Continuous production of hepatitis A virus in PLC/PRF/5 cell cultures: use of antigen for serology

The strain CF53 of hepatitis A virus (HAV) previously adapted to growth in PLC/PRF/5 cells was grown in 175 cm2 flasks, at different passages. After infection, cells were incubated at 32 degrees C in RPMI 1640 medium supplemented with 2.5% foetal calf serum (FCS) for 6-12 months. HAV which was released continuously in the culture medium was harvested weekly. Hepatitis A virus antigen (HAAg) and infectious virus production was stable during each passage. The antigen titre, determined by radioimmunoassay, was about 50 for each passage whereas the infectious virus titre increased from 10(3.7) (passage 7) to 10(6.0) TCID50/ml (passage 13). Virus production was not influenced by the FCS concentration (0-2.5%) in the maintenance medium. The cell culture produced HAAg was used for detection of total anti-HAV antibodies, anti-HAV titration and IgM antibody capture assay and the results were identical to those obtained with commercial kits. HAAg produced by this practical and cheap method could easily replace primate derived antigen for the detection of anti-HAV antibodies.     

Effects of chicken anaemia virus and infectious bursal disease virus-induced immunodeficiency on infectious bronchitis virus replication and genotypic drift

We followed changes in a portion of the S1 gene sequence of the dominant populations of an infectious bronchitis virus (IBV) Arkansas (Ark) vaccine strain during serial passage in chickens infected with the immunosuppressive chicken anaemia virus (CAV) and/or infectious bursal disease virus (IBDV) as well as in immunocompetent chickens. The IBV-Ark vaccine was applied ocularly and tears were collected from infected chickens for subsequent ocular inoculation in later passages. The experiment was performed twice. In both experiments the dominant S1 genotype of the vaccine strain was rapidly and negatively selected in all chicken groups (CAV, IBDV, CAV + IBDV and immunocompetent). Based on the S1 genotype, the same IBV subpopulations previously reported in immunocompetent chickens and named component (C) 1 to C5 emerged both in immunocompetent and immunodeficient chickens. During the first passage different subpopulations emerged, followed by the establishment of one or two predominant populations after further passages. Only when the subpopulation designated C2 became established in either CAV-infected or IBDV-infected chickens was IBV maintained for more than four passages. These results indicate that selection does not cease in immunodeficient chickens and that phenotype C2 may show a distinct adaptation to this environment. Subpopulations C1 or C4 initially became established in immunocompetent birds but became extinct after only a few succeeding passages. A similar result was observed in chickens co-infected with CAV + IBDV. These results suggest that the generation of genetic diversity in IBV is constrained. This finding constitutes further evidence for phenotypic drift occurring mainly as a result of selection.     

Studies on avian infectious bronchitis virus (IBV)

Summary The growth of ten strains of avian infectious bronchitis virus (IBV) in several cultured cells was examined. The cultured cells used were chick kidney (CK), chick embryo (CE), HeLa and BHK-21 cells. The results obtained can be summarized as follows. 1. All the strains showed similar growth curves in CK cells. Progeny viruses appeared in the culture medium 4 to 6 hours after inoculation and peak virus titers of 10^6.5–10^8.5 TCID₅₀ per 0.1 ml were obtained after 36 hours. A cytopathogenic effect (CPE) was detected within 24 hours. No distinct CPE and low multiplicities were observed on culturing at 30° C. 2. All strains replicated in CE cells, although only small amounts of virus were produced. No CPE was observed. 3. Only Beaudette-42 and Holte strains grew in BHK-21 cells. 4. No IBV strains grew in HeLa cells.With 6 Figures     

Primary isolation and serial passage of hepatitis A virus strains in primate cell cultures

Although several primate cell types have been reported to support replication of hepatitis A virus, optimal conditions for the isolation and production of quantities of virus have not been defined. We therefore examined seven different primate cell types for their ability to support replication of primate-passaged and wild-type virus as reflected by intracytoplasmic accumulation of viral antigen (direct immunofluorescence and radioimmunoassay) and propagation of cell culture-adapted virus. Of the cells tested, low-passage African green monkey kidney (AGMK) cells were most sensitive for initial isolation. Viral replication was documented after inoculation of AGMK cells with seven of nine hepatitis A virus antigen-positive fecal specimens (from seven epidemiologically distinct sources). With six inocula, virus was successfully passed in serial cultures. AGMK-adapted virus was readily propagated in continuous AGMK (BS-C-1) cells. The optimal temperature for the growth of virus in BS-C-1 cells was 35 degrees C. Viral release into supernatant fluids was documented in the absence of any cytopathic effect, and infectivity titers in supernatant fluids 21 days after inoculation (50% tissue culture infective does [TCID50], 10(6.0)/ml) equalled or exceeded those in the cell fraction (TCID50, 10(5.5)/ml). Cells maintained in serum-free media readily supported viral growth, with yields of virus (TCID50, 10(6.5)/ml) equal to or greater than those obtained with cells maintained in 2% fetal bovine serum.     

Effect of pH on the growth and cytopathogenicity of avian infectious bronchitis virus in chick kidney cells

The growth of avian infectious bronchitis virus (IBV) in chick kidney cells at different pH values in the range 6.0-9.0 demonstrated that although the virus was released at a much faster rate at the higher pH values the titre tended to drop more quickly. At the acid pH values the virus was released more slowly but reached a maximum titre similar to that at the higher pH values and showed only minimum reduction in infectivity up to 49 hours post inoculation. The stability of virus in tissue culture medium was shown to be directly related to pH 6.0-8.0, being more stable at the acid pH values. The degree of cytopathogenicity induced in chick kidney cells following infection with IBV was directly related to the pH at which the cells were incubated, occurring earlier and more extensively in cells at the higher pH values. Cell macromolecule synthesis in chick kidney cells was inhibited following infection with IBV and was apparently due to cell damage and death.     

Replication and morphogenesis of avian coronavirus in Vero cells and their inhibition by monensin

Avian infectious bronchitis virus (IBV) was adapted to Vero cells by serial passage. No significant inhibition of IBV replication was observed when infected Vero cells were treated with α-amanitin or actinomycin D. In thin sections of infected cells, assembly of IBV was observed at the rough endoplasmic reticulum (RER), and mature IBV particles were located in dilated cisternae of the RER as well as in smooth cytoplasmic vesicles. In addition to typical IBV particles, enveloped particles containing numerous ribosomes were identified at later times postinfection. Monensin, a sodium ionophore which blocks glycoprotein transport to plasma membranes at the level of the Golgi complex, was found to inhibit the formation of infectious IBV. In thin sections of infected Vero cells treated with the ionophore, IBV particles were located in dilated cytoplasmic vesicles, but fewer particles were found when compared to controls. A similar pattern of virus-specific proteins was detected in control or monensin-treated IBV-infected cells, which included two glycoproteins (170000 and 24000 daltons) and a polypeptide of 52000 daltons. These results suggesl lhal the ionophore inhibits assembly of a virus which malures at intracellular membranes.     

Alterations in virus protein synthesis and capsid production in infection with DI particles of herpesvirus

High multiplicity, undiluted passage of equine herpesvirus type 1 (EHV-1) in L-M cells resulted in the rapid production of virus particles whose genome was genetically less complex, contained more reiterated DNA sequences and exhibited a greater buoyant density (rho = 1.724 g/ml) than the DNA (rho = 1.716 g/ml) of standard virus. These data and the finding that these particles inhibited the replication of standard virus in interference assays confirmed that these were defective interfering (DI) particles (Henry et al. 1979). Additional evidence for this has been obtained from the pattern of cyclic fluctuation in infectious virus titre through 17 serial passages as well as from the pronounced variation in the particle to plaque ratio for each passage. Total particle production was markedly reduced in cells infected with virus preparations containing DI particles and quantification of major cell-associated EHV-1 capsid species by electron microscopy and analysis in Renografin density gradients indicated that this reduction occurred at the level of capsid assembly. Although total capsid production was reduced in cells infected with DI particle preparations, the synthesis of I (immature) capsids increased relative to that of L (empty) capsids and these alterations in the assembly of capsid species could be related to changes in the synthesis of capsid proteins. In cells infected with EHV-1 preparations rich in DI particles, the synthesis of major capsid protein 150000 was greatly reduced, whereas core protein 46000, a major component of I capsids, was overproduced as compared to standard virus infection. Capsids produced in cells infected with virus preparations rich in DI particles were identical in polypeptide composition to those made in standard virus infection.     

Cell cycle arrest and apoptosis induced by the coronavirus infectious bronchitis virus in the absence of p53

Manipulation of the cell cycle and induction of apoptosis are two common strategies used by many viruses to regulate their infection cycles. In cells infected with coronaviruses, cell cycle perturbation and apoptosis were observed in several reports. However, little is known about how these effects are brought out, and how manipulation of the functions of host cells would influence the replication cycle of coronavirus. In this study, we demonstrate that infection with coronavirus infectious bronchitis virus (IBV) imposed a growth-inhibitory effect on cultured cells by inducing cell cycle arrest at S and G(2)/M phases in both p53-null cell line H1299 and Vero cells. This cell cycle arrest was catalyzed by the modulation of various cell cycle regulatory genes and the accumulation of hypophosphorylated RB, but was independent of p53. Proteasome inhibitors, such as lactacystin and NLVS, could bypass the IBV-induced S-phase arrest by restoring the expression of corresponding cyclin/Cdk complexes. Our data also showed that cell cycle arrest at both S- and G(2)/M-phases was manipulated by IBV for the enhancement of viral replication. In addition, apoptosis induced by IBV at late stages of the infection cycle in cultured cells was shown to be p53-independent. This conclusion was drawn based on the observations that apoptosis occurred in both IBV-infected H1299 and Vero cells, and that IBV infection did not affect the expression of p53 in host cells.     

Multiplicity-dependent host range restriction of human adenovirus in human embryonal carcinoma cells

The ability of human adenovirus type 5 to replicate and produce infectious virus was examined in two different cell lines, HeLa cells and Tera I, a human embryonal carcinoma cell line. High titers of infectious adenovirus were produced (10⁹ PFU/ml) by growth in HeLa cell cultures independent of the input multiplicity of infection (by 96 hr postinfection). On the other hand, 3–4 logs less infectious virus were synthesized in Tera I cells when low (0.01–0.1 PFU/cell) input multiplicities of infection were used. At input multiplicities of 1, 10, or 100 PFU/cell, $\text{1?1}\text{1}\text{2}$ logs less infectious virus was produced by Tera I cells than HeLa cell cultures at late times (96–144 hr) postinfection. These results demonstrate an adenovirus input multiplicity-dependent, host range restriction in human embryonal carcinoma cells. These observations may provide a means of examining the differences between cells with distinct developmental potentials.     

Non-cytopathic infection of rhabdomyosarcoma cells by coxsackie B5 virus

Summary Infection of rhabdomyosarcoma (RD) cells by coxsackie B5 virus (CBV₅) was non-cytopathic, although low titres of infectious virus were produced after 24 h post-infection. The extent of CBV₅ replication in RD cells increased after sequential passage of the virus in these cells. The RD cells from the first cycle of CBV₅ infection were recovered and maintained in culture for 3 months (equivalent to 21 passages) releasing infectious virus throughout this period; these cells were considered to be persistently infected with CBV₅ and were designated piRD cells. Coxsackie virus antigen was demonstrated in a small proportion of piRD cells by immunofluorescence staining. High resolution two-dimensional polyacrylamide gel electrophoresis was used to analyse the intracellular proteins prepared from piRD cells, three proteins were detected which were absent in uninfected RD cells. These new proteins were similar in charge to virus proteins induced during CBV₅ lytic infection of HEp-2 cells. Quantitative densitometry of 2-dimensional protein profiles of piRD and uninfected cells showed no significant disruption of RD cell protein synthesis by the persistent virus infection. Three cloned cell lines were recovered from piRD cells, none of which showed evidence of infectious virus or virus-induced protein synthesis suggesting that the parental cell line was a carrier culture for CBV₅.     

Molecular and biological changes associated with HeLa cell attenuation of wild-type yellow fever virus.

Six passages of the mosquito-borne flavivirus yellow fever (YF) wild-type strain Asibi in HeLa cells attenuated the virus for monkeys and newborn mice and resulted in loss of mosquito competence. Attenuation after the passage in HeLa cells was not unique to YF virus strain Asibi as demonstrated by the HeLa passage attenuation of wild-type YF virus strain French viscerotropic virus and YF vaccine virus 17D-204 for newborn mice. In contrast, wild-type strain Dakar 1279 and the French neurotropic vaccine virus remained virulent for newborn mice after six passages in HeLa cells. Thus not all strains of YF virus can be attenuated by passage in HeLa cells. Attenuation of YF virus strains Asibi and French viscerotropic virus was accompanied by alterations in the antigenic and biological properties of the viruses, including changes to envelope protein epitopes. Attenuation for newborn mice was coincidental with the acquisition by the HeLa-passaged viruses of the vaccine-specific envelope protein epitope recognized by monoclonal antibody H5. This suggests that this conformational change may play a role in the attenuation process. Wild-type Dakar 1279, which remained virulent for newborn mice after passage in HeLa cells, retained its wild-type antigenic character. The genome of Asibi HeLa p6 virus differed from wild-type Asibi virus by 29 nucleotides that encoded 10 amino acid substitutions: 5 in the envelope protein, 1 in NS2A, 3 in NS4B, and 1 in NS5. The substitution at NS4B-95 is seen in three different attenuation processes of wild-type YF virus, leading us to speculate that it is involved in the attenuation of virulence of wild-type strain Asibi.     

Influence of early passages on the character of freshly isolated strains of Tahyna virus.

The plaque size, thermostability, virulence, and average lethal time of uncloned freshly isolated strains of Tahyna virus were studied in the course of 3 passages in suckling mice. All of the 10 strains were in the 1st passage thermostable (indices 4 degrees/50 degrees C less than 1.0 log10) and highly virulent (indices ic/sc less than 1.0 log10). They differed significantly in plaque size only. The effect of 3 passages in suckling mice was manifested by titre increase and plaque size reduction. The differences in thermostability and virulence were too small as to be distinctly significant, but at least they indicated the heterogeneity of the strains. The variability in behaviour of uncloned strains and significance of changes observed during passages are discussed.     

Lithium chloride inhibits the coronavirus infectious bronchitis virus in cell culture.

The avian coronavirus infectious bronchitis virus (IBV) is a major economic pathogen of domestic poultry that, despite vaccination, causes mortality and significant losses in production. During replication of the RNA genome there is a high frequency of mutation and recombination, which has given rise to many strains of IBV and results in the potential for new and emerging strains. Currently the live-attenuated vaccine gives poor cross-strain immunity. Effective antiviral agents may therefore be advantageous in the treatment of IBV. Lithium chloride (LiCl) is a potent inhibitor of the DNA virus herpes simplex virus but not RNA viruses. The effect of LiCl on the replication of IBV was examined in cell culture using two model cell types; Vero cells, an African Green monkey kidney-derived epithelial cell line; and DF-1 cells, an immortalized chicken embryo fibroblast cell line. When treated with a range of LiCl concentrations, IBV RNA and protein levels and viral progeny production were reduced in a dose-dependent manner in both cell types, and the data indicated that inhibition was a cellular rather than a virucidal effect. Host cell protein synthesis still took place in LiCl-treated cells and the level of a standard cellular housekeeping protein remained unchanged, indicating that the effect of LiCl was specifically against IBV.     

Detection of infectious bronchitis virus in experimentally infected chickens by an antigen-competitive ELISA

An antigen-competitive enzyme-linked immunosorbent assay (Ag-C-ELISA) was developed for the detection of infectious bronchitis virus (IBV) antigens, M41 strain, in tissues from experimentally infected chickens, or in allantoic fluid harvested from inoculated embryonated eggs. The detection limit of IBV in the Ag-C-ELISA was 10^4.1 median embryo infective doses (EID50)/well. Tracheal and lung samples from chickens vaccinated with 10^2.5 EID50 of live attenuated infectious bronchitis (H120) vaccine were negative in the direct detection Ag-C-ELISA. The results indicate that the Ag-C-ELISA has the potential to detect IBV, either directly in tissue samples or when combined with the passage of material in embryonated eggs, thereby constituting an alternative method for the diagnosis of IBV.     

Compatibility of lyotropic liquid crystals with viruses and mammalian cells that support the replication of viruses

We report a study that investigates the biocompatibility of materials that form lyotropic liquid crystals (LCs) with viruses and mammalian cells that support the replication of viruses. This study is focused on aqueous solutions of tetradecyldimethyl-amineoxide (C(14)AO) and decanol (D), or disodium cromoglycate (DSCG; C(23)H(14)O(11)Na(2)), which can form optically birefringent, liquid crystalline phases. The influence of these materials on the ability of vesicular stomatitis virus (VSV) to infect human epitheloid cervical carcinoma (HeLa) cells was examined by two approaches. First, VSV was dispersed in aqueous C(14)AO+ D or DSCG, and then HeLa cells were inoculated by contacting the cells with the aqueous C(14)AO + D or DSCG containing VSV. The infectivity of VSV to the HeLa cells was subsequently determined. Second, VSV was incubated in LC phases of either C(14)AO + D or DSCG for 4 h, and the concentration (titer) of infectious virus in the LC was determined by dilution into cell culture medium and subsequent inoculation of HeLa cells. Using these approaches, we found that the LC containing C(14)AO + D caused inactivation of virus as well as cell death. In contrast, we determined that VSV retained its infectivity in the presence of aqueous DSCG, and that greater than 74-82% of the HeLa cells survived contact with aqueous DSCG (depending on concentration of DSCG). Because VSV maintained its function (and we infer structure) in LCs formed from DSCG, we further explored the influence of the virus on the ordering of the LC. Whereas the LC formed from DSCG was uniformly aligned on surfaces prepared from self-assembled monolayers (SAMs) of HS(CH(2))(11)(OCH(2)CH(2))(4)OH on obliquely deposited films of gold in the absence of VSV, the introduction of 10(7)-10(8) infectious virus particles per milliliter caused the LC to assume a non-uniform orientation and a colorful appearance that was readily distinguished from the uniformly aligned LCs. Control experiments using cell lysates with equivalent protein concentrations but no virus did not perturb the uniform alignment of the LC.     

Variation in susceptibility of HeLa cell lines to coxsackievirus A 9

Summary In the course of serial passages for several years a line of uncloned HeLa cells (A line) showed a gradual decrease in plaquing efficiency by coxsackievirus A 9 (CA 9 virus), while subcultures prepared from the same line kept frozen at an early passage level (A original line) did not show any change. However, it was observed later that the plaque-forming ability of the A original line (A orig. line) also decreased after serial passages as was observed with the A line. Comparing the characteristics of the same cell line at two different passage levels, it was found that the efficiency of adsorption of virus to cells was nearly the same, while virus yield at 8 hours after infection was different. The activity of alkaline phosphatase of cells was also different between these two passage levels, suggesting that a high enzymatic activity is associated with the susceptibility of cell cultures to CA 9 virus. Magnesium chloride at 25 mM enhanced plaque formation by CA 9 virus in highly passaged less susceptible cell cultures, and a possible role of the chemical as a stabilizer of alkaline phosphatase was discussed.With 2 FiguresThe following abbreviations are used in the text: YLE-Earle's solution supplemented with 0.5 per cent lactalbumin hydrolysate and 0.1 per cent yeast extract; PBS-phosphate buffered saline; UK-primary cynomolgus monkey kidney.     

体外传代培养成体大鼠骨髓间充质干细胞生物学特性的观察

目的观察成体大鼠骨髓间充质干细胞（MSC）体外培养过程中生物学特性的改变,为骨髓间充质干细胞的应用研究提供实验依据。方法成体大鼠骨髓MSC的分离培养,流式细胞仪观察MSC免疫表型及细胞周期,检测细胞的诱导分化能力以及细胞的生长曲线,TRAP—ELISA方法检测其端粒酶活性。结果体外培养的成体大鼠MSC,随着传代次数的增加,长梭形细胞的体积逐渐增大。第4代时免疫表型阳性细胞率分别为CD29：（94．75±3．68）％,CD71：（95．43±2．23）％,CD90：（98．08±3．88）％;当传到第7代时,阳性细胞率仅为CD29：（50．00±3．35）％,CD71：（50．70±2．43）％,CD90：（48．60±2．83）％;第9代时MSC检测不到任何阳性免疫表型。前5代MSC增殖较快,第3代时处于S期和G2／M期的细胞比例为（38．36±2．01）％,处于G0／G1期细胞为（61．64±2．13）％;第7代以后细胞增殖能力明显降低,第12代时处于S期和G2／M期的细胞为（10．83±1．63）％,而G0／G1期的细胞为（89．17±1．96）％,此时MSC已经基本停止增长。当体外培养的MSC传到第9代以后,在成骨和成脂肪诱导体系作用下,细胞丧失了分化为Von Kossa法染色和油红O染色阳性细胞的能力;同时其端粒酶活性也随着传代次数的增多由最初的（52．7±0．78）％逐渐降低为阴性。结论体外培养的成体大鼠骨髓MSC随着传代次数的增加,其生物学特性发生明显改变。     

Characteristics of Parker's rat coronavirus (PRC) replicated in L-2 cells

Summary Parker's rat coronavirus (PRC) is a naturally-occurring viral infection of the laboratory rat. On the first passage, ATCC strain 8190 of PRC replicated in L-2 cells. Using the tenth passage of PRC in L-2 cells, the characteristics of the virus were compared with previous studies of sialodacryoadenitis virus (SDAV) replicated in L-2 cells. Based on light and immunofluorescence microscopic examination of control and inoculated cell cultures, PRC-associated CPE was frequently confined primarily to individual cells, and there were relatively few syncytial giant cells. Maximum titers were recovered at 36h post inoculation (pi). Infectious virus was demonstrated at pH values ranging from 6.0 to 9.0 and a pH of 7.5 was determined to produce the highest titers of PRC. The optimum temperature for viral replication was 33°C. Up to 15 passages of PRC in L-929 cells failed to produce detectable virus. However, after adaptation in L-2 cells (20th passage), PRC replicated to high titers in L-929 cells. Previously, in vitro studies of rat coronaviruses have been hampered by the lack of an identified continuous cell line to replicate these viruses in the laboratory. L-2 cells represent a readily-available continuous cell line that can support the replication of relatively high titers of PRC.