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.     

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.     

Optimization of environmental factors for the production and handling of recombinant retrovirus

Certain steps from the production to infection of the amphotrophic retroviral vector, MFG-LacZ, were optimized and the factors that affect retroviral titers were analyzed. Retroviral vector titers were highest when the culture supernatant was harvested 3 days after the producer cells had reached confluence. About a 2-fold increase in vector production was achieved at 32°C compared to that at 37°C. Low serum concentrations had no significant effect on the titers of virus produced by the CRIP cell line. Retroviral vectors were stable at 4°C but very unstable at 37°C and were quite sensitive to freezing and thawing. About 30%–50% of viral infectivity was lost during the thawing step and the loss was not recovered by the addition of commonly used cryoprotectants. Increase in viral exposure time for infection to target NIH3T3 cells was linearly proportional to the retroviral titer for up to 15 h. In addition, using DEAE-dextran in place of polybrene as a polycation during infection enhanced infection efficiency about 3-fold. The retrovirus was robust to simple ultrafiltration and its titer could be easily concentrated 16-fold. Taken together, our data suggest that at least a 100-fold increase in titer can be achieved with simple optimization.     

In Vitro Studies with Modoc Virus in Vero Cells: Plaque Assay and Kinetics of Growth, Neutralization, and Thermal Inactivation

A sensitive and quantitative assay system is described for plaquing Modoc virus in Vero cells. Neutralizing antibodies to Modoc virus could be detected by using this in vitro system by their interference with viral plaque formation. Virus was readily neutralized within 30 min at 37 C by a 1:10 dilution of hyperimmune hamster serum. The rate of neutralization and the total amount of virus neutralized was not altered significantly by the addition of 20 U of guinea pig complement to the hyperimmune hamster serum. A study of the growth of Modoc virus in Vero cells is also presented. After an initial latent period of 20 h, viral titer increased exponentially for 20 h. By 83 h after infection, 8,000 plaque-forming units of virus were detected per cell. The stability of viral infectivity in phosphate-buffered saline at pH 7.4 was evaluated. No reduction in viral titer was detected after 3 days at 7 or 22 C. A continuous decrease in infectivity at 37 C was observed, however, throughout the observation period.     

Persistent infectivity of a disease-associated herpesvirus in green turtles after exposure to seawater

Herpesviruses are associated with several diseases of marine turtles including lung-eye-trachea disease (LETD) and gray patch disease (GPD) of green turtles (Chelonia mydas) and fibropapillomatosis (FP) of green, loggerhead (Caretta caretta), and olive ridley turtles (Lepidochelys olivacea). The stability of chelonian herpesviruses in the marine environment, which may influence transmission, has not been previously studied. In these experiments, LETD-associated herpesvirus (LETV) was used as a model chelonian herpesvirus to test viral infectivity after exposure to seawater. The LETV virus preparations grown in terrapene heart (TH-1) cells were dialyzed for 24 to 120 hr against aerated artificial or natural seawater or Hank's balanced salt solution (HBBS). Fresh TH-1 cells were inoculated with dialyzed LETV, and on day 10 post-infection cells were scored for cytopathic effect. Virus samples dialyzed up to 120 hr were positive for the herpesvirus DNA polymerase gene by polymerase chain reaction. Electron microscopy revealed intact LETV nucleocapsids after exposure of LETV to artificial seawater or HBSS for 24 hr at 23 C. LETV preparations remained infectious as long as 120 hr in natural and artificial seawater at 23 C. Similar results were obtained with a second culturable chelonian herpesvirus, HV2245. LETV infectivity could not be detected after 48 hr exposure to artificial seawater at 30 C. Since LETV and HV2245 remain infectious for extended periods of time in the marine environment, it is possible that FP-associated and GPD-associated herpesviruses also may be stable. These findings are significant both for researchers studying the epidemiological association of herpesviruses with diseases of marine turtles and for individuals who handle turtles in marine turtle conservation efforts.     

Survival of human immunodeficiency virus (HIV), HIV-infected lymphocytes, and poliovirus in water.

The potential for human immunodeficiency virus (HIV) to enter domestic sewers via contaminated body fluids such as blood has spurred interest in the survival of this virus in water and wastewater. This study focused on establishing the inactivation of HIV and productively infected lymphocytes in dechlorinated tap water. In addition, HIV survival was compared with that of poliovirus. Results indicated that either free HIV or cell-associated HIV was rapidly inactivated, with a 90% loss of infectivity within 1 to 2 h at 25 degrees C and a 99.9% loss by 8 h. In comparison, poliovirus showed no loss of infectivity over 24 h. The presence of human serum in tap water slowed the rate of HIV inactivation through 8 h but did not stabilize the virus through 24 h. In addition, blood from stage IV AIDS patients was introduced into tap water, and the recovery of HIV was monitored by using both an infectivity assay and polymerase chain reaction amplification of viral sequences. Virally infected cells were no longer detectable after 5 min in dechlorinated tap water, while little diminution in amplifiable sequences was observed over 2 h. Thus, detection of viral sequences by polymerase chain reaction technology should not be equated with risk of exposure to infectious HIV.     

Adaptation of BHK-21 Cells to Growth in Shaker Culture and Subsequent Challenge by Japanese Encephalitis Virus

Baby hamster kidney (BHK-21) cells were adapted to grow in shaker culture using Waymouth medium 752/1 containing 20 mM N-2-hydroxyethyl-piperazine-N'-2'-ethanesulfonic acid buffer and supplemented with 2.5% (vol/vol) calf serum, 0.002% (wt/vol) sodium oleate, and 0.2% fatty acid-free bovine serum albumin (WO2.5). Infectivity of Japanese encephalitis virus grown in the cells adapted to WO2.5 approached 2 x 10(8) plaque-forming units per ml. The culture volume of infected cells was reduced fivefold 12 h after infection. This step resulted in a 10-fold increase in infectivity over that obtained from infected cultures not subjected to volume reduction.     

Initial fast reaction of bromine on reovirus in turbulent flowing water.

An apparatus is described for precise observation of the kinetics of the initial fast reaction of bromine with reovirus in turbulent flowing water. When quantitative electron microscopy shows that virus suspensions are essentially all single particles, the loss of infectivity follows first-order kinetics, the plaque titer falling at the rate of 3 log10 units/s at pH 7, 2 C, and at a 3-muM bromine concentration. Virus suspensions containing small aggregates (2 to 10/clump) exhibit a constantly decreasing disinfection rate with bromine. At a survival level of 10(-3) for single virions, the aggregated preparations have lost only 99% of their plaque titer and 10(-4) is reached only after 4 s of exposure. The disinfection rate does not appear to be a simple function of the size and frequency of aggregates in the virus suspension even when the aggregates contain no foreign material. Unpurified virus preparations (crude freeze-thaw lysates of infected cells) are shown, by zonal centrifugation, to contain 50% to over 90% of the infectivity in large, fast sedimenting aggregates. Such aggregates would strongly influence the bromine resistance of virus in polluted water.     

Initial fast reaction of bromine on reovirus in turbulent flowing water.

An apparatus is described for precise observation of the kinetics of the initial fast reaction of bromine with reovirus in turbulent flowing water. When quantitative electron microscopy shows that virus suspensions are essentially all single particles, the loss of infectivity follows first-order kinetics, the plaque titer falling at the rate of 3 log10 units/s at pH 7, 2 C, and at a 3-muM bromine concentration. Virus suspensions containing small aggregates (2 to 10/clump) exhibit a constantly decreasing disinfection rate with bromine. At a survival level of 10(-3) for single virions, the aggregated preparations have lost only 99% of their plaque titer and 10(-4) is reached only after 4 s of exposure. The disinfection rate does not appear to be a simple function of the size and frequency of aggregates in the virus suspension even when the aggregates contain no foreign material. Unpurified virus preparations (crude freeze-thaw lysates of infected cells) are shown, by zonal centrifugation, to contain 50% to over 90% of the infectivity in large, fast sedimenting aggregates. Such aggregates would strongly influence the bromine resistance of virus in polluted water.     

Stability of snakehead (Ophiocephalus striatus) rhabdovirus under different environmental conditions

Snakehead rhabdovirus stored in cell culture medium was found to be relatively temperature resistant with ≥ 99.9% reduction in infectivity only occurring after 6 months at 15°C, 6 weeks at 25°C and 10 days at 35°C. At 25°C, virus stability was greatest in the range pH 5–7. Infectivity was less well retained at pH9 and rapidly lost at pH3 and pH11. Stability was poor in serum-free media with a 99.9% reduction in infectivity occurring in less than 48 hours at 25°C in de-ionized water, tap water, balanced salt solutions and artificial seawater.     

Diagnosis of bovine respiratory syncytial virus infection by complement fixation test.

The complement fixation test by the microtiter method was applied to the serological diagnosis of bovine respiratory syncytial (RS) virus infection. When used as complement fixing antigens, untreated infected cell culture fluid, fluorocarbon-treated, and ether-treated materials showed no differences in antigenicity among them. The complement fixing antigenicity of bovine RS virus appeared in bovine kidney and Vero cell cultures for the first time 4 days after inoculation. Both the infectivity and complement fixing antigenicity reached a maximum 6 days after inoculation. In detecting complement fixing antibody from infected cattle, the most outstanding specific reaction was obtained when 5% fresh normal calf serum had been added to the diluent of complement. Neutralizing and complement fixing antibodies were examined in serum samples from two cattle in the course of experimental infection. It was found that both antibodies turned to be positive 2 weeks after inoculation. There was a linear correlation between neutralizing and complement fixing antibody titers, when serum samples from 40 natural cases were tested in the acute and convalescent stages. In addition, common antigenicity was demonstrated between the virus of bovine origin and the Long strain of human RS virus by complement fixation test.     

Toxoplasma gondii antigens: recovery analysis of tachyzoites cultivated in Vero cell maintained in serum free medium

Vero cells have been used successfully in Toxoplasma gondii maintenance. Medium supplementation for culture cells with fetal bovine serum is necessary for cellular growth. However, serum in these cultures presents disadvantages, such as the potential to induce hypersensitivity, variability of serum batches, possible presence of contaminants, and the high cost of good quality serum. Culture media formulated without any animal derived components, designed for serum-free growth of cell lines have been used successfully for different virus replication. The advantages of protozoan parasite growth in cell line cultures using serum-free medium remain poorly studied. Thus, this study was designed to determine whether T. gondii tachyzoites grown in Vero cell cultures in serum-free medium, after many passages, are able to maintain the same antigenic proprieties as those maintained in experimental mice. The standardization of Vero cell culture in serum-free medium for in vitro T. gondii tachyzoite production was performed establishing the optimal initial cell concentration for the confluent monolayer formation, which was 1×10(6) Vero cell culture as initial inoculum. The total confluent monolayer formatted after 96 h and the best amount of harvested tachyzoites was 2.1×10(7) using parasite inoculum of 1.5×10(6) after 7 days post-infection. The infectivity of tachyzoites released from Vero cells maintained in serum-free medium was evaluated using groups of Swiss mice infected with cell-culture tachyzoites. The parasite concentrations were similar to those for mice infected with tachyzoites collected from other infected mice. The data from both in vivo and in vitro experiments showed that in at least 30 culture cell passages, the parasites maintained the same infectivity as maintained in vivo. Another question was to know whether in the several continued passages, immunogenic progressive loss could occur. The nucleotide sequences studied were the same between the different passages, which could mean no change in their viability in the lysate antigen. Thus, the antigen production by cell culture has clear ethical and cost-saving advantages. Moreover, the use of culture media formulated without any human or animal derived components, designed for serum-free growth of cell lines, successfully produced tachyzoites especially for antigen production.     

Persistence of human rhinovirus infectivity under diverse environmental conditions

The persistence of human rhinovirus type 2 and type 14 infectivity was studied under various laboratory conditions designed to mimic those commonly found in the environment. The effects of temperature, ionic strength, protein content, and evaporation were compared. Both viruses were stable (less than 0.3-log decrease in titer) at 6 and 23 degrees c for 24 h in the liquid state regardless of salt or protein additives; a titer decrease of less than 1.0 log was noted at 37 degrees C. However, evaporation at 37 degrees C reduced virus infectivity by 3.2 to 4.5 logs in buffered water, an effect which could be significantly lessened by the addition of bovine serum albumin in saline (2.0- to 2.9-log decrease in titer). These studies support and extend observations by others that the human rhinoviruses retain sufficient infectivity after drying on hard surfaces to permit their transmission to susceptible persons upon contact.     

Persistence of human rhinovirus infectivity under diverse environmental conditions.

The persistence of human rhinovirus type 2 and type 14 infectivity was studied under various laboratory conditions designed to mimic those commonly found in the environment. The effects of temperature, ionic strength, protein content, and evaporation were compared. Both viruses were stable (less than 0.3-log decrease in titer) at 6 and 23 degrees c for 24 h in the liquid state regardless of salt or protein additives; a titer decrease of less than 1.0 log was noted at 37 degrees C. However, evaporation at 37 degrees C reduced virus infectivity by 3.2 to 4.5 logs in buffered water, an effect which could be significantly lessened by the addition of bovine serum albumin in saline (2.0- to 2.9-log decrease in titer). These studies support and extend observations by others that the human rhinoviruses retain sufficient infectivity after drying on hard surfaces to permit their transmission to susceptible persons upon contact.     

绵羊卵泡液和次黄嘌呤对卵母细胞体外减数分裂的影响

目的　利用在培养液中添加绵羊卵泡液和次黄嘌呤 ,抑制卵母细胞GVBD发生 ,延长转录活性 ,从而使卵母细胞真正成熟 ,提高胚胎质量及生产效率。方法　利用体外成熟技术对有屠宰采集的绵羊卵母细胞进行培养 ,培养液中添加卵泡液及次黄嘌呤 ,检查成熟效果。结果　将卵母细胞培养在 5 0 %和 10 0 %的卵泡液中 ,2 4h后处于GV期的卵母细胞分别为 19% (8 4 2 )和 33 3% (13 39)。在含有 4mmol L次黄嘌呤的培养液中 ,2 4h后有2 1 6 % (16 74 )的卵母细胞处GV期 ,而对照组中只有 6 % (3 5 0 ) ,经过次黄嘌呤处理的卵母细胞多数都停滞于PⅠ期(44 6 % ,33 74 )。在 4mmol L次黄嘌呤培养液中添加FSH并未使受到抑制的卵母细胞诱导成熟。结论　卵泡液和次黄嘌呤只能在有限的程度上抑制减数分裂的重新启动 ,并对减数分裂的全过程都有影响 ,这种影响程度与抑制因子的浓度相关 ,存在明显的剂量效应。     

Influence of osmoregulation processes on starvation survival of Escherichia coli in seawater.

The adaptation of enteric bacteria in seawater has previously been described in terms of nutrient starvation. In the present paper, we bring experimental arguments suggesting that survival of these microorganisms could also depend on their ability to overcome the effects of osmotic stress. We analyzed the influence of osmoregulatory mechanisms (potassium transport, transport and accumulation of organic osmolytes) on the survival of Escherichia coli in seawater microcosms by using mutants lacking components of the osmotic stress response. Long-term protection was afforded to cells by growth in a medium whose osmotic pressure was increased by either NaCl, LiCl, or saccharose. Achievement of the protection state depended at least partly on osmoregulatory mechanisms, but differed when these were activated or induced during prior growth or in resting cells suspended in phosphate buffer or in seawater. When achieved during growth, K+ transport, glycine-betaine (GBT) synthesis or transport, and trehalose synthesis helped increase the ability to survive in seawater. Protection by GBT was also obtained with resting cells in a phosphate buffer at high osmotic pressure. However, when added only to the seawater, GBT did not change the survival ability of cells no matter what their osmoregulation potential. These results showed that the survival of E. coli cells in seawater depends, at least partly, on whether they possess certain genes which enable them to regulate osmotic pressure and whether they can be stimulated to express those genes before or after their release into the environment. This expression requires nutrients as the substrates from which the corresponding gene products are made.     

An analysis of factors influencing the isolation rate of herpes simplex virus.

Attempts were made to improve the rate of isolation of herpes simplex virus (HSV) from clinical specimens by minimizing loss of virus infectivity during transportation and employing the most sensitive cells for isolation. Basical analyses using standard strains of type 1 and type 2 HSV indicated that virus titer decrease was marked even at low temperatures in environments free of proteinous stabilizer such as normal serum or tissue extract, negating the generally held concept that HSV is stable in distilled water. YLE (Earle-lactalbumin HYDROLYSATE-YEAST EXTRACT) medium containing 20% inactivated calf serum was determined to be a transport medium of choice, because degradation of suspended virus during storage and freeze-thawing was negligible and loss of virus during Millipore filtration was minimal. Special coating of the membrane could also be obviated by the use of this solution. In a cell susceptibility test using clinical specimens, secondary rabbit kidney (SRK) cells were the most sensitive, showing a quick development of cytopathic effect. Vero and RK-13 cells were the second best, whereas monkey kidney, HeLa and L cells were far less sensitive. A total of 136 specimens from suspected cases, sent by dermatologists, were tested using SRK cells, and 99 strains of type 1 and 15 strains of type 2 HSV were isolated. Excluding one case from which vaccinia virus was isolated, the isolation rate of HSV was 84.4%.     

In vitro culture technique for Cryptocaryon irritans, a parasitic ciliate of marine teleosts

A medium for the in vitro culture of Cryptocaryon irritans, which is an obligatorily parasitic ciliate of marine teleosts and causes 'white spot disease', was developed. The medium consisted of a layer of cultured fish cells (FHM), with an agarose gel layer covering the cell layer. The agarose gel contained 0.22% agarose, 10% fetal calf serum, 100 I.U. ml(-1) Penicillin G potassium and 100 microg ml(-1) streptomycin sulphate. Theronts of C. irritans transformed to trophonts and grew to 180 microm in mean length in the medium, although they gradually decreased in number. When trophonts fully developed in medium were transferred into seawater 4 d after inoculation, approximately 70% of them transformed to encysted tomonts and released theronts. When fish were challenged with theronts obtained from in vitro-raised parasites, approximately 40% of the theronts were recovered from fish, indicating comparative infectivity of in vitro-raised theronts to those of in vivo-raised theronts. This is the first report that C. irritans fully developed in vitro and its entire life cycle was completed without a host fish.     

Quantity and infectivity of embryo-associated bovine viral diarrhea virus and antiviral influence of a blastocyst impede in vitro infection of uterine tubal cells

In previous studies, bovine viral diarrhea virus (BVDV) remained associated with IVF embryos after viral exposure and washing. However, uterine tubal cells (UTC) were not infected when exposed embryos were washed and individually co-cultured with them. The objective of this study was to evaluate quantity and infectivity of embryo-associated virus and antiviral influence of a blastocyst as possible explanations for failure to infect the UTC in vitro. Morulae and blastocysts were produced in vitro and washed. A portion of the embryos were incubated for 2 h in medium containing 10(6) to 10(8) cell culture infective doses (50%, CCID50) of a genotype I, noncytopathic BVDV per milliliter and then washed again. Virus isolation was attempted on sonicated negative (virus unexposed) and positive (virus exposed) control embryo groups after washing. The influence of quantity and infectivity of embryo-associated virus was evaluated by transferring exposed, washed embryo groups (2, 5, and 10 embryos/group) or sonicate fluid of exposed, washed, sonicated embryo groups (2, 5, and 10 embryos/group) to cultures containing bovine UTC in IVC medium that was free of BVDV neutralizing activity. The antiviral influence of an embryo was evaluated by adding 1 to 10(5) CCID50 of BVDV to UTC in the presence or absence of a single unexposed blastocyst in IVC medium. After 2 d in co-culture, the UTC, IVC medium and washed embryos (when present) were tested separately for the presence of BVDV using virus isolation. Virus was isolated from sonicate fluids of all positive but no negative controls. Virus was not isolated from any UTC following 2 d of culture with virally exposed groups of intact embryos. However, virus was isolated from UTC cultured with sonicate fluids from some groups of 5 (60%) and 10 (40%) embryos. Infective virus also remained associated with some groups of 2 (20%), 5 (40%) and 10 (60%) intact embryos after 48 h of post-exposure culture. Finally, primary cultures of UTC were more susceptible to infection with BVDV in the absence of a blastocyst (P = 0.01). Results indicate that insufficient quantity and reduced infectivity of embryo-associated virus as well as an antiviral influence of intact IVF blastocysts may all contribute to failure of embryo-associated virus to infect UTC in vitro.     

Rolling round bottle culture of HmLu-1 cells and the production of bovine ephemeral fever virus.

An attempt was made to cultivate HmLu-1 cells in a rolling round bottle. As a result, the optimum conditions of cultivation were found to consist in the number of cells transplanted per bottle being 1 X 10(8), the volume of growth medium per bottle being 250 ml, and the velocity of rolling being 6 revolutions per hour. It was possible to make a monolayer of cells develop all over the glass surface under these conditions. A preliminary experiment was carried out to clarify the production of virus in the tube culture. In it, the highest virus titer was obtained two days after inoculation of a 4-day-old culture with a 1:100 dilution of stock virus. On the other hand, when the 4-day-old culture cells in the rolling round bottle were inoculated with virus suspension and when 100, 500, or 800 ml of maintenance medium was added to each bottle, there was little difference in virus titer obtained among the culture bottles. Then the virus yield per cell was compared between the rolling round bottle culture method and the stationary square bottle culture method. The highest virus titer was reached two days after virus inoculation, regardless of the culture method. The virus yield was 1.9 times as high in the rolling method as in the stationary method. From the results mentioned above, it was clarified that the rolling round bottle culture method made it possible to obtain a large amount of bovine ephemeral fever virus at a high titer in a labor-saving manner.