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.     

Adaptation to the cell line PLC/PRF/5 of hepatitis A virus released in the cell culture medium

The propagation of hepatitis A virus (HAV), CF53 strain, released without any cytopathic effect into the PLC/PRF/5 cells supernatant, was studied in the course of six serial passages (6th to 11th). The decrease (from 5 to 1 week) of incubation time required to detect HAV, by RIA, in culture supernatant, the increase in Hepatitis A antigen (from 777 to 10,038 c.p.m./50 microliter) and infectivity titre (from 10(3.0) TCID 50/ml to 10(4.5) TCID 50/ml) were consistent with the adaptation of this virus to the cell line PLC/PRF/5.     

Grape seed extract for control of human enteric viruses

Grape seed extract (GSE) is reported to have many pharmacological benefits, including antioxidant, anti-inflammatory, anticarcinogenic, and antimicrobial properties. However, the effect of this inexpensive rich source of natural phenolic compounds on human enteric viruses has not been well documented. In the present study, the effect of commercial GSE, Gravinol-S, on the infectivity of human enteric virus surrogates (feline calicivirus, FCV-F9; murine norovirus, MNV-1; and bacteriophage MS2) and hepatitis A virus (HAV; strain HM175) was evaluated. GSE at concentrations of 0.5, 1, and 2 mg/ml was individually mixed with equal volumes of each virus at titers of ～7 log(10) PFU/ml or ～5 log(10) PFU/ml and incubated for 2 h at room temperature or 37°C. The infectivity of the recovered viruses after triplicate treatments was evaluated by standardized plaque assays. At high titers (～7 log(10) PFU/ml), FCV-F9 was significantly reduced by 3.64, 4.10, and 4.61 log(10) PFU/ml; MNV-1 by 0.82, 1.35, and 1.73 log(10) PFU/ml; MS2 by 1.13, 1.43, and 1.60 log(10) PFU/ml; and HAV by 1.81, 2.66, and 3.20 log(10) PFU/ml after treatment at 37°C with 0.25, 0.50, and 1 mg/ml GSE, respectively (P < 0.05) in a dose-dependent manner. GSE treatment of low titers (～5 log(10) PFU/ml) at 37°C also showed viral reductions. Room-temperature treatments with GSE caused significant reduction of the four viruses, with higher reduction for low-titer FCV-F9, MNV-1, and HAV compared to high titers. Our results indicate that GSE shows promise for application in the food industry as an inexpensive novel natural alternative to reduce viral contamination and enhance food safety.     

Removal and inactivation of hepatitis A virus during manufacture of urokinase from human urine

The purpose of the present study was to examine the efficacy and mechanism of the PAB (para-amino benzamidine) affinity column chromatography, Viresolve NFP virus filtration, pasteurization (60°C heat treatment for 10 h), and lyophilization steps employed in the manufacture of urokinase from human urine as regards the removal and/or inactivation of the hepatitis A virus (HAV). Samples from the relevant stages of the production process were spiked with HAV and subjected to scale-down processes mimicking the manufacture of urokinase. Samples were collected at each step, immediately titrated using a 50% tissue culture infectious dose (TCID₅₀), and the virus reduction factors evaluated. PAB chromatography was found to be an effective step for removing HAV with a log reduction factor of 3.24. HAV infectivity was rarely detected in the urokinase fraction, while most of the HAV infectivity was recovered in the unbound and wash fractions. HAV was completely removed during the vire solve NFP filtration with a log reduction factor of ≥4.60. Pasteurization was also found to be an effective step in inactivating HAV, where the titers were reduced from an initial titer of 7.18 log₁₀ TCID₅₀ to undetectable levels within 10 h of treatment. The log reduction factor achieved during pasteurization was ≥4.76. Lyophilization revealed the lowest efficacy for inactivating HAV with a log reduction factor of 1.48. The cumulative log reduction factor was ≥14.08. Accordingly, these results indicate that the production process for urokinase exhibited a sufficient HAV reducing capacity to achieve a high margin of virus safety.     

Comparative pathology in ferrets infected with H1N1 influenza A viruses isolated from different hosts

Virus replication and pulmonary disease pathogenesis in ferrets following intranasal infection with a pandemic influenza virus strain (A/California/4/09 [CA09]), a human seasonal influenza H1N1 virus isolate (A/New Caledonia/20/99 [Ncal99]), a classical swine influenza H1N1 virus isolate (A/Swine/Iowa/15/30 [Sw30]), or an avian H1N1 virus isolate (A/Mallard/MN/A108-2355/08 [Mal08]) were compared. Nasal wash virus titers were similar for Ncal99 and Sw30, with peak virus titers of 10(5.1) 50% tissue culture infectious doses (TCID(50))/ml and 10(5.5) TCID(50)/ml occurring at day 3 postinfection (p.i.), respectively. The mean peak titer for CA09 also occurred at day 3 p.i. but was higher (10(7) TCID(50)/ml). In contrast, the peak virus titers (10(3.6) to 10(4.3) TCID(50)/ml) for Mal08 were delayed, occurring between days 5 and 7 p.i. Disease pathogenesis was characterized by microscopic lesions in the nasal turbinates and lungs of all ferrets; however, Sw30 infection was associated with severe bronchointerstitial pneumonia. The results demonstrate that although CA09 is highly transmissible in the human population and replicates well in the ferret model, it causes modest disease compared to other H1N1 viruses, particularly Sw30 infection.     

Evaluation of the serum-free medium MDSS2 for the production of poliovirus on vero cells in bioreactors

The serum-free medium MDSS2 (Merten et al., 1994), was used for cultivating Vero cells as well as for producing poliovirus (Sabin type 1) in static and in perfused micro-carrier cultures. At slightly different growth rates of 0.0120/h and 0.0106/h, respectively, static cultures in serum-containing (SCM) and serum-free (SFM) medium produced titers of ^106.75 and 10^6.67 TCID50 per 50 μl; signifying a specific productivity of 0.89 and 1.07 TCID50/c. Serum-free bioreactor cultures of Vero cells on DEAE-dextran microcarriers at 6.25 g/l produced cell densities of about 1.5×10⁶c/ml. After infection with virus (multiplicity of infection (MOI) 0.1–0.3) titers of about 6.3×10⁸ TCID50/ml were obtained, signifying an average specific productivity of 7.1 TCID50/c.h. Although these values were 4 and 2 fold, respectively, higher than in classical resum-based production processes (Montagnon et al. Dev. biol. Stand. 1981, 47, 55), a reference culture, for which cell growth was done in SCM and only virus production was done in SFM, produced 2×10⁹ TCID/ml with an average specific virus production rate of 18.9 TCID50/c.h. The differences between the fully serum-free and our reference process were mainly due to physiological differences of cells grown in SCM and SFM and also due to strongly modified consumption kinetics after virus infection leading to limitations of one or several essential medium compounds, like glucose and amino acids. Avoiding these limitations by increasing the residual concentration of glucose, glutamine, histidine, and SH-amino acids, led to specific virus production rates (of about 17.9 TCID59/c.h.) comparable to those found in the reference virus production process. The optimisation of the production of the poliovirus (Sabin 1) will be described with respect to the modification of the medium composition. This revised version was published online in June 2006 with corrections to the Cover Date.     

Solar UV radiation does not inactivate marine birnavirus in coastal seawater

We examined the inactivation kinetics of marine birnavirus (MABV) in a coastal sea, in seawater samples collected from 50 cm depth. MABV was added to both natural and autoclaved seawater at a concentration of 6 x 10(6.43) TCID50 (50% tissue culture infectious dose) ml(-1), put in dialysis tubes and incubated at the original depth. The inactivation of MABV by solar UV radiation was examined using light and dark tubes. The infectivity titer of MABV was measured by the TCID50 method using CHSE-214 cells. Virus infectivity in natural seawater decreased quickly and was below the detection limit by 270 min in both light and dark conditions; however, virus infectivity was maintained in the autoclaved seawater until 420 min. These results suggest that the loss of virus infectivity is not caused by sunlight UV radiation.     

Anti-HIV-1 activity of an ionically modified porous polypropylene membrane determined by filtration of a viral suspension

We describe here a unique anti-HIV-1 membrane, derived from a chemically modified porous polypropylene (PP) membrane, which lowers viral infectivity upon the filtration of HIV-1 suspension. A cationic polymer, polyethyleneimine (PEI) was graft-polymerized onto the PP filter membrane (PP-PEI), and infectious HIV-1(HTLV-IIIB) derived from MOLT-4/HIV-1(HTLV-IIIB) cells (HIV-1(HTLV-IIIB(MOLT-4)) was applied. When a viral suspension of high titer (10(3.93) TCID50 ml(-1) was filtered, efficient reduction (>99%) of gag p24 antigen levels and infectious titer resulted. In a viral suspension of medium titer (10(2.37) TCID50 ml(-1), a significant decrease in the p24 antigen did not occur, although the titer was markedly reduced (>95%). Electron microscopic observation suggested that PEI induced viral aggregations under high titer conditions, and under medium titer conditions, PEI deprived HIV-1(HTLV-IIIB(MOLT-4)) of its infectivity alone to avoid virus adsorption. In contrast, HIV-1 propagated in human peripheral blood mononuclear cells (PBMC) such as HIV-1(HTLV-III(PBMC)) was more efficiently trapped by PP-PEI at lower titers as compared with HIV-1(HTLV-IIIB(MOLT-4)) from MOLT-4/HIV-1(HTLV-IIIB) cells. These data suggest host cell modification in the interactions between PP-PEI and HIV-1 strains. Since HIV-1(HTLV-IIIB(MOLT-4)) and HIV-1(HTLV-IIIB(PBMC)) were almost electrically neutral and negative, respectively, we concluded that the divergent effect of PEI on each HIV-1(HTLV-IIIB) resulted from their different electrical characteristics.     

Rubella Virus: Continuous and Concurrent Production of Complement-fixing Antigen and Infectious Virus in Suspension Cultures

Rubella complement-fixing (CF) antigen and infectious virus were produced continuously and concurrently for as long as 63 days in suspension cultures of BHK-21 cells prepared from uncloned monolayer stock cultures. CF titers ranged from 1:4 to 1:32, and the peak infectivity titer was greater than 8.0 (TCID(50) log(10)) per ml. Suspension cultures could be recultivated after prolonged storage in liquid nitrogen. The resulting monolayer or suspension cultures also produced CF antigen. Suspension cultures provide an effective system for the long-term continuous and concurrent production of rubella virus diagnostic reagents.     

Characterization of a virus obtained from snakeheads Ophicephalus striatus with epizootic ulcerative syndrome (EUS) in the Philippines

This is the first report of the isolation and characterization of a fish virus from the Philippines. The virus was isolated using snakehead spleen cells (SHS) from severely lesioned epizootic ulcerative syndrome (EUS)-affected snakehead Ophicephalus striatus from Laguna de Bay, in January 1991. The virus induced cytopathic effects (CPE) in SHS cells yielding a titer of 3.02 x 10(6) TCID50 ml(-1) at 25 degrees C within 2 to 3 d. Other susceptible cell lines included bluegill fry (BF-2), catfish spleen (CFS) and channel catfish ovary (CCO) cells. Replication in chinook salmon embryo cells (CHSE-214) was minimal while Epithelioma papulosum cyprini cells (EPC) and rainbow trout gonad cells (RTG-2) were refractory. Temperatures of 15 to 25 degrees C were optimum for virus replication but the virus did not replicate at 37 degrees C. The virus can be stored at -10 and 8 degrees C for 30 and 10 d, respectively, without significant loss of infectivity. Viral replication was logarithmic with a 2 h lag phase; viral assembly in the host cells occurred in 4 h and release of virus occurred 8 h after viral infection. A 1-log difference in TCID50 titer between the cell-free virus and the total virus was noted. Freezing and thawing the virus caused a half-log drop in titer. Viral exposure to chloroform or heating to 56 degrees C for 30 min inactivated the virus. Exposure to pH 3 medium for 30 min resulted in a more than 100-fold loss of viral infectivity. The 5-iododeoxyuridine (IUdR) did not affect virus replication, indicating a RNA genome. Neutralization tests using the Philippine virus, the ulcerative disease rhabdovirus (UDRV) and the infectious hematopoietic necrosis virus (IHNV) polyvalent antisera showed slight cross-reaction between the Philippine virus antiserum and UDRV but established no serological relationship with SHRV and IHN virus. Transmission electron microscopy (TEM) of SHS cells infected with the virus showed virus particles with typical bullet morphology and an estimated size of 65 x 175 nm. The Philippine virus was therefore a rhabdovirus, but the present study did not establish its role in the epizootiology of EUS.     

Effect of relative humidity and air temperature on survival of hepatitis A virus on environmental surfaces.

Stainless steel disks (diameter, 1 cm) were contaminated with fecally suspended hepatitis A virus (HAV; strain HM-175) and held at low (25% +/- 5%), medium (55% +/- 5%), high (80% +/- 5%), or ultrahigh (95% +/- 5%) relative humidity (RH) at an air temperature of 5,20, or 35 degrees C. HAV survival was inversely proportional to the level of RH and temperature, and the half-lives of the virus ranged from greater than 7 days at the low RH and 5 degrees C to about 2 h at the ultrahigh RH and 35 degrees C. In parallel tests with fecally suspended Sabin poliovirus (PV) type 1 at the low and ultrahigh RH, all PV activity was lost within 4 h at the low RH whereas at the ultrahigh RH it remained detectable up to 12 h. HAV could therefore survive much better than PV on nonporous environmental surfaces. Moreover, the ability of HAV to survive better at low levels of RH is in direct contrast to the behavior of other enteroviruses. These findings should help in understanding the genesis of HAV outbreaks more clearly and in designing better measures for their control and prevention.     

Effect of relative humidity and air temperature on survival of hepatitis A virus on environmental surfaces.

Stainless steel disks (diameter, 1 cm) were contaminated with fecally suspended hepatitis A virus (HAV; strain HM-175) and held at low (25% +/- 5%), medium (55% +/- 5%), high (80% +/- 5%), or ultrahigh (95% +/- 5%) relative humidity (RH) at an air temperature of 5,20, or 35 degrees C. HAV survival was inversely proportional to the level of RH and temperature, and the half-lives of the virus ranged from greater than 7 days at the low RH and 5 degrees C to about 2 h at the ultrahigh RH and 35 degrees C. In parallel tests with fecally suspended Sabin poliovirus (PV) type 1 at the low and ultrahigh RH, all PV activity was lost within 4 h at the low RH whereas at the ultrahigh RH it remained detectable up to 12 h. HAV could therefore survive much better than PV on nonporous environmental surfaces. Moreover, the ability of HAV to survive better at low levels of RH is in direct contrast to the behavior of other enteroviruses. These findings should help in understanding the genesis of HAV outbreaks more clearly and in designing better measures for their control and prevention.     

Susceptibility of zona-intact and zona-free in vitro-produced bovine embryos at different stages of development to infection with bovine herpesvirus-1

The aim of the present study was to determine if BHV-1 is able to replicate within in vitro produced embryos and to investigate the degree to which the zona pellucida (ZP) is able to protect in vitro produced embryos against infection with BHV-1. Both ZP-intact and ZP-free matured oocytes, zygotes (1 d post insemination; 1dpi), 8-cell stage embryos (3 dpi), morulae (6 dpi) were incubated for 1 h in 1 ml of MEM containing 10(7.7) TCID(50)/ml BHV-1 (Cooper strain). Three titers (10(5.7), 10(6.7) and 10(7.7) TCID(50)/ml) of the Cooper strain were used for incubation of hatched blastocysts (9 dpi). Bovine embryonic lung cells (BEL) on microcarriers were inoculated following the same protocol as for the embryos. At 0, 12, 24, 36 and 48 h post inoculation (hpi), groups of embryos and BEL cells were collected for virus titration and for the determination of the percentage of viral antigen positive cells by immunofluorescence. For the 3 developmental stages in ZP-free embryos, similar maximal intracellular virus progeny titers were obtained at 24 to 48 hpi ranging from 10(1.32) to 10(1.43) TCID(50)/ 100 embryonic cells. The intracellular virus titer in the BEL cells peaked at 10(3.08) TCID(50)/ 100 BEL cells. The percentage of cells which expressed viral antigens was 13% in ZP-free hatched blastocysts, 17% in ZP-free morulae and 100% in BEL cells. In ZP-intact embryos, no replication of BHV-1 was detected. These results clearly show that only after removal of the zona pellucida, BHV-1 is able to replicate within the in vitro produced embryos, with only a subset of embryonic cells being fully susceptible.     

Novel mutations in a tissue culture-adapted hepatitis C virus strain improve infectious-virus stability and markedly enhance infection kinetics

Hepatitis C virus (HCV) establishes persistent infections and leads to chronic liver disease. It only recently became possible to study the entire HCV life cycle due to the ability of a unique cloned patient isolate (JFH-1) to produce infectious particles in tissue culture. However, despite efficient RNA replication, yields of infectious virus particles remain modest. This presents a challenge for large-scale tissue culture efforts, such as inhibitor screening. Starting with a J6/JFH-1 chimeric virus, we used serial passaging to generate a virus with substantially enhanced infectivity and faster infection kinetics compared to the parental stock. The selected virus clone possessed seven novel amino acid mutations. We analyzed the contribution of individual mutations and identified three specific mutations, core K78E, NS2 W879R, and NS4B V1761L, which were necessary and sufficient for the adapted phenotype. These three mutations conferred a 100-fold increase in specific infectivity compared to the parental J6/JFH-1 virus, and media collected from cells infected with the adapted virus yielded infectious titers as high as 1 × 10(8) 50% tissue culture infective doses (TCID(50))/ml. Further analyses indicated that the adapted virus has longer infectious stability at 37°C than the wild type. Given that the adapted phenotype resulted from a combination of mutations in structural and nonstructural proteins, these data suggest that the improved viral titers are likely due to differences in virus particle assembly that result in significantly improved infectious particle stability. This adapted virus will facilitate further studies of the HCV life cycle, virus structure, and high-throughput drug screening.     

Immunization of pigs with the attenuated S- strain of Japanese encephalitis virus.

The attenuated S- strain of Japanese encephalitis virus was produced from a wild strain of this virus by serial cultivation in primary bovine kidney cell cultures at 30 degrees C. Pigs were inoculated with it and examined for ability to produce antibody and protect themselves from infection with a wild strain used for challenge. In pigs inoculated with a single dose of 10(6.5) approximately 10(7.5) TCID50 of the S- strain, the neutralizing antibody titer or hemagglutination-inhibiting antibody (HI) titer increased to 10 approximately 320. An antibody titer exceeding 10 was maintained for 2 approximately 9 weeks. In pigs inoculated twice with 10(6.5) approximately 10(7.0) TCID50 of the S- strain, HI titer increased to 80 approximately 640. In many of these pigs, HI titers of 80 approximately 160 persisted for more than 6 weeks. Pigs inoculated once or twice with 10(7.0) approximately 10(7.5) TCID50 of the S- strain were challenged by inoculation with 10(4.5) approximately 10(5.5) TCID50 of a wild strain and examined for the occurrence of viremia. As a result, an ability to protect from infection was demonstrated in pigs which showed an antibody titer surpassing 10 at the time of challenge. Pregnant sows inoculated with 10(7.0) TCID50 of the S- strain were challenged by inoculation with 10(7.0) TCID50 of a wild strain. Neither death nor infection occurred to any fetus harbored by them. From these results, it is concluded that the S- strain can be used as live virus vaccine for porcine practice.     

Growth of Chikungunya Virus in Baby Hamster Kidney Cell (BHK-21-Clone 13) Suspension Cultures

This report describes the methods used to obtain high titers of chikungunya virus with suspension cultures of BHK-21-clone 13 cells. The cells were grown at 37 C to a cell concentration of 10(6) to 2 x 10(6) per ml. After maximum cell growth, the cells were inoculated with chikungunya virus at a multiplicity of 1 to 2 50% suckling mouse intracerebral lethal doses (SMICLD(50)) per cell in the spent Eagle's minimum essential medium for suspension cultures (MEMS), or the cell cultures were centrifuged at 200 x g and resuspended in either fresh MEMS or medium 199 prior to inoculation. The medium used had no effect on virus titer. The inoculated cultures were incubated at 34 C until the cell viability dropped to 30%, which usually occurred 28 to 30 hr postinoculation. After these procedures, chikungunya virus titers of log(10) 10.3 to 11.8 SMICLD(50) per ml were obtained.     

Enzyme-linked immunosorbent assay-format tissue culture infectious dose-50 test for titrating dengue virus

A dengue nonstructural protein 1 (NS1) antigen capture enzyme-linked immunosorbent assay (ELISA)-based tissue culture infectious dose-50 (TCID(50)) test (TCID(50)-ELISA) was developed as an alternative to the standard plaque assay for titrating dengue virus. Virus titers obtained by TCID(50)-ELISA were comparable to those obtained by the plaque assay and by the traditional TCID(50)-cytopathic effect (CPE) test (TCID(50)-CPE), with a better reproducibility and a lower coefficient of variation. Quantitative comparison of TCID(50)-ELISA and TCID(50)-CPE resulted in a correlation coefficient of 0.976. Moreover, this new method showed a wider application to C6/36, Vero E6, BHK-21, and Vero cells compared with other titration methods. In summary, the novel TCID(50)-ELISA method described here provides a more reliable and more accurate alternative compared to the plaque assay and TCID(50)-CPE for titration of dengue virus.     

Antiviral activity of carrageenan on hepatitis A virus replication in cell culture

Sulphated polysaccharides such as iota-, lambda- and kappa-carrageenans showed a potent inhibitory effect on the replication of hepatitis A virus (HAV) in the human hepatoma cell line PLC/PRF/5. No cytotoxic effects were detected with concentrations of carrageenans up to 200 μg/ml. The selectivity indices of these substances, calculated as the ratio of the dose that reduced the number of viable cells to 50 % (CD₅₀) to the effective dose that inhibited 50 % of viral antigen expression (ED₅₀), were > 400 with iota-carrageenan, > 222 with lambda-carrageenan and > 10 with kappa-carrageenan. The selectivity index of ribavirin (reference substance) was only 5. The 3 types of carrageenans resulted in concentration-dependent reduction of HAV-antigen expression and HAV infectivity. Iota-and lambda-carrageenan emerged, from the present study, as promising candidates for chemotherapy of acute hepatitis A.