In Vitro Chick Embryo Cell Response to Strain MC29 Avian Leukosis Virus

Strain MC29 avian leukosis (myelocytomatosis) virus induced infection, elaboration of virus, and morphological alteration in chick embryo cells in vitro. Virus liberation began within 18 hr, morphological change was detectable at about 40 hr, and the cultures could be completely altered within 80 hr after infection. Altered cells were about half the volume and grew at approximately twice the rate of uninfected elements. The output of virus estimated by electron microscopy was about 140 particles per cell per hr. Deoxyribonucleic acid remained constant, but ribonucleic acid increased in both infected and control cells in adjustment to culture environment. The rates of uptake and incorporation of ³H-uridine and the incorporation of ³H-thymidine increased in the infected cells with onset of morphological change but were unaffected by processes of infection and virus elaboration per se. Incorporation of a ¹⁴C-amino acid mixture was slightly greater in the infected than in control cells. The speed of continuity of infection and massive morphological alteration constitute a unique response to avian tumor viruses, and the system gives promise of singular value for detailed studies of the processes of infection and morphological change.     

Enhanced Growth and Plaquing of Rabies Virus in Static Chick Embryo Cell Culture

The 7-day egg passage line of HEP Flury strain of rabies virus was inoculated to primary chick embryo (CE) cells prepared in different ways to compare efficiencies of viral growth and plaquing. Special care to minimize cellular damage due to trypsin at the step of monodispersion and sowing a comparatively large number of cells for monolayer preparation were required for rabies plaquing, whereas such cares were not necessary for plaquing of vesicular stomatitis virus. Plaque number and size were increased by incorporation of a high concentration of thymidine into cell growth medium. Various other means to produce a static state of CE cells were tested, and a maximal plaquing efficiency was obtained when dishes receiving a massive number of dispersed cells in MEM plus 1% calf serum were incubated at 37 C for 1 day without any buffering for monolayer preparation and postinfection incubation was done at 32 C in a CO₂-incubator. Bottle cultures of CE cells prepared in a similar manner, when infected with HEP Flury virus, yielded a markedly higher titer of virus than CE cells prepared by our previous standard method.     

Characterization of Wolbachia Host Cell Range via the In Vitro Establishment of Infections

Maternally transmitted bacteria of the genus Wolbachia are obligate, intracellular symbionts that are frequently found in insects and cause a diverse array of reproductive manipulations, including cytoplasmic incompatibility, male killing, parthenogenesis, and feminization. Despite the existence of a broad range of scientific interest, many aspects of Wolbachia research have been limited to laboratories with insect-rearing facilities. The inability to culture these bacteria outside of the invertebrate host has also led to the existing bias of Wolbachia research toward infections that occur in host insects that are easily reared. Here, we demonstrate that Wolbachia infections can be simply established, stably maintained, and cryogenically stored in vitro using standard tissue culture techniques. We have examined Wolbachia host range by introducing different Wolbachia types into a single tissue culture. The results show that an Aedes albopictus (Diptera: Culicidae) cell line can support five different Wolbachia infection types derived from Drosophila simulans (Diptera: Drosophilidae), Culex pipiens (Culicidae), and Cadra cautella (Lepidoptera: Phycitidae). These bacterial types include infection types that have been assigned to two of the major Wolbachia clades. As an additional examination of Wolbachia host cell range, we demonstrated that a Wolbachia strain from D. simulans could be established in host insect cell lines derived from A. albopictus, Spodoptera frugiperda (Lepidoptera: Noctuidae), and Drosophila melanogaster. These results will facilitate the development of a Wolbachia stock center, permitting novel approaches for the study of Wolbachia infections and encouraging Wolbachia research in additional laboratories.     

Host Adaptation of a Wolbachia Strain after Long-Term Serial Passage in Mosquito Cell Lines

The horizontal transfer of the bacterium Wolbachia pipientis between invertebrate hosts hinges on the ability of Wolbachia to adapt to new intracellular environments. The experimental transfer of Wolbachia between distantly related host species often results in the loss of infection, presumably due to an inability of Wolbachia to adapt quickly to the new host. To examine the process of adaptation to a novel host, we transferred a life-shortening Wolbachia strain, wMelPop, from the fruit fly Drosophila melanogaster into a cell line derived from the mosquito Aedes albopictus. After long-term serial passage in this cell line, we transferred the mosquito-adapted wMelPop into cell lines derived from two other mosquito species, Aedes aegypti and Anopheles gambiae. After a prolonged period of serial passage in mosquito cell lines, wMelPop was reintroduced into its native host, D. melanogaster, by embryonic microinjection. The cell line-adapted wMelPop strains were characterized by a loss of infectivity when reintroduced into the original host, grew to decreased densities, and had reduced abilities to cause life-shortening infection and cytoplasmic incompatibility compared to the original strain. We interpret these shifts in phenotype as evidence for genetic adaptation to the mosquito intracellular environment. The use of cell lines to preadapt Wolbachia to novel hosts is suggested as a possible strategy to improve the success of transinfection in novel target insect species.     

The Tumorgenicity of Glioblastoma Cell Line U87MG Decreased During Serial In Vitro Passage

Established cancer cell lines are routinely used to study cancer. Several factors such as serial passage may affect the reproducibility of experiments with cancer cell lines, but few researches focused on these changes. In the present study, different morphology and decreased tumorigenicity were observed in late passage U87MG cells. In vitro experiments further revealed that late passage U87MG cells possessed lower invasion properties than early passage, whereas no significant differences of proliferation and migration were found between early and late passage U87MG cells. In particular, we confirmed that late passage U87MG cells exhibited more epithelial phenotype with decreased PI3K/Akt pathway and TGF-β pathway expressions at protein level. In summary, our results focused on the changes of U87MG cells during serial in vitro passage, suggested that passage-induced changes may lead to notable changes of biological characteristics and several molecular transitions in cancer cell lines, indicating the necessity to shorten experiment-span and accomplish experiments with the same or similar passage cancer cell strains.     

Enhanced Virus Transformation of Hamster Embryo Cells In Vitro

Since transformation of hamster cells in vitro by simian virus 40 (SV40) is a rare event in a homogeneously infected cell population, physiochemical studies of the events of virus transformation are difficult. Similarly, other deoxyribonucleic acid-containing oncogenic viruses produce transformed-cell foci in vitro with low efficiency. Sublethal doses of X-ray irradiation, as well as preincubation of hamster embryo cells with the radiomimetic analogue, 5-iododeoxyuridine, markedly sensitized hamster embryo cells to SV40 in vitro. Agents were used at dosages which neither produced lethality nor caused neoplastic transformation in the absence of virus. Embryo cells maintained in vitro for long periods of time became increasingly more sensitive to SV40 transformation. Radiation also stimulated transformation by adenovirus 31. Delay in the addition of virus to preirradiated cells reduced the susceptibility to transformation by SV40 which was observed for cells infected immediately after irradiation, suggesting that radiation repair mechanisms or, possibly, release from radiation-induced "mitotic delay" may interfere with the process of neoplastic conversion by SV40.     

Biosynthesis and Expression of Gangliosides During Differentiation of Chick Embryo Retina Cells In Vitro

Cells from neural retina from 7-day chick embryos were cultured on polylysine-coated dishes up to 7 days. The small, round-shaped cells at seeding differentiated progressively, and after 4 days in vitro the majority had enlarged bodies and abundant processes. The content of protein and DNA was essentially unchanged during the entire period of culture. The incorporation of radioactivity from [3H]glucosamine into gangliosides declined slightly, reaching about 65% of the initial values at the end of the culture period. The proliferating activity measured by the incorporation of [3H]thymidine into DNA decreased to 10% or less of the initial value after 3 days in vitro. Almost at the same chronological times as in ovo, the synthesis of GD3 and of a ganglioside partially identified as GT3 decreased from 70 and 19% of the total incorporation into gangliosides in the first 20 h of culture to about 7 and 5%, respectively, after 3 days in vitro. Conversely, the synthesis of GD1a increased from about 6% at the beginning to about 70% at the end of the culture times. Immunocytochemical analyses of the expression of gangliotetraosyl gangliosides in cultured cells showed that these gangliosides appeared in the bodies and processes of cells having neuronal morphology; very little immunostaining of the scarce flattened cells, probably Müller cells, was found. The results indicate that the changes in ganglioside metabolism, which lead to decreased synthesis of gangliosides lacking the galactosyl-N-acetyl-galactosaminyl disaccharide end and to increased synthesis of gangliotetraosyl gangliosides, occur in cells that in culture differentiate into neurons.     

Titration and Extensive Serial Passages of Yaba Virus In Vitro

A sensitive assay system of Yaba virus (YV) was established in a cynomolgus monkey kidney cell line, JINET, in which the virus caused multilayered cellular foci countable even with the unaided eye. The specificity of the foci induced by YV in these cells was demonstrated by (1) the focus-forming ability was destroyed by heating at 60 C for 12 min; (2) the focus formation was inhibited by specific antiserum; (3) specific fluorescence was detected only in cells composing the foci when tested by fluorescent antibody technique; (4) a linear relationship was observed between the virus concentration and the number of foci formed; (5) YV preparation passed 20 times in JINET cells still possessed “tumorigenicity” in cynomolgus monkeys. The sensitivity of JINET cells to YV was comparable to that of cynomolgus monkeys, and YV was successively propagated in JINET cells with 2 log increase in infectivity titer during over 40 serial passages. Application of this assay system to growth kinetic studies of YV and quantitation of neutralizing antibody to YV is also discussed.     

Fate of Tobacco Mosaic Virus after Entering the Host Cell

Diminutive viral RNAs recovered from tobacco leaves inoculated with ³²P-TMV were investigated. At 3.5 hr after inoculation, most of the viral RNA without coat protein revealed two peaks after sucrose density gradient analysis of SDS-extract from 12,000 × g leaf pellet. The first peak appeared between bacterial ribosomal RNA of 16 S and 5 S and the second peak was around 5 S. These two peaks were digestible with RNase and they appeared as early as 5 min after inoculation. These diminutive RNAs seemed to be derived from partially uncoated parental virus by abscission of their naked RNA tails. The active formation of these diminutive RNAs and their early appearance after inoculation seemed to indicate that most of the inoculated TMV received incomplete uncoating.     

Fate of Tobacco Mosaic Virus after Entering the Host Cell

Tobacco leaves were inoculated with tobacco mosaic virus labeled with ³²P or ³⁵S. After various intervals, extracts of the leaves were prepared. In extracts from leaves infected for 5 to 360 min, about 40 to 60% of the virus retained on leaves was recovered in the pellet of the homogenate centrifuged at 12 000 × g. The virus associated with the 12 000 × g pellet was dissociable by treatment with pancreatic RNase, alkali or sodium dodecyl sulfate (SDS). The parental virus extracted by SDS from the pellet at 12 000 × g had a large amount of partially uncoated virus possessing naked RNA. Analysis by density gradient centrifugation suggested that, in addition to partially uncoated virus, some fragmented RNA was also associated with the 12 000 × g pellet. This fragmented RNA seemed to be derived from partially uncoated virus. Density gradient analysis of SDS extracts from the 12 000 × g pellet suggested that some of the virus underwent uncoating at the internal regions of the virus particle.     

An Avirulent Mutant of Rabies Virus Is Unable To Infect Motoneurons In Vivo and In Vitro

An antigenic double mutant of rabies virus (challenge virus standard [CVS] strain) was selected by successive use of two neutralizing antiglycoprotein monoclonal antibodies, both specific for antigenic site III. This mutant differed from the original virus strain by two amino acid substitutions in the ectodomain of the glycoprotein. The lysine in position 330 and the arginine in position 333 were replaced by asparagine and methionine, respectively. This double mutant was not pathogenic for adult mice. When injected intramuscularly into the forelimbs of adult mice, this virus could not penetrate the nervous system, either by the motor or by the sensory route, while respective single mutants infected motoneurons in the spinal cord and sensory neurons in the dorsal root ganglia. In vitro experiments showed that the double mutant was able to infect BHK cells, neuroblastoma cells, and freshly prepared embryonic motoneurons, albeit with a lower efficiency than the CVS strain. Upon further incubation at 37°C, the motoneurons became resistant to infection by the mutant while remaining permissive to CVS infection. These results suggest that rabies virus uses different types of receptors: a molecule which is ubiquitously expressed at the surface of continuous cell lines and which is recognized by both CVS and the double mutant and a neuron-specific molecule which is not recognized by the double mutant.     

狂犬病毒CTN—1株在Vero细胞上的适应传代研究

本文报导了用我国狂犬病毒固定毒人二倍体细胞适应株（ＣＴＮ－１）进行Ｖｅｒｏ细胞适应传代研究。通过连续传代培养,滴度可达８．０１ｏｇＬＤ５０／ｍｌ,达到了ＷＨＯ规定的不需浓缩的标准。病毒用０．０１ＭＯＩ感染细胞其产量与１Ｍｏｌ感染量相仿。病毒增殖高峰在４－５天,维持达１５天无明显下降,且可连续收获４－５次。因此,该毒种符合ＷＨＯ提出的疫苗生产毒种要求,可用于狂犬病疫苗生产。     

Parainfluenza Virus 5 Expressing the G Protein of Rabies Virus Protects Mice after Rabies Virus Infection

Rabies remains a major public health threat around the world. Once symptoms appear, there is no effective treatment to prevent death. In this work, we tested a recombinant parainfluenza virus 5 (PIV5) strain expressing the glycoprotein (G) of rabies (PIV5-G) as a therapy for rabies virus infection: we have found that PIV5-G protected mice as late as 6 days after rabies virus infection. PIV5-G is a promising vaccine for prevention and treatment of rabies virus infection.     

Mature Form of the Deoxyribonucleic Acid from Chick Embryo Lethal Orphan Virus

The deoxyribonucleic acid (DNA) of chick embryo lethal orphan (CELO) virus, an oncogenic avian adenovirus, had a biphasic denaturation profile indicating intramolecular base composition heterogeneity. This was confirmed by shearing the DNA and centrifuging it to equilibrium in Cs(2)SO(4) in the presence of HgCl(2) when two bands were formed. No circular molecules formed when CELO virus DNA was annealed, although lambda DNA formed circles under the same conditions. No circular molecules were found by sedimentation or electron microscopy when the DNA was digested with exonuclease III and then annealed, but 30 to 40% of T7 DNA molecules became circular under similar conditions. The complementary strands of CELO virus DNA both appeared to be continuous, and, when CELO DNA was denatured and then annealed under appropriate conditions, all of the renatured molecules were linear. It is concluded that CELO virus DNA consists of a unique rather than permuted collection of linear molecules that lack exposed single-strand complementary ends or duplex terminal repetitions. These results are discussed in relation to the replication of viral DNA and the transformation of host cells.     

鸡胚早期发育过程中细胞迁移的基因调控

鸡胚是发育生物学研究的经典动物模型,通过基因导入技术调节胚胎发育的基因功能,研究鸡胚早期发育过程中的细胞迁移,有助于更好地诠释相关先天性疾病的发生发展过程。在早期胚胎发育的过程中,原肠胚期三胚层的形成、心管的发生及神经嵴的发育都伴随着显著的细胞迁移过程。该文将结合近年来国内外对该过程的研究进展,介绍这三个不同时期细胞的迁移及相关基因调控。     

Acquisition of Sequences Homologous to Host DNA by Closed Circular Simian Virus 40 DNA II. Further Studies on the Serial Passage of Virus Clones

Three plaque isolates of SV40 strain 777 and 1 plaque isolate of strain 776 were grown to high-titer stocks and serially passaged, undiluted, in monkey BS-C-1 cells. In each case, the serial passaging procedure resulted in the accumulation of closed-circular SV40 DNA molecules containing covalently linked sequences homologous to reiterated host cell DNA (called substituted virus DNA). The relative yields, at a given passage level, of SV40 DNA with measurable homology to host DNA varied in different sets of serial passages, including passages of the same virus clone. More reproducible yields of substituted viral DNA progeny were obtained when the serial passaging procedure was initiated from earlier passages rather than from the original plaque-purified stock. Fractionation of closed-circular SV40 DNA molecules on alkaline sucrose gadients indicated that the majority of substituted virus DNA molecules are not plaque producers and are slightly smaller in size than plaque-forming DNA molecules which display no detectable homology to host DNA. Evidence that substituted SV40 DNA molecules replicate during serial undiluted passage was obtained from experiments which demonstrated (i) the presence of host sequences in replicative forms of the viral DNA and (ii) the incorporation of (3)H-thymidine into host sequences isolated from the mature substituted virus DNA molecule.