Mousepox in inbred mice innately resistant or susceptible to lethal infection with ectromelia virus. III. Experimental transmission of infection and derivation of virus-free progeny from previously infected dams

The incidence and duration of transmission of infection with ectromelia virus strain NIH-79 was tested in innately resistant (C57BL/6) and innately susceptible (BALB/c) inbred mice. Transmission by C57BL/6 index mice occurred through 3 weeks and by BALB/c index mice through 4 weeks, although the duration of infection in individual index mice was often shorter. Soiled caging that previously housed infected mice was inconsistently infectious. Transmission was high in cages where infected mice died and were cannibalized by cagemates, but was low to moderate in cages where there was no cannibalism. Infected mice that were bred 6 weeks after they were infected, delivered virus-free progeny and did not transmit infection to their non-immune breeding partners. Sentinel mice housed in the room with experimentally infected mice were seronegative for antibody to ectromelia virus and to other murine viruses. These results support the view that infection with NIH-79 virus is typically short-lived. They also indicate that breeding of recovered mice can save valuable colonies that have been exposed to ectromelia virus.     

Long-term survival of human rotavirus in raw and treated river water

This study was aimed at assessing the role of water as a vehicle for rotavirus spread by determining how well these viruses survive in the water environment. A cell culture adapted strain of human rotavirus subgroup 2, grown in MA-104 cells, was used as a model. Virus survival was tested in the following types of water samples, derived from the Ottawa River, at two different times of the year: (i) raw water (RW), (ii) muncipally treated tap water (TW), and (iii) raw water that had been filtered (FW) through a membrane (0.22 micron). The water samples, with approximately 5.0 X 10(4) plaque-forming units (PFU) of the virus, were held at either 4 or 20 degrees C and tested for infectious virus over a period of 64 days. The TW samples had a total and free chlorine content of 0.05 and less than 0.05 mg/L, respectively. The chlorine in these samples was not neutralized before virus contamination. Irrespective of the holding temperature, the virus titre in FW remained essentially unaltered throughout the test period. In TW held at 4 degrees C, there was no significant drop in the virus titre even after 64 days, whereas at 20 degrees C the titre in TW was reduced by about 2 log10 over the same period. Even though the loss of virus infectivity was most rapid in RW held at 20 degrees C, it took about 10 days for a 99.0% reduction in the plaque titre of the virus. These findings, therefore, indicate that rotaviruses can survive for several days in raw and treated river water thus making recreational and potable waters potential vehicles for the transmission of rotavirus infections.     

Mousepox in inbred mice innately resistant or susceptible to lethal infection with ectromelia virus. II. Pathogenesis

The pathogenesis of mousepox due to infection with ectromelia virus strain NIH-79 was characterized in genetically susceptible (BALB/cAnNCr) and genetically resistant (C57BL/6NCr) mice. BALB/c mice inoculated subcutaneous (s.c.) or intranasally (i.n.) had high mortality. Most mice died within 7 days from severe necrosis of the spleen and liver. Necrotic foci in livers of BALB/c mice that survived beyond 7 days often were accompanied by mononuclear cell infiltrates and by hyperplasia of lymphoid tissues. C57BL/6 mice inoculated by either route remained asymptomatic and necrotic lesions were mild or absent, whereas focal non-suppurative hepatitis and lymphoid hyperplasia were prominent. Infectious virus and viral antigen were distributed widely in tissues of BALB/c mice, but had limited distribution in C57BL/6 mice. Both mouse strains had infection of the respiratory tract, genital tract, oral tissues and bone marrow, and BALB/c mice also had infection of the intestines. Both strains also developed serum antibody to vaccinia virus antigen after infection. The results show that ectromelia virus occurs in tissues conducive to mouse to mouse transmission and that the severity and character of mousepox lesions correlate directly with resistance and susceptibility to infection. They also support the concept that cellular immunity contributes to survival from infection.     

Survival of spumavirus,a primate retrovirus,in laboratory media and water

The persistence of a previously characterized spumavirus strain (strain SV-522) was investigated utilizing various laboratory media and waters, including Eagle's minimal essential medium (EMEM) plus 0% fetal bovine serum (EMEM-0%), EMEM-2%, EMEM-10%, Chlamydia transport medium (CTM), phosphate-buffered saline, distilled, estuarine, and marine water, human serum, and the germicides, ethyl alcohol (70%) and sodium hypochlorite (10%). Experiments were performed at 4 degrees C and/or 23 degrees C. Infectivity endpoints were determined in stock aliquots upon initiation of testing and then after 3, 5, 7, and 10 days. The virus was reisolated from all diluents after 5 days at 23 degrees C and in EMEM-10% after 7 days. The virus was detected in CTM, EMEM-2%, EMEM-10%, and estuarine and marine waters after 7 days at 4 degrees C. Differences in the persistence of the virus may be ascribed to temperature and organic load. Water ionic strengths (e.g., estuarine vs. marine water) had no effect on modifying persistence of viral particles. Infectivity of spumavirus was undetectable after 30 s in 70% ethanol or 10% sodium hypochlorite. After 30 min at 23 degrees C, spumavirus infectivity in normal but not heat-inactivated human serum increased by almost 100-fold. Persistence of infectivity of primate spumavirus after 7 days in media and waters, and the agent's infectious potential in the human host, emphasize a need for cautious recognition during the manipulation of primate cells/organs and in the handling of primates themselves.     

Viricidal effects of Lactobacillus and yeast fermentation

The survival of selected viruses in Lactobacillus- and yeast-fermented edible waste material was studied to determine the feasibility of using this material as a livestock feed ingredient. Five viruses, including Newcastle disease virus, infectious canine hepatitis virus, a porcine picornavirus, frog virus 3, and bovine virus diarrhea, were inoculated into a mixture of ground food waste (collected from a school lunch program) containing Lactobacillus acidophilus. Mixtures were incubated at 20, 30, and 40 degrees C for 216 h. In a second trial, four viruses, including Newcastle disease virus, infectious canine hepatitis virus, frog virus 3, and a porcine picornavirus, were inoculated into similar edible waste material containing Saccharomyces cerevisiae. Mixtures were incubated at 20 and 30 degrees C for 216 h. Samples were obtained daily for quantitative (trial 1) and qualitative (trial 2) virus isolation. Temperature, pH, and redox potential were monitored. Controlled pH and temperature studies were also done and compared with the inactivation rates in the fermentation processes. In trial 1 (Lactobacillus fermentation), infectious canine hepatitis virus survived the entire test period in the fermentation process but was inactivated below pH 4.5 in the controlled studies. Newcastle disease virus was inactivated by day 8 in the fermentation process and appeared to be primarily heat sensitive and secondarily pH sensitive in the controlled studies. The porcine picornavirus survived the fermentation process for 8 days at 20 degrees C but was inactivated more rapidly at 30 and 40 degrees C. The controlled studies verified these findings. Frog virus 3 was inactivated by day 3 in the fermentation process and appeared to be sensitive to low pH in the controlled studies. Bovine virus diarrhea was rapidly inactivated in the fermentation process (less than 2 h) and was pH and temperature sensitive. In trial 2 (yeast fermentation), infectious hepatitis virus survived the entire test period in the fermentation process. Newcastle disease virus was inactivated by day 7 at 20 degrees C and day 6 at 30 degrees C. The porcine picornavirus was inactivated by day 7 at 30 degrees C but survived the entire test period at 20 degrees C. Frog virus 3 was inactivated by day 3 at 20 degrees C and day 2 at 30 degrees C.     

The effect of media, serum and temperature on in vitro survival of bovine blastocysts after Open Pulled Straw (OPS) vitrification

The recently introduced Open Pulled Straw (OPS) vitrification technique has successfully been used for cryopreserving porcine embryos as well as for bovine embryos and oocytes. The aim of this work is to investigate several factors on the in vitro survival of bovine blastocysts. In 5 experiments, a total of 862 in vitro produced blastocysts and expanded blastocysts was vitrified and warmed using the OPS technology, then cultured in vitro for an additional 3 days. The culture medium in Experiments 1 to 4 was SOFaa with supplements and 5% calf serum (CS). In Experiment 1, the replacement of TCM-199 + 20% CS with PBS + 20% CS in the holding medium during vitrification and warming did not result in significant differences in the re-expansion (92 vs 95%) and hatching rates (79 vs 72%). In Experiment 2, the PBS holding medium was supplemented with either 20% CS, 5 mg/mL bovine serum albumin (BSA) or 3 mg/mL polyvinylalcohol (PVA). Although the re-expansion rates did not differ (98, 95 and 93%, respectively), there was a decrease in the hatching rate after vitrification with PVA (77 and 78 vs 51%, respectively). In Experiment 3, the influence of temperature of equilibration media prior to and rehydration media after the vitrification was investigated. When the temperature of these media was adjusted to 20 degrees C instead of the standard 35 degrees C, both the re-expansion and the hatching rates decreased markedly. However, increasing the time of equilibration with the diluted cryoprotectant solution at 20 degrees C eliminated these differences. In Experiment 4, the ethylene-glycol and dimethyl sulfoxide cryoprotectant mixture was replaced with ethylene glycol-ficoll-trehalose solution. No difference in the re-expansion (89 vs 96%, respectively) or hatching rate (79 vs 84%, respectively) was detected. In Experiment 5, the vitrified-warmed blastocysts were cultured in SOFaa medium supplemented with 5% CS or 5 mg/mL BSA. Although the re-expansion rates were identical in the 2 groups (95%), the hatching rates were lower when embryos were cultured in BSA (71 and 47%, respectively). These findings indicated the possible broader application for OPS, as they demonstrated that the physical advantages of rapid cooling and warming may be accompanied by different chemical composition (holding media, cryoprotective additives) according to the requirements of the biological structure. Our study also shows the need for serum supplementation of the medium for hatching to occur after OPS vitrification.     

Protamine precipitation of two reovirus particle types from polluted waters.

Two forms of virus particle are released from reovirus-infected cell cultures, infectious reovirus and potentially infectious reovirus (PIV). PIV particle forms have a complete outer coat and are not infectious until the outer coat is altered or removed. The PIV concentration in polluted waters, however, has not been determined. Protamine sulfate precipitation, using 0.25% fetal bovine serum and 0.005% protamine sulfate for the first precipitation of the sample and 0.0025% for the second, was employed to concentrate infectious reovirus and PIV from water and sewage. Infectious reovirus and PIV particles were concentrated over 500-fold from river water inoculated with virus, and virus recoveries of between 80 and 100% were achieved. Virus precipitates stored at -20 degrees C as a protamine-virus concentrate showed a 5% loss of PIV after 14 days. Virus preparations were assayed, before and after treatment, with 200 micrograms of chymotrypsin per ml, using a fluorescent-antibody procedure. Protamine sulfate precipitation and fluorescent-antibody detection are effective ways to recover and assay reoviruses present in raw sewage.     

Tissue culture of the deep-sea eel<Emphasis Type="Italic"> Simenchelys parasiticus</Emphasis> collected at 1,162 m

We successfully cultivated fin cells of the deep-sea eel Simenchelys parasiticus (collected at 1,162 m) in L-15 medium supplemented with fetal bovine serum (FBS) and additional NaCl. We found that the pectoral fin cells proliferated in L-15 medium enriched with 4 g/l of NaCl salt (pH 7.3) containing 10% FBS at 10 degrees C and 15 degrees C. No cells were attached to the plastic culture plates when Dulbecco's modified Eagle's medium (pH 7.8) or 0-2 g/l of NaCl was added to the medium or when incubation was carried out at 4 degrees C. The majority of the explant outgrowth cells were detached when temperature increased to higher than 15 degrees C. The rate of proliferation of the fin cells was extremely slow and was dependent on the FBS concentration. Cell growth was enhanced by approximately 2.2-fold, and doubling time decreased from 170 h to 77 h when the FBS concentration was increased from 10% to 20% (v/v). Our established deep-sea eel cells were passaged 16 times over a 1-year period under atmospheric pressure conditions.     

Thermoinactivation of human cytomegalovirus

Vonka, Vladimir (Baylor University College of Medicine, Houston, Tex.), and Matilda Benyesh-Melnick. Thermoinactivation of human cytomegalovirus. J. Bacteriol. 91:221-226. 1966.-The inactivation at 4 and 37 C of several strains of human cytomegalovirus was studied. The preliminary findings that freshly harvested cytomegalovirus was inactivated more rapidly at 4 C than at higher temperatures was confirmed. Intracellular virus still within infected cells was found to be more stable at 4 C than virus released by sonic treatment just before incubation at 4 C. The composition of the diluent played an important role. In tris(hydroxymethyl)-aminomethane buffer, virus was unstable at both 4 and 37 C, with the rate of inactivation faster at 4 than at 37 C. Similar results were obtained when bicarbonate-phosphate buffer or Eagle's medium when bicarbonate was used as virus diluent. Calf serum stabilized the virus at 37 C, but not at 4 C. The deletion of bicarbonate from Eagle's medium had a stabilizing effect at both temperatures. An even greater stabilizing effect at both 4 and 37 C was obtained when distilled water was used as virus diluent. Inactivation rates varied from one strain to the next at 4 C but not at 37 C. Differences were found also with virus progeny derived from a single strain, but harvested at different stages during virus multiplication. Virus harvested early was more labile at 4 than at 37 C, whereas the late virus was more labile at the higher temperature. Intracellular and extracellular virus preparations were inactivated at the same rates at either 4 or 37 C.     

Development of water buffalo (Bubalus bubalis) embryos from in vitro matured oocytes reconstructed with fetal skin fibroblast cells as donor nuclei

The present study was carried out to explore the feasibility of using buffalo fetal skin fibroblasts as donor nuclei and to find out the developmental competence of embryos following transfer of these nuclei to in vitro matured enucleated buffalo oocytes. Skin cells were isolated from 1 to 2-month-old fetuses obtained from slaughterhouse, by enzymatic digestion (0.5% w/v trypsin +0.05% w/v collagenase in Dulbecco's PBS) for 15-20 min. The cells were washed 4 times with Dulbecco's PBS and then once with RPMI-1640+10% FBS by centrifugation at 600 x g. The cells were then cultured in the same medium in a CO2 incubator (5% CO2 in air) at 38.5 degrees C for 2-3 days. Cumulus-oocyte complexes (COCs) collected from slaughterhouse buffalo ovaries were subjected to IVM in the IVM medium (TCM-199 + 5 microg/ml FSH-P + 10 microg/ml LH+10% FBS) for 20-22 h in a CO2 incubator (5% CO2 in air) at 38.5 degrees C. Oocytes were denuded with 0.1% trypsin followed by repeated pipetting and then enucleated by aspirating the first polar body with 10-15% of nearby cytoplasm with a micromanipulator. Two different types of donor cells (growing cells and those arrested with cytochalasin-B) were used for reconstruction of oocytes. The reconstructs were electro fused and incubated in the activation medium (TCM-199 + 8 microg/ml cytochalasin-B+10% FBS) for 4 h. These were then cultured in IVC medium (TCM-199+10% FBS) in a CO2 incubator (5% CO2 in air) at 38.5 degrees C for 48 h. The cleaved embryos were then co-cultured with buffalo oviduct cells in embryo development media (EDM). Out of 119 denuded matured oocytes which were enucleated and reconstructed with growing cells, 78 (65.5%) were electro fused, activated and cultured, out of which 4 (5.1%) reconstructs cleaved and developed to 2-cell stage, 3 (3.8%) reached to 4-cell stage and 3 (3.8%) reached to 8-cell stage. In the synchronized group, out of 62 denuded matured oocytes which were reconstructed with cytochalasin-B blocked cells, 40 (65%) were electrofused, activated and cultured, out of which 4 (10%) developed to 2-cell stage, 3 (7.50%) to 4-cell stage, 2 (5.0%) to early morula stage and 1 (2.50%) to blastocysts stage. These results suggest that buffalo fetal skin fibroblasts could be used as donor nuclei for the production of buffalo embryos after nuclear transfer to enucleated in vitro matured buffalo oocytes.     

A simplified multiwell plate assay for the measurement of hepatitis A virus infectivity.

A standardized multiwell plate assay (MWPA) was developed to provide a simple in situ measurement of hepatitis A virus (HAV) infectivity titers. Following attachment (4 h, 35 degrees C) of serial 10-fold dilutions of HAV strain CR326 F (variant F') to confluent MRC-5 monolayers in 24-well plates, cultures were overlaid with maintenance medium and incubated for 35 days at 35 degrees C with weekly medium replacement. Cells were fixed with 90% acetone and HAV antigen was quantitated by reaction with a radio-iodinated anti-HAV serum. Measurement of virus infectivity was based on the amounts of specifically bound and eluted radiolabelled antibody. Virus titers (50% tissue culture infectious doses/ml, TCID50/ml) were calculated using the method of Reed & Muench (Am J Hyg. 1938; 27: 493-497), with wells producing cpm values greater than 2.1-times that of uninoculated controls considered positive. The use of a virus standard provided an estimate of the test variability (+/- 5% in Log10 TCID50 units). The MWPA offers a significant savings in time and reagents, and is proposed as a standard method for the simple and reliable measurement of the potency of HAV vaccine preparations.     

Survival of human parainfluenza viruses in the South Polar environment

The survival of human parainfluenza virus types 1, 2, and 3 was measured in both indoor and outdoor environments at South Pole Station, Antarctica, in an effort to determine the long-term survival of these viruses in this environment and to identify the possible source of respiratory tract illnesses which occurred in this isolated population in 1978 after 10 and 27 weeks of total social isolation. Viruses were applied to plastic petri plate surfaces which were then stored in indoor (21.4 degrees C; water vapor density, 1.50 g of water per m3) and outdoor environments (-22.4 to -33.2 degrees C; water vapor density, 0.706 and 0.247 g of water per m3). Parainfluenza virus type 1 at an initial titer of 3.75 log10 50% tissue culture infective doses per ml was inactivated after 4 days at room temperature and after 7 days outside. Parainfluenza virus type 2 and 3 at initial titers of 5.58 and 5.38 log10 50% tissue culture infective doses per ml were inactivated after 7 and 12 days, respectively, at room temperature and after 17 days of storage outside. Results indicate that the long-term survival of parainfluenza virus in either environment for up to 10 weeks is unlikely and probably did not provide the source of infectious virus responsible for the midisolation outbreaks of parainfluenza virus-related respiratory tract illnesses observed in this population during the 1978 winter season.     

Survival of human parainfluenza viruses in the South Polar environment.

The survival of human parainfluenza virus types 1, 2, and 3 was measured in both indoor and outdoor environments at South Pole Station, Antarctica, in an effort to determine the long-term survival of these viruses in this environment and to identify the possible source of respiratory tract illnesses which occurred in this isolated population in 1978 after 10 and 27 weeks of total social isolation. Viruses were applied to plastic petri plate surfaces which were then stored in indoor (21.4 degrees C; water vapor density, 1.50 g of water per m3) and outdoor environments (-22.4 to -33.2 degrees C; water vapor density, 0.706 and 0.247 g of water per m3). Parainfluenza virus type 1 at an initial titer of 3.75 log10 50% tissue culture infective doses per ml was inactivated after 4 days at room temperature and after 7 days outside. Parainfluenza virus type 2 and 3 at initial titers of 5.58 and 5.38 log10 50% tissue culture infective doses per ml were inactivated after 7 and 12 days, respectively, at room temperature and after 17 days of storage outside. Results indicate that the long-term survival of parainfluenza virus in either environment for up to 10 weeks is unlikely and probably did not provide the source of infectious virus responsible for the midisolation outbreaks of parainfluenza virus-related respiratory tract illnesses observed in this population during the 1978 winter season.     

Mousepox in inbred mice innately resistant or susceptible to lethal infection with ectromelia virus. I. Clinical responses

Clinical responses to infection with ectromelia virus strain NIH-79 were determined in several strains of inbred mice. All mice were equally susceptible to infection, but mortality was strain dependent. BALB/c AnNCr, A/JNCr, DBA/2NCr and C3H/He/NCr MTV- mice were highly susceptible to lethal infection whereas AKR/NCr and SJL/NCr mice were moderately susceptible and C57BL/6NCr mice were highly resistant. Death rates were influenced strongly by virus dose and by route of inoculation. High doses were associated with early and high mortality. For a given dose, intraperitoneal inoculation resulted in the highest mortality and death rates were progressively reduced in mice inoculated by the footpad, subcutaneous and intranasal routes. Footpad swelling was prominent in resistant mice and in survivors among susceptible strains. Deaths among AKR and SJL mice were sporadic and often occurred late irrespective of virus dose. It is suggested that this pattern could be influenced by secondary contact infections or by immunologic injury associated with host responses to ectromelia virus.     

Formulation and production of a blood-free and chemically defined virus production media for VERO cells

Vaccines provide effective protection against many infectious diseases as well as therapeutics for select pathologies, such as cancer. Many viral vaccines require amplification of virus in cell cultures during manufacture. Traditionally, cell cultures, such as VERO, have been used for virus production in bovine serum-containing culture media. However, due to concerns of potential adventitious agents present in fetal bovine serum (FBS), regulatory agencies suggest avoiding the use of bovine serum in vaccine production. Current serum-free media suitable for VERO-based virus production contains high concentrations of undefined plant hydrolysates. Although these media have been extensively used, the lack of chemical definition has the potential to adversely affect cell growth kinetics and subsequent virus production. As plant hydrolysates are made from plant raw materials, performance variations could be significant among different lots of production. We developed a chemically defined, serum-free medium, OptiVERO, which was optimized specifically for VERO cells. VERO cell growth kinetics were demonstrated to be equivalent to EMEM-10% FBS in this chemically defined medium while the plant hydrolysate-containing medium demonstrated a slower doubling time in both two-dimensional (2D) and 3D cultures. Virus production comparisons demonstrated that the chemically defined OptiVERO medium performed at least as good as the EMEM-10%FBS and better than the plant hydrolysate-containing media. We report the success in using recombinant proteins to replace undefined plant hydrolysates to formulate a chemically defined medium that can efficiently support VERO cell expansion and virus production.     

Development of bovine embryos in a cell-free culture medium: Effects of type of serum, timing of its inclusion and heat inactivation

Bovine embryos, derived from in vitro matured (IVM)/in vitro fertilized (IVF) ova, were used to investigate the effects of timing of serum inclusion in the culture medium and different types of blood sera and heat inactivation of the serum on embryo development. In Experiment 1, oocytes at 18 h post insemination were allocated to 1 of the following 4 treatments: 1) TCM-199 + 0.1 mg/ml polyvinylalcohol (PVA), 2) TCM-199 supplemented with 10% bovine calf serum (BCS), 3) PVA medium followed by BCS medium at 47 h, or 4) PVA medium followed by BCS medium at 82 h. Supplementation with BCS at 18 h post insemination suppressed (P<0.05) development of morulae/blastocysts (17.6%) when compared with PVA (30.5%) or with serum supplementation at 47 or 82 h post insemination (32.4 and 27.6%, respectively). However, inclusion of BCS at 18, 47 or 82 h post insemination produced more blastocysts (16.8, 29.3 and 22.1%, respectively; P<0.05) than medium +PVA (8.8%). In Experiment 2, ova were cultured from 18 h to 42 h post insemination in PVA-medium, then >/=2-cell embryos were transferred into serum-supplemented medium for another 168 h. Fetal bovine serum (FBS) +/- heat-inactivation (56 degrees C for 30 min, = heated FBS) suppressed morula/blastocyst development compared with medium + PVA, medium + BCS or medium + heated BCS (P<0.05). Bovine calf serum was superior to FBS in supporting blastocyst development (35.1 and 15.2%, respectively), but there was no difference between BCS and heated BCS. However, heated FBS increased the proportion of blastocysts/>/=8-cell embryos compared with that of FBS (51.0 and 31.4%, respectively; P<0.05). These results indicate that the type of serum supplementation and the timing of its inclusion in the culture medium markedly affect bovine embryo development in vitro, and that heat inactivation of serum with high embryotrophic properties is not necessary.     

Prolongation of herpes simplex virus latency in cultured human cells by temperature elevation.

Treatment of herpes simplex virus type 2 (HSV-2)-infected human fibroblast cells with cytosine arabinoside (ara-C) at 25 microgram/ml resulted in complete inhibition of virus replication. Removal of ara-C after 7 days of treatment ultimately resulted in renewed virus replication, but after a delay of at least 5 days. If however, the temperature was elevated from 37 degrees C to 39.5 to 40 degrees C at the time of ara-C reversal, infectious HSV-2 did not reappear. As long as the cultures were maintained at 39.5 to 40 degrees C (up to at least 128 days), HSV-2 was latent and infectious virus was undetectable. If the temperature was reduced to 37 degrees C at any time during the latent period, infectious virus was always reactivated, but only after a period of incubation at 37 degrees C of a least 11 days. Infectious-center assays performed with latent cultures indicated that only a very small fraction of cells could reactivate virus. The infectious-center titer did not show significant changes during much of the period of latency. This seemed to argue against the possibility that the latent cultures were synthesizing very small amounts of infectious virus. Additional studies were aimed at determining the minimum incubation period at 37 degrees C required to reactivate infectious HSV-2. Latent cultures reduced from 39.5 to 40 degrees C to 37 degrees C for less than 96 h did not yield infectious HSV-2, but those incubated at 37 degrees C for 96 h or more did.     

Replacement of serum and hormone additives with follicular fluid in the IVM medium: effects on maturation, fertilization and subsequent development of buffalo oocytes in vitro

Buffalo follicular fluid was used in the IVM medium in place of serum and hormone additives for stimulating nuclear and cytoplasmic maturation of buffalo oocytes in vitro. Follicular fluid (buFF) was aspirated from visible surface follicles from buffalo ovaries. Cumulus oocyte complexes (COCs) were matured for 24 to 26 h at 38.5 degrees C, 5% CO(2) in air in the maturation medium (TCM-199). When used, the concentration of fetal bovine serum (FBS) was 10% and that of FSH-P was 5 mug/ml. In Experiment 1 TCM-199 was supplemented with 1) FBS, 2) FBS + FSH-P, 3) 20% buFF and 4) 40% buFF. The matured oocytes were denuded and stained with Giemsa stain to study nuclear maturation. The proportion of oocytes which completed nuclear maturation was similar in medium containing FSH (74%) and 20 or 40% buFF (67%), which was higher (P < 0.05) than in medium with FBS but without FSH or buFF (47%). In Experiment 2, which was aimed at examining the effects of buFF on cumulus expansion and rates of fertilization and subsequent development to the blastocyst stage after IVF, the maturation medium was supplemented with 1) FBS + FSH-P, 2) 20% buFF and 3) 40% buFF. The COCs matured in medium containing 20 or 40% buFF had significantly higher (P < 0.01) cumulus expansion than those matured in medium with FBS + FSH-P. Of the COCs matured in medium with FBS + FSH-P and 20 or 40% buFF, the fertilization rates indicated by the incidence of cleavage (56, 51 and 52%, respectively) and the proportion of cleaved COCs developing to morula (58, 54 and 57%, respectively) and blastocyst stage (30, 31 and 35%, respectively) were not significantly different. In Experiment 3, supplementation of the maturation medium with 1) FBS + FSH-P and 2) FBS + FSH-P + 20% buFF resulted in similar rates of morulae (41 and 38%, respectively) and blastocysts (31 and 25%, respectively), indicating that simultaneous presence of FBS, FSH-P and buFF did not have an additive effect on embryo yield. The results show that the gonadotropin and serum source in the IVM medium can be replaced by buFF at the 20% level to achieve comparable morula and blastocyst yields.     

A型流感病毒持续感染细胞系来源的IVpi-189病毒生物学性状的初步研究

为明确E61-24-P15 A型重组流感病毒的第189代传代子病毒(IVpi-189)是否具备流感病毒温度敏感减毒活疫苗候选株的特点,将IVpi-189病毒感染MDCK细胞,并于不同培养温度条件下培养,观察其致细胞病变效应,病毒合成、释放情况,以及不同温度条件下病毒存活时间。结果显示32℃培养温度下,IVpi-189病毒具有等同于亲代野生病毒株的诱导细胞病变能力,而当培养温度上调至38℃,IVpi-189病毒致细胞病变效果出现缓慢且程度明显减轻。空斑形成单位实验发现IVpi-189病毒在38℃培养条件下增殖能力明显下降,其原因与病毒灭活速度及子病毒释放无关,但与感染细胞病毒合成能力下降有关。上述实验结果初步证实流感病毒持续感染细胞系来源的IVpi-189病毒具有温度敏感减毒活疫苗的生物学特性,在许可培养温度条件下具有良好的增殖能力,而在非许可培养温度下,病毒增殖活性受到明显抑制。本研究为流感病毒减毒活疫苗的开发研制提供实验佐证。