Detection of marine birnavirus in the Japanese pearl oyster Pinctada fucata and seawater from different depths

This study examines the seasonal changes of marine birnavirus (MABV) in seawater and the Japanese pearl oyster Pinctada fucata reared at different depths (2 and 15 m). Oysters and seawater were collected in 1998, and a 2-step PCR was carried out to detect MABV. Virus isolation was performed on the PCR-positive samples in the oyster. The detection rate of the MABV genome in the oyster was low during June, but increased after July at both 2 and 15 m depths. MABV was not isolated until after September, when isolation rates of 10 to 28.6% were recorded. The results suggest that growth of MABV in the oyster is similar at 2 and 15 m depth. In contrast, the MABV genome in seawater was present through the year at 15 m depth, but was not detected in summer at 2 m. This suggests that the virus is destroyed by UV and/or other factors at 2 m in summer, but is stable in deeper waters.     

Low-pressure UV inactivation and DNA repair potential of Cryptosporidium parvum oocysts.

Because Cryptosporidium parvum oocysts are very resistant to conventional water treatment processes, including chemical disinfection, we determined the kinetics and extent of their inactivation by monochromatic, low-pressure (LP), mercury vapor lamp UV radiation and their subsequent potential for DNA repair of UV damage. A UV collimated-beam apparatus was used to expose suspensions of purified C. parvum oocysts in phosphate-buffered saline, pH 7.3, at 25 degrees C to various doses of monochromatic LP UV. C. parvum infectivity reductions were rapid, approximately first order, and at a dose of 3 mJ/cm(2) (=30 J/m(2)), the reduction reached the cell culture assay detection limit of approximately 3 log(10). At UV doses of 1.2 and 3 mJ/cm(2), the log(10) reductions of C. parvum oocyst infectivity were not significantly different for control oocysts and those exposed to dark or light repair conditions for UV-induced DNA damage. These results indicate that C. parvum oocysts are very sensitive to inactivation by low doses of monochromatic LP UV radiation and that there is no phenotypic evidence of either light or dark repair of UV-induced DNA damage.     

Occurrence of Marine Birnavirus Through the Year in Coastal Seawater in the Uwa Sea

This study aims to determine the seasonal occurrence of marine birnavirus (MABV) at a coastal site in the Uwa Sea, Japan, in 1997 and 1998. To detect MABV from seawater, a simple method was developed for concentrating MABV by dialysis and ethanol precipitation. The concentrated virus was used for polymerase chain reaction (PCR), enzyme-linked immunosorbent assay, and virus isolation. Viral genome was detected through the experimental period. The amount of PCR product varied; it was small in summer, but increased from fall to winter. Viral protein was also detected, and the amount in the January sample was equivalent to approximately 10² TCID₅₀ (50% tissue culture infectious dose) of the virus. However, infectious viruses were not isolated. This suggested that MABV was released from hosts to environmental seawater in winter and possibly degraded after release. Received May 7, 1999; accepted September 16, 1999.     

Virus Decay and Its Causes in Coastal Waters

Recent evidence suggests that viruses play an influential role within the marine microbial food web. To understand this role, it is important to determine rates and mechanisms of virus removal and degradation. We used plaque assays to examine the decay of infectivity in lab-grown viruses seeded into natural seawater. The rates of loss of infectivity of native viruses from Santa Monica Bay and of nonnative viruses from the North Sea in the coastal seawater of Santa Monica Bay were determined. Viruses were seeded into fresh seawater that had been pretreated in various ways: filtration with a 0.2-(mu)m-pore-size filter to remove organisms, heat to denature enzymes, and dissolved organic matter enrichment to reconstitute enzyme activity. Seawater samples were then incubated in full sunlight, in the dark, or under glass to allow partitioning of causative agents of virus decay. Solar radiation always resulted in increased rates of loss of virus infectivity. Virus isolates which are native to Santa Monica Bay consistently degraded more slowly in full sunlight in untreated seawater (decay ranged from 4.1 to 7.2% h(sup-1)) than nonnative marine bacteriophages which were isolated from the North Sea (decay ranged from 6.6 to 11.1% h(sup-1)). All phages demonstrated susceptibility to degradation by heat-labile substances, as heat treatment reduced the decay rates to about 0.5 to 2.0% h(sup-1) in the dark. Filtration reduced decay rates by various amounts, averaging 20%. Heat-labile, high-molecular-weight dissolved material (>30 kDa, probably enzymes) appeared responsible for about 1/5 of the maximal decay. Solar radiation was responsible for about 1/3 to 2/3 of the maximal decay of nonnative viruses and about 1/4 to 1/3 of that of the native viruses, suggesting evolutionary adaptation to local light levels. Our results suggest that sunlight is an important contributing factor to virus decay but also point to the significance of particles and dissolved substances in seawater.     

Inactivation of Foot-and-Mouth Disease Virus by Interaction of Dye and Visible Light

The inactivation of foot-and-mouth disease virus was studied by means of the interaction of neutral red, Toluidine Blue, and methylene blue with visible light. The virus, Type A, strain 1, CANEFA of Argentine origin, was grown in tissue culture and tested in the crude and clarified state. Virus and dye were mixed and incubated together at 4 C for 45 min in the dark, or were mixed and immediately exposed to the visible light source without prior incubation together. Mixtures of crude virus and dye, under any of the experimental conditions used, did not inactivate more than 1 to 2 logs of viral infectivity when held in the dark or when exposed to light during a period of 45 min. Complete inactivation of virus was achieved when clarified virus and dye were mixed and immediately exposed to the visible light source for 15 min. Prior incubation of clarified virus and dye permitted inactivation by methylene blue only, whereas no incubation prior to exposure resulted in three of the dyes contributing to inactivation. A concentration of 6 μg of neutral red, Toluidine Blue, methylene blue, and crystal violet was used per milliliter of virus suspension. Crystal violet was not a good viral inactivator under the conditions of the experimentation. Inactive virus induced the formation of neutralizing antibodies in adult chickens and mice. The antibody titer stimulated by the antigen treated with methylene blue and visible light was probably significant.     

Determination of pyrimidine dimers in Escherichia coli and Cryptosporidium parvum during UV light inactivation, photoreactivation, and dark repair

UV inactivation, photoreactivation, and dark repair of Escherichia coli and Cryptosporidium parvum were investigated with the endonuclease sensitive site (ESS) assay, which can determine UV-induced pyrimidine dimers in the genomic DNA of microorganisms. In a 99.9% inactivation of E. coli, high correlation was observed between the dose of UV irradiation and the number of pyrimidine dimers induced in the DNA of E. coli. The colony-forming ability of E. coli also correlated highly with the number of pyrimidine dimers in the DNA, indicating that the ESS assay is comparable to the method conventionally used to measure colony-forming ability. When E. coli were exposed to fluorescent light after a 99.9% inactivation by UV irradiation, UV-induced pyrimidine dimers in the DNA were continuously repaired and the colony-forming ability recovered gradually. When kept in darkness after the UV inactivation, however, E. coli showed neither repair of pyrimidine dimers nor recovery of colony-forming ability. When C. parvum were exposed to fluorescent light after UV inactivation, UV-induced pyrimidine dimers in the DNA were continuously repaired, while no recovery of animal infectivity was observed. When kept in darkness after UV inactivation, C. parvum also showed no recovery of infectivity in spite of the repair of pyrimidine dimers. It was suggested, therefore, that the infectivity of C. parvum would not recover either by photoreactivation or by dark repair even after the repair of pyrimidine dimers in the genomic DNA.     

Comparative Inactivation of Murine Norovirus, Human Adenovirus, and Human JC Polyomavirus by Chlorine in Seawater

Viruses excreted by humans affect the commercial and recreational use of coastal water. Shellfish produced in contaminated waters have been linked to many episodes and outbreaks of viral gastroenteritis, as well as other food-borne diseases worldwide. The risk can be reduced by appropriate treatment following harvesting and by depuration. The kinetics of inactivation of murine norovirus 1 and human adenovirus 2 in natural and artificial seawater by free available chlorine was studied by quantifying genomic copies (GC) using quantitative PCR and infectious viral particles (PFU). Human JC polyomavirus Mad4 kinetics were evaluated by quantitative PCR. DNase or RNase were used to eliminate free genomes and assess potential viral infectivity when molecular detection was performed. At 30 min of assay, human adenovirus 2 showed 2.6- and 2.7-log(10) GC reductions and a 2.3- and 2.4-log(10) PFU reductions in natural and artificial seawater, respectively, and infectious viral particles were still observed at the end of the assay. When DNase was used prior to the nucleic acid extraction the kinetic of inactivation obtained by quantitative PCR was statistically equivalent to the one observed by infectivity assays. For murine norovirus 1, 2.5, and 3.5-log(10) GC reductions were observed in natural and artificial seawater, respectively, while no viruses remained infectious after 30 min of contact with chlorine. Regarding JC polyomavirus Mad4, 1.5- and 1.1-log(10) GC reductions were observed after 30 min of contact time. No infectivity assays were conducted for this virus. The results obtained provide data that might be applicable to seawater used in shellfish depuration.     

Relative effects of ultraviolet and visible light on the activities of corn earworm and beet armyworm (Lepidoptera: Noctuidae) nucleopolyhedroviruses

Five different combinations of fluorescent tubes (UV-B/UV-B, UV-B/UV-A, UV-A/ UV-A, UV-B/White, White/White) were used to determine relative effects of UV and visible light on the nucleopolyhedroviruses (NPV) of Helicoverpa zea and Spodoptera exigua. For both viruses, the greatest inactivation occurred with exposure to UV-B radiation. Both virus concentration and radiation exposure time influenced the rate and degree of inactivation. In the case of the UV-A/UV-A and White/White combinations inactivation occurred only with the longest exposure (24 h) and the lowest virus concentration (0.747 PIB/mm2). The NPV from H. zea was found to be more sensitive to UV radiation than the NPV from S. exigua.     

Low-Pressure UV Inactivation and DNA Repair Potential of Cryptosporidium parvum Oocysts

Because Cryptosporidium parvum oocysts are very resistant to conventional water treatment processes, including chemical disinfection, we determined the kinetics and extent of their inactivation by monochromatic, low-pressure (LP), mercury vapor lamp UV radiation and their subsequent potential for DNA repair of UV damage. A UV collimated-beam apparatus was used to expose suspensions of purified C. parvum oocysts in phosphate-buffered saline, pH 7.3, at 25°C to various doses of monochromatic LP UV. C. parvum infectivity reductions were rapid, approximately first order, and at a dose of 3 mJ/cm² (=30 J/m²), the reduction reached the cell culture assay detection limit of ~3 log₁₀. At UV doses of 1.2 and 3 mJ/cm², the log₁₀ reductions of C. parvum oocyst infectivity were not significantly different for control oocysts and those exposed to dark or light repair conditions for UV-induced DNA damage. These results indicate that C. parvum oocysts are very sensitive to inactivation by low doses of monochromatic LP UV radiation and that there is no phenotypic evidence of either light or dark repair of UV-induced DNA damage.     

Improving baculovirus resistance to UV inactivation: increased virulence resulting from expression of a DNA repair enzyme

The use of baculoviruses as biological control agents is hampered by their susceptibility to inactivation by ultraviolet (UV) light. In an attempt to reduce UV inactivation, an algal virus pyrimidine dimer-specific glycosylase, cv-PDG, was expressed in the baculovirus Autographa californica M nucleopolyhedrovirus (AcMNPV), and the infectivity of recombinant viruses expressing cv-PDG was measured after exposure to UV light. Expression of cv-PDG resulted in a 3-fold decrease in inactivation of budded virus by UV as measured by plaque assay in Spodoptera frugiperda Sf21 cells. However, occluded viruses expressing cv-PDG were not more resistant to UV inactivation than wild type AcMNPV when fed to either S. frugiperda or Trichoplusia ni neonate larvae. Surprisingly, however, viruses expressing cv-PDG showed a significant decrease in both the dose of occluded virus required for oral lethality and the time required for lethality compared to control virus, but these effects were only seen in S. frugiperda and not in T. ni larvae.     

Solar UV reduces Cryptosporidium parvum oocyst infectivity in environmental waters

Aims: To determine the effect of solar radiation on Cryptosporidium parvum in tap and environmental waters. Methods and Results: Outdoor tank experiments and a cell culture infectivity assay were used to measure solar inactivation of C. parvum oocysts in different waters. Experiments conducted on days with different levels of solar insolation identified rapid inactivation of oocysts in tap water (up to 90% inactivation within the first hour). Increased dissolved organic carbon content in environmental waters decreased solar inactivation. The role of solar ultraviolet (UV) in inactivation was confirmed by long-pass filter experiments, where UV-B was identified as the most germicidal wavelength. Reductions in oocyst infectivity following solar radiation were not related to a loss of excystation capacity. Conclusions: Solar UV can rapidly inactivate C. parvum in environmental waters. Significance and Impact of the Study: This is the first study to assess natural sunlight inactivation of C. parvum oocysts in surface waters and drinking water using an infectivity measure and determines the wavelengths of light responsible for the inactivation. The findings presented here provide valuable information for determining the relative risks associated with Cryptosporidium oocysts in aquatic environments and identify solar radiation as a critical process affecting the oocyst survival in the environment.     

Inactivation of viruses in red cell and platelet concentrates with aluminum phthalocyanine (AIPc) sulfonates.

Aluminum phthalocyanine tetrasulfonates (AIPcS) are photoactive compounds with absorption maxima at 665-675 nm. The inactivation of viruses (vesicular stomatitis virus, VSV; human immunodeficiency virus, HIV) added to either whole blood or red blood cell concentrates (RBCC) and platelet concentrates (PC) on treatment with tetrasulfonated AIPc (AIPcS4) was evaluated. Treatment of RBCC with 10 microM AIPcS4 and 44 J/cm2 visible light resulted in the inactivation of greater than or equal to 10(5.5) infectious doses (TCID50) of cell-free VSV, greater than or equal to 10(5.6) TCID50 of cell-associated VSV, and greater than or equal to 10(4.7) TCID50 of cell-free sindbis virus. Both greater than or equal to 10(4.2) TCID50 of cell-free and greater than or equal to 10(3.6) TCID50 of cell-associated forms of HIV were also shown to be inactivated. Encephalomyocarditis virus, used as a model for nonenveloped viruses, was not inactivated. Equivalent virus kill with Photofrin II required a substantially higher concentration of dye and longer exposure to visible light. Following AIPcS4 treatment, red cell integrity was well maintained as judged by the low level (less than 2%) of hemoglobin release immediately following treatment and on subsequent storage, by measurements of erythrocyte osmotic fragility, and by the normal recovery and circulatory survival on infusion of treated, autologous red blood cells in baboons. Treatment of PC with 10 microM AIPcS4 and 44 J/cm2 visible light also resulted in effective virus kill (greater than or equal to 10(5.5) TCID50) of VSV; however, both the rate and extent of platelet aggregation in response to collagen addition declined by at least 50%. Based on these results, further characterization of AIPcS4-treated RBCC is justified.     

Infectivity of RNA from inactivated poliovirus

During inactivation of poliovirus type 1 (PV-1) by exposure to UV, hypochlorite, and heat (72 degrees C), the infectivity of the virus was compared with that of its RNA. DEAE-dextran (1-mg/ml concentration in Dulbecco's modified Eagle medium buffered with 0.05 M Tris, pH 7.4) was used to facilitate transfecting PV-1 RNA into FRhK-4 host cells. After interaction of PV-1 RNA with cell monolayer at room temperature (21 to 22 degrees C) for 20 min, the monolayers were washed with 5 ml of Hanks balanced salt solution. The remainder of the procedure was the same as that for the conventional plaque technique, which was also used for quantifying the PV-1 whole-particle infectivity. Plaque formation by extracted RNA was approximately 100,000-fold less efficient than that by whole virions. The slopes of best-fit regression lines of inactivation curves for virion infectivity and RNA infectivity were compared to determine the target of inactivation. For UV and hypochlorite inactivation the slopes of inactivation curves of virion infectivity and RNA infectivity were not statistically different. However, the difference of slopes of inactivation curves of virion infectivity and RNA infectivity was statistically significant for thermal inactivation. The results of these experiments indicate that viral RNA is a primary target of UV and hypochlorite inactivations but that the sole target of thermal inactivation is the viral capsid.     

Determination of Pyrimidine Dimers in Escherichia coli and Cryptosporidium parvum during UV Light Inactivation, Photoreactivation, and Dark Repair

UV inactivation, photoreactivation, and dark repair of Escherichia coli and Cryptosporidium parvum were investigated with the endonuclease sensitive site (ESS) assay, which can determine UV-induced pyrimidine dimers in the genomic DNA of microorganisms. In a 99.9% inactivation of E. coli, high correlation was observed between the dose of UV irradiation and the number of pyrimidine dimers induced in the DNA of E. coli. The colony-forming ability of E. coli also correlated highly with the number of pyrimidine dimers in the DNA, indicating that the ESS assay is comparable to the method conventionally used to measure colony-forming ability. When E. coli were exposed to fluorescent light after a 99.9% inactivation by UV irradiation, UV-induced pyrimidine dimers in the DNA were continuously repaired and the colony-forming ability recovered gradually. When kept in darkness after the UV inactivation, however, E. coli showed neither repair of pyrimidine dimers nor recovery of colony-forming ability. When C. parvum were exposed to fluorescent light after UV inactivation, UV-induced pyrimidine dimers in the DNA were continuously repaired, while no recovery of animal infectivity was observed. When kept in darkness after UV inactivation, C. parvum also showed no recovery of infectivity in spite of the repair of pyrimidine dimers. It was suggested, therefore, that the infectivity of C. parvum would not recover either by photoreactivation or by dark repair even after the repair of pyrimidine dimers in the genomic DNA.     

Infectivity of RNA from Inactivated Poliovirus

During inactivation of poliovirus type 1 (PV-1) by exposure to UV, hypochlorite, and heat (72°C), the infectivity of the virus was compared with that of its RNA. DEAE-dextran (1-mg/ml concentration in Dulbecco's modified Eagle medium buffered with 0.05 M Tris, pH 7.4) was used to facilitate transfecting PV-1 RNA into FRhK-4 host cells. After interaction of PV-1 RNA with cell monolayer at room temperature (21 to 22°C) for 20 min, the monolayers were washed with 5 ml of Hanks balanced salt solution. The remainder of the procedure was the same as that for the conventional plaque technique, which was also used for quantifying the PV-1 whole-particle infectivity. Plaque formation by extracted RNA was approximately 100,000-fold less efficient than that by whole virions. The slopes of best-fit regression lines of inactivation curves for virion infectivity and RNA infectivity were compared to determine the target of inactivation. For UV and hypochlorite inactivation the slopes of inactivation curves of virion infectivity and RNA infectivity were not statistically different. However, the difference of slopes of inactivation curves of virion infectivity and RNA infectivity was statistically significant for thermal inactivation. The results of these experiments indicate that viral RNA is a primary target of UV and hypochlorite inactivations but that the sole target of thermal inactivation is the viral capsid.     

Inactivation of poliovirus 1 and F-specific RNA phages and degradation of their genomes by UV irradiation at 254 nanometers

Several models (animal caliciviruses, poliovirus 1 [PV1], and F-specific RNA bacteriophages) are usually used to predict inactivation of nonculturable viruses. For the same UV fluence, viral inactivation observed in the literature varies from 0 to 5 logs according to the models and the methods (infectivity versus molecular biology). The lack of knowledge concerning the mechanisms of inactivation due to UV prevents us from selecting the best model. In this context, determining if viral genome degradation may explain the loss of infectivity under UV radiation becomes essential. Thus, four virus models (PV1 and three F-specific RNA phages: MS2, GA, and Qbeta) were exposed to UV radiation from 0 to 150 mJ.cm-2. PV1 is the least-resistant virus, while MS2 and GA phages are the most resistant, with phage Qbeta having an intermediate sensitivity; respectively, 6-log, 2.3-log, 2.5-log, and 4-log decreases for 50 mJ.cm-2. In parallel, analysis of RNA degradation demonstrated that this phenomenon depends on the fragment size for PV1 as well as for MS2. Long fragments (above 2,000 bases) for PV1 and MS2 fell rapidly to the background level (>1.3-log decrease) for 20 mJ.cm-2 and 60 mJ.cm-2, respectively. Nevertheless, the size of the viral RNA is not the only factor affecting UV-induced RNA degradation, since viral RNA was more rapidly degraded in PV1 than in the MS2 phage with a similar size. Finally, extrapolation of inactivation and UV-induced RNA degradation kinetics highlights that genome degradation could fully explain UV-induced viral inactivation.     

Inactivation of murine norovirus, feline calicivirus and echovirus 12 as surrogates for human norovirus (NoV) and coliphage (F+) MS2 by ultraviolet light (254 nm) and the effect of cell association on UV inactivation

Aims: To determine inactivation profiles of three human norovirus (NoV) surrogate viruses and coliphage MS2 by ultraviolet (UV) irradiation and the protective effect of cell association on UV inactivation. Methods and Results: The inactivation rate for cell‐free virus or intracellular echovirus 12 was determined by exposure to 254‐nm UV light at fluence up to 100 mJ cm^?2. The infectivity of murine norovirus (MNV), feline calicivirus (FCV) and echovirus 12 was determined by cell culture infectivity in susceptible host cell lines, and MS2 infectivity was plaque assayed on Escherichia coli host cells. The UV fluencies to achieve 4‐log₁₀ inactivation were 25, 29, 30 and 70 (mJ cm^?2) for cell‐free FCV, MNV, echovirus 12 and MS2, respectively. However, a UV fluence of 85 mJ cm^?2 was needed to inactivate intracellular echovirus 12 by 4 log₁₀. Conclusions: Murine norovirus and echoviruses 12 are more conservative surrogates than FCV to predict the UV inactivation response of human NoV. Intracellular echovirus 12 was 2·8‐fold more resistant to UV irradiation than cell‐free one. Significance and Impact of the Study: Variation in UV susceptibilities among NoV surrogate viruses and a likely protective effect of cell association on virus susceptibility to UV irradiation should be considered for effective control of human NoV in water.     

Mechanisms and Rates of Decay of Marine Viruses in Seawater

Loss rates and loss processes for viruses in coastal seawater from the Gulf of Mexico were estimated with three different marine bacteriophages. Decay rates in the absence of sunlight ranged from 0.009 to 0.028 h^-1, with different viruses decaying at different rates. In part, decay was attributed to adsorption by heat-labile particles, since viruses did not decay or decayed very slowly in seawater filtered through a 0.2-μm-pore-size filter (0.2-μm-filtered seawater) and in autoclaved or ultracentrifuged seawater but continued to decay in cyanide-treated seawater. Cyanide did cause decay rates to decrease, however, indicating that biological processes were also involved. The observations that decay rates were often greatly reduced in 0.8- or 1.0-μm-filtered seawater, whereas bacterial numbers were not, suggested that most bacteria were not responsible for the decay. Decay rates were also reduced in 3-μm-filtered or cycloheximide-treated seawater but not in 8-μm-filtered seawater, implying that flagellates consumed viruses. Viruses added to flagellate cultures decayed at 0.15 h^-1, corresponding to 3.3 viruses ingested flagellate^-1 h^-1. Infectivity was very sensitive to solar radiation and, in full sunlight, decay rates were 0.4 to 0.8 h^-1. Even when UV-B radiation was blocked, rates were as high as 0.17 h^-1. Calculations suggest that in clear oceanic waters exposed to full sunlight, most of the virus decay, averaged over a depth of 200 m, would be attributable to solar radiation. When decay rates were averaged over 24 h for a 10-m coastal water column, loss rates of infectivity attributable to sunlight were similar to those resulting from all other processes combined. Consequently, there should be a strong diel signal in the concentration of infectious viruses. In addition, since sunlight destroys infectivity more quickly than virus particles, a large proportion of the viruses in seawater is probably not infective.     

Survival of the North American strain of viral hemorrhagic septicemia virus (VHSV) in filtered seawater and seawater containing ovarian fluid, crude oil and serum-enriched culture medium

The North American strain of viral hemorrhagic septicemia virus (NA-VHSV) could be recovered for up to 40 h in natural filtered seawater (27 ppt) with a 50% loss of infectivity after approximately 10 h at 15 degrees C. Addition of 10 ppb North Slope crude oil to the seawater had no effect on virus survival. However, when various concentrations of teleost ovarian fluid were added to seawater, virus could be recovered after 72 h at 0.01% ovarian fluid and after 96 h at 1.0%. When cell culture medium supplemented with 10% fetal bovine serum was added to the seawater, 100% of the virus could be recovered for the first 15 d and 60% of the virus remained after 36 d. These findings quantify NA-VHSV infectivity in natural seawater and demonstrate that ovarian fluid, which occurs naturally during spawning events, significantly prolongs the survival and infectivity of the virus. The extended stabilization of virus in culture medium supplemented with serum allows for low titer field samples to be collected and transported in an unfrozen state without significant loss of virus titer.