGM-27-specific residues near both ends of the 2C protein were required for virulence since a chimera containing AGM-27 sequences in the carboxy-terminal half of 2C was partially attenuated for tamarins while one containing AGM-27 sequences only in the amino-terminal half of 2C was even more attenuated. Chimeras containing either the entire or only the 3′ half of the simian virus 2C gene in the HAV/7 background were attenuated for chimpanzees.