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PubMed2000

Approximately 150 nucleotides from the 5' end of an influenza A segment 1 defective virion RNA are needed for genome stability during passage of defective virus in infected cells.

Defective influenza A virus RNAs analyzed in two studies so far possess at least 80-90 nucleotides from the 5' end of the virion RNA segment and more typically around 200 nucleotides, whereas the 3' sequence could be as short as 25 nucleotides (P. A. Jennings et al., Cell 34, 619-627; 1983; S. D. Duhaut and N. J. Dimmock, Virology 247, 241-253, 1998). To determine the biological significance of the highly conserved 5' sequence, we constructed plasmids that expressed a naturally occurring defective segment 1 RNA from A/equine/Newmarket/7339/79 (EQV, H3N8) or modified RNAs with lesser amounts of the 5' end. These had terminal 5' sequences of 220 nucleotides (POLI-220), 150 nucleotides (POLI-150), 80 nucleotides (POLI-80), and 30 nucleotides (POLI-30). Their remaining sequence came from the 3' end of virion RNA, and all were exactly 445 nucleotides in length. After transfection with one of the RNA-expressing POLI plasmids and plasmids encoding PB1, PB2, PA, and NP proteins, Vero cells were infected with a helper influenza virus of one of three different subtypes (the parental H3N8, an H2N2, or an H1N1 virus). Progeny infectious and presumptive progeny defective virus in the resulting tissue culture fluids were then passaged serially to new cultures up to 10 times. We found that POLI-220 and POLI-150 RNAs proved stable on passage and POLI-80 RNA was detected intermittently, while POLI-30 was not detected beyond passage three. Data were essentially reproducible with the three helper viruses and in two cell lines. It thus appears that the terminal 5' 150 nucleotides are necessary for influenza virion RNA molecules to be replicated and packaged consistently in cell culture. The possible functional significance of the 5' sequence is discussed.

Animals↗

PubMed2002

Detection of Neorickettsia (Ehrlichia) risticii in tissues of mice experimentally infected with cercariae of trematodes by in situ hybridization.

Neorickettsia (Ehrlichia) risticii was demonstrated to occur in cercariae developing in Juga yrekaensis snails by experimental transmission, genetic detection and histopathology. Cercariae were isolated from the digestive glands of snails collected in a fresh stream water area of Siskiyou County, CA, and inoculated into CF1 mice. Mice developed clinical signs, splenomegaly and histopathologic abnormalities. The agent was maintained by serial passages of whole blood in CF1 mice. A 527-bp product of the 16S rRNA gene of N. risticii was serially detected by nested PCR in blood, feces, salivary gland, suprarenal gland, spleen, intestine and bone marrow of inoculated mice. N. risticii DNA was detected by in situ hybridization with DIG-labeled probe in PCR-positive salivary gland, intestine and spleen tissue sections of experimental mice on day 30 after inoculation. Infection in mice was established when cercariae were inoculated by either IP or SC routes but not established following intraoral route. N. risticii was detected by PCR in spleen, intestine and bone marrow even after 73 days post-inoculation whereas blood from the same animals became negative at 58 days. N. risticii was observed by in situ hybridization in salivary gland, spleen and intestine of mice infected by IP or SC inoculation. This ISH protocol should aid investigations on the host range of the Neorickettsiosis and pathogenesis of neorickettiosis in vector, animal or human.

Animals↗

PubMed1987

Production in Vero cells of an inactivated rabies vaccine from strain FRV/K for animal and human use.

A new concentrated and purified rabies vaccine was produced in Vero cells. Two rabies virus strains, the fixed rabies virus Pasteur (FRV) and Pittman Moore (PM) were adapted to Vero cells by 20 cycles of alternating passages in the brain of weaning mice. Intracerebral (i.c.) inoculation of weaning mice was followed then by 17 and 20 serial passages in Vero cells of RFV and PM strains, respectively. The adapted strains designated as FRV/K and PM/K gave titres of 10(6) +/- 1.5 log (LD50/ml for i.c. inoculated mice) in several harvests taken from one infected cell culture. Pooled harvests were concentrated 20-fold by ultrafiltration and were tested as animal vaccine after inactivation with beta-propiolactone (BPL). Another vaccine preparation destined for human use, in addition to concentration and inactivation, was also purified by gel filtration. Control tests revealed that the antigenic content of different strain FRV/K harvests was very high in comparison with that of strain PM/K and the reference tissue culture vaccine (RIV, Netherland). In sheep the antibody response induced by the FRV/K strain was very high; serum neutralizing index (NI) higher than 4 was reached 40 days after the second vaccine dose, whereas the vaccine preparation from strain PM/K gave NI of 2.3 and the reference vaccine NI of 3.8, respectively. Safety tests in rabbits and guinea pigs showed neither pyrogenicity nor toxicity.

Animals↗

PubMedDate not supplied

Mutations in the human immunodeficiency virus type 1 reverse transcriptase gene observed in stavudine and didanosine strains obtained by in vitro passages.

We have selected a human immunodeficiency virus type 1 (HIV1) using the technique of in vitro selection to generate variants that are resistant to didanosine and/or stavudine. After serial passages of the Lai strain of HIV1 in MT-2 cells in increased concentrations of didanosine-stavudine association, 2 novel mutations in reverse transcriptase at codon 57 (Asp-->His) and at codon 98 (AIa-->Val) were observed. These mutations were associated with an 11.5-fold increase in the didanosine and a 4.5-fold increase in the stavudine 50% inhibitory concentration.

Anti-HIV Agents↗

PubMed1975

Selection for plaque variants of two California group arboviruses (Jamestown canyon and La Crosse) by passage in natural vertebrate hosts.

The plaque size and distribution of prototype La Crosse (LAC) and Jamestown Canyon (JC) viruses were investigated in Vero cell cultures. The effect of serial passage of the viruses in their natural vertebrate hosts - the chipmunk and grey squirrel for LAC and the white-tailed deer for JC virus - was studied. Prototype JC virus was predominately a pinpoint plaque type (about 0.3 mm in diameter). A large plaque variant (about 1.0 mm in diameter), which was only 1 percent of prototype JC virus, increased to over 80 percent of the resultant virus after one passage in white-tailed deer. The large plaque type (about 1.0 mm in diameter) also predominated in JC isolates from mosquitoes, biting flies, and a white-tailed deer in Wisconsin. Prototype LAC virus consisted of a variety of plaque sizes, ranging in diameter from 0.3 mm to 3.0 mm. Passage through chipmunks increased the mean plaque diameter almost twofold, whereas passage in the grey squirrel resulted in a more uniform small plaque population practically eliminating the largest plaques. Preliminary results suggest that the resultant viruses are antigenically different. If these selective processes occur in nature, they man explain how four serologically related California group arboviruses with distinct vector-host cycles in nature could have evolved and have sympatric distribution.

Animals↗

PubMed1993

Attenuation of a reactivated cardiovirulent coxsackievirus B3: The 5'-nontranslated region does not contain major attenuation determinants.

To investigate the molecular basis of pathogenicity of Coxsackieviruses, a virus was reactivated by transfection from a full-length cDNA clone derived from cardiovirulent Coxsackievirus B3 (CVB3). The reactivated virus, rCVB3, was passaged serially in human dermatofibroblasts (HDF). No cytopathic effect was observed up to 12 days after inoculation with rCVB3 or early-passage virus, although disintegration of the monolayers was observed with late-passage virus (10th to 14th passages). Approximately 10% of HDF inoculated with rCVB3 were positive for viral antigens by immunofluorescence using enterovirus- or CVB3-specific monoclonal antibodies. These observations, together with the low infectivity titre of rCVB3 in HDF, suggests that HDF initially support only carrier state infection. After the 14th passage, the cardiovirulence of passaged virus (p14V) in mice was attenuated by a factor of > 10(4). Phenotypic changes of plaque size were also noticed in p14V: An attenuated variant (p14V-1) that produced larger plaques than rCVB3 in Vero cells has been plaque purified. The 5'-terminus of the genome of attenuant p14V-1 was amplified by polymerase chain reaction (PCR) and its sequence determined. Only one point mutation was found within the 5'-nontranslated region (5'NTR) at position 690 (A to U) compared to the viral RNA sequence obtained for rCVB3. An intertypic chimeric virus was reactivated from a cDNA clone after replacing the 5'-terminal 891 nucleotides of the wild-type genome with the corresponding region of the attenuant p14V-1. This chimeric virus, CB3/p14V-1/1, produced wild-type plaques in Vero cells and showed cardiovirulence similar to that of rCVB3 in mice.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed2006

Apoptotic cell death increases with senescence in normal human dermal fibroblast cultures.

Normal human dermal fibroblasts have a limited life-span in vitro and stop proliferation after a fixed number of cell divisions. This process by which cells stop proliferation is called senescence. Senescence is also characterized by a decrease in the total cell number. In this study, we characterized an increase in cell death in normal human dermal fibroblasts in vitro as a function of increasing cell passage. With increasing passage, human fibroblasts showed an increase in the number of dead cells and increased DNA fragmentation as determined by flow cytometry. Serial passage of human fibroblasts also resulted in mitochondrial dysfunction, represented by a loss of mitochondrial membrane potential. The apoptotic markers caspase-3 and cytochrome c were both found to increase in senescent cells. These results suggest the activation of an apoptotic pathway within a population of human fibroblasts as a function of cell passage.

Apoptosis↗

PubMed1986

Malignant catarrhal fever: experimental transmission of the 'sheep-associated' form of the disease from cattle and deer to cattle, deer, rabbits and hamsters.

Attempts to transmit malignant catarrhal fever (MCF) from 16 bovine cases of the 'sheep-associated' form of the disease are described. On two occasions disease was transmitted to bovine calves but transmission to red deer (Cervus elaphus) was not achieved. In addition, MCF was transmitted from one experimentally affected calf to a rabbit and on another occasion directly to rabbits with material from a field case which failed to transmit to a bovine calf or red deer. Subsequently each of these isolates was readily passaged through rabbits and one was also passaged to Syrian hamsters. Tissue from MCF-affected red deer consistently produced disease on inoculation into rabbits and deer but failed to cause disease in bovine calves. Contact infection between red deer occurred once and roe deer (Capreolus capreolus) were also shown to be susceptible to infection by inoculation. Passage of MCF in rabbits with an isolate from red deer failed to produce evidence of further adaptation even after 125 serial passages. Despite the failure to transmit disease from cattle to deer or from deer to cattle it is considered probable that there is only one sheep-associated agent which causes MCF in both species. The reasons for the anomalies in transmission of this form of the disease are discussed.

Animals↗

PubMed1995

Experimental evidence of recombination in coronavirus infectious bronchitis virus.

Embryonated eggs were coinfected with two strains of the coronavirus avian infectious bronchitis virus (IBV), IBV-Beaudette and IBV-M41, to investigate whether recombination between the two strains would occur. Virions were isolated from the allantoic fluid of the coinfected eggs and putative hybrid RNAs were detected by polymerase chain reaction (PCR), using strain-specific oligonucleotides. PCR products, of the expected sizes, were obtained as predicted from potential recombination events between the nucleoprotein (N) gene and the 3'-untranslated region of the two IBV genomes. Sequencing confirmed that they corresponded to hybrid RNAs. Virus produced as a result of the mixed infection was treated with an M41-specific neutralizing monoclonal antibody and passaged in Vero cells, in which IBV-Beaudette, but not IBV-M41, replicated. Hybrid RNA was still detectable after three serial passages. Since no IBV-M41 was detectable this confirmed that infectious recombinant genomes had been produced in the embryonated eggs. These findings not only support the circumstantial evidence, from sequencing studies of IBV field strains, that recombination occurs during replication of IBV and contributes to the diversity of IBV, but also show that coronavirus RNA recombination is not limited to mouse hepatitis virus.

Animals↗

PubMed1998

Genetic stability of Sabin 1 strain of poliovirus: implications for quality control of oral poliovirus vaccine.

The Sabin vaccine strains of poliovirus, like all RNA viruses, exist as a quasispecies of genomic sequences whose composition can be altered during virus propagation. Since changes in vaccine virus during manufacture can enhance the neurovirulent potential of the vaccine, each monovalent lot of oral poliovirus vaccine (OPV) undergoes several tests to ensure consistency of manufacture, including the monkey neurovirulence test (MNVT). Recently, we proposed a new molecular approach for direct quantification of vaccine variants with neurovirulent potential as an alternative way to monitor consistency of OPV production. Analysis of the Sabin 1 genome allowed us to identify a limited number of specific loci that exhibit significant change during viral propagation in vitro and in vivo. Here we explore the possible roles of these changes and show that 7427-U-->C and 7441-G-->A alterations in the 3'-UTR of the Sabin 1 virus do not increase monkey neurovirulence. These, as well as our previous results, suggest that only mutations in the 5'-UTR play a significant role in the limited increase in Sabin 1 monkey neurovirulence observed after extended propagation of the virus beyond the passage level used in vaccine production. Our studies with high-passage batches of the Sabin 1 strain confirmed the stability of this strain, which retains acceptable levels of monkey neurovirulence even after serial passages at elevated temperature. Compared to the MNVT, molecular analysis of the genetic composition of Sabin 1 poliovirus provides a more sensitive analytical approach to monitor consistency of vaccine production.

Animals↗

PubMed1968

Induction of bone marrow colony-stimulating activity by a filterable agent in leukemic and normal mouse serum.

1. Leukemic Swiss mice of ICR/Ha strain which had been injected at birth with a lymphoid-leukemia-inducing virus preparation yielded sera which produced elevations of serum colony-stimulating activity within 16 hr and significant plasma-LDH-enzyme elevation at 4 days when injected intraperitoneally into normal ICR/Ha Swiss mice. Colony-stimulating activity was assayed in vitro by the stimulation of hemopoietic colony formation by DBA/1 bone marrow cells. 2. The inducing agent in leukemic serum was passageable, filterable, sedimentable, and heat-, ether-, and UV-labile. 3. A similar agent was recovered from normal Swiss serum after blind serial passages through normal mice. 4. LDH elevating virus induced a similar elevation of serum colony-stimulating activity when injected at high titers, and cross-resistance was demonstrated between LDH virus and the passaged leukemic serum agent.

Animals↗

PubMed1985

Virulence of chick embryo fibroblast-passaged flury HEP rabies virus and its revertants in mice.

Chick embryo fibroblast-passaged Flury high egg passage (HEP) rabies virus failed to kill nude mice or cyclophosphamide-treated mice when inoculated intracerebrally. The virus regained neurovirulence for adult mice after three passages in mouse neuroblastoma C1300 cells (NA cells). However, even after 20 passages in NA cells, the virulence could not be increased to the level shown by the virus passaged several times in suckling mice. Some physiological and biological properties of the virus showing and not showing mouse virulence after five serial passages and after one single passage in NA cells, respectively, were compared.

Animals↗

PubMed1996

Chimeric simian/human immunodeficiency virus that causes progressive loss of CD4+ T cells and AIDS in pig-tailed macaques.

By animal-to-animal passage of simian/human immunodeficiency virus (SHIV) in pig-tailed macaques, we have developed a macaque model of human immunodeficiency virus type 1 (HIV-1) disease in humans. Passaging was begun with a chimeric virus containing the env gene of HIV-1 HXBc2 and the gag and pol genes of simian immunodeficiency virus SIVmac239. SHIV was passaged serially in cohorts of two macaques each, using bone marrow-to-bone marrow transfers at 5, 5, and 16 weeks for passages 2, 3, and 4, respectively. The fifth passage was done by using cell-free virus isolated from cerebrospinal fluid of a passage 4 macaque. The virus became more virulent with each passage. Virus replication was restricted in all three animals in passages 1 and 2 but not in five of the six animals in passages 3, 4, and 5. In these animals, intense virus replication in the lymphoid tissues resulted in almost total elimination of CD4+ T cells within weeks of inoculation, and three of these animals developed AIDS in less than 1 year. The more uniform virus-host interaction initiated by the cell-free virus in the passage 5 animals contrasted with a more variable pattern of disease initiated by infectious bone marrow cells during earlier passages. The virulent cell-free SHIV can now be used to screen the efficacy of vaccines directed against the envelope of HIV-1.

Acquired Immunodeficiency Syndrome↗

PubMed1989

Spontaneously hypertensive rat vascular smooth muscle cells in culture exhibit increased growth and Na+/H+ exchange.

The cellular mechanisms responsible for abnormalities in spontaneously hypertensive rat (SHR) vascular smooth muscle cell (VSMC) growth and vasoreactivity are not defined. Because Na+/H+ exchange, which we have previously demonstrated in cultured VSMC, plays an essential role in mediating growth factor responses, we hypothesized that abnormalities in SHR growth regulation might be reflected in the activity of this transporter. To test this hypothesis, we studied DNA synthesis and Na+/H+ exchange (measured as the rate of amiloride-sensitive intracellular alkalinization or Na+ influx) in early subcultures (less than 6) of aortic VSMC from 12-wk-old SHR and Wistar Kyoto (WKY) animals. Serum-deprived SHR VSMC grew more rapidly in response to 10% serum with an increase in [3H]thymidine incorporation of 439% compared with 191% in WKY controls. Basal intracellular pH (pHi) values determined by fluorescent pH measurements were 7.37 +/- 0.04 and 7.27 +/- 0.03 (P less than 0.05) in early passage SHR and WKY, respectively. Acid recovery (initial pHi = 6.8) by SHR VSMC was faster than by WKY VSMC as measured by alkalinization (1.8 +/- 0.6 vs. 0.8 +/- 0.2 mmol H+/liter.min, P less than 0.05) or by amiloride-sensitive 22Na+ influx (14.5 +/- 1.2 vs. 4.0 +/- 0.5 nmol Na+/mg protein.min, P less than 0.05). In comparison to WKY cells early passage SHR VSMC exhibited 2.5-fold greater alkalinization and amiloride-sensitive 22Na+ influx in response to 100 nM angiotensin II. During serial passage, WKY cells acquired enhanced Na+/H+ exchange and growth rates so that by passage 6, these differences were no longer present. These findings in early cultures of SHR VSMC, removed from the in vivo neurohumoral milieu, suggest that increased Na+/H+ exchange in SHR may reflect alterations in Na+ homeostasis that might contribute to altered SHR VSMC function such as enhanced growth and vasoreactivity.

Amiloride↗

PubMed1983

Characterization of early B lymphocyte precursors present in long-term bone marrow cultures.

Lymphoid precursor cells are present in long-term bone marrow cultures (LTBMC), but their differentiation into mature lymphocytes is blocked. A quantitative assay for B cell precursors in LTBMC, which gives a linear relationship between the number of grafted LTBMC cells and the frequency of B cell colony forming units (CFU-B) in the spleen and bone marrow of immunodeficient CBA/N mice 19 days after reconstitution, is described. Characterization of the B cell precursor indicates that this assay is detecting a very early precursor and not a B lymphocyte or a late pre-B cell. This conclusion is based on the observations that a) pre-B cells transformable by Abelson murine leukemia virus are not present in LTBMC by 3 days postrecharge and CFU-B are absent by 6 days postrecharge; b) late B cell progenitors capable of rapid repopulation of irradiated CBA/N mice are not present in LTBMC, since a lag in the kinetics of B cell reconstitution in animals grafted with LTBMC cells is observed compared with fresh bone marrow cells; c) the B cell precursors in LTBMC have high proliferative potential, since they can stably repopulate recipient mice for at least 8 wk postreconstitution and through two serial passages in irradiated CBA/N recipients; and d) the B cell precursors are large, rapidly sedimenting cells as determined by velocity sedimentation. The serial transplantation experiment further shows that a split is often observed between lymphoid and myeloid reconstituting ability of LTBMC cells. The LTBMC B cell precursor may be a pluripotent stem cell or a lymphoid stem cell, although its differentiative potential remains to be determined.

Abelson murine leukemia virus↗

PubMed1987

Temporary and permanent modifications to a single strain of mouse scrapie on transmission to rats and hamsters.

The interspecies transmission of scrapie is frequently associated with exceptionally long incubation periods at first passage in the new host compared to later passages (the species barrier effect). The basis of this was investigated using the 139A strain of scrapie which had been cloned by three serial passages in mice at limiting infectious doses. Cloned scrapie was passaged through hamsters (twice) or rats (thrice) and then reisolated in mice. Large species barrier effects were encountered on mouse-to-hamster and hamster-to-mouse passage resulting in the isolation of a mutant strain, 139-H/M, with properties very different from 139A. In contrast, the strain reisolated from rats was indistinguishable from 139A. However, a large species barrier was encountered at the mouse-to-rat passage but not at the rat-to-mouse passage. It is suggested that the transmission of scrapie between species may be associated with no change in properties or a permanent change in the scrapie genome due to the selection of mutants. A third possibility, the donor species effect, is a temporary change occurring only at first passage in the new host species which is largely or entirely caused by the introduction of material from the previous host. We speculate that the donor species effect could be explained if some host protein forms a functional part of the infectious agent.

Animals↗

PubMed1980

Changes in virulence, M protein and IgG Fc receptor activity in a type 12 group A streptococcal strain during mouse passages.

A type 12 group A strain (1800) was passaged serially through mice 25 times. The ability to servive in normal human blood dropped from a growth index of 52 after the first passage to 1 after four passages. After 14 passages the growth index increased again and stabilized above 30. The virulence for mice increased from a LD100 of 10(8) colony forming units (CFU) to 10-100 CFU after 7 passages and then remained constant. The Mqw antigen disappeared after 4 passages as tested by immunodiffusion, electroimmunoassay and indirect bactericidal tests. Three antisera, raised in rabbits against strains originally belonging to types M3, M12 and M46 but devoid of type antigens after mouse passages showed high bactericidal indices against the 1800 strain after 14 or more passages on mice. Anti-type M1 serum was also found bactericidal for the passaged strains. The IgG Fc-receptor activity of the strain isolated after each mouse passage was tested in hemagglutination experiments with human red blood cells coated with "incomplete" anti-Rh and hot hydrochloric acid extracts of the strains. The capacity to agglutinate "Ripley"-coated cells increased gradually during the first 12 passages and subsequently the titres of the extracts stabilized between 1:160 and 1:320. The HUN coat, useful for detection of the G3m (5) maraker gave titraes increasing with the number of passages while the titres for IgG1 coats kept at 1:4 or below. On background of these results, the possible role of the IgG Fc-receptor as a virulence factor is discussed.

Animals↗

PubMed1976

Characterization of miniature pig kidney cells and their resistance to chlamydial infection.

A miniature pig kidney cell line has been established from porcine fetuses taken aseptically by hysterectomy and maintained for more than 50 passages in Eagle's minimal essential medium containing 10% heat-inactivated newborn calf serum. Cell transfers were performed each week. Primary and serially passaged cells were found to be highly refractory to infection by Chlamydia trachomatis strains TW-3, Bour, and LGV 440L and Chlamydia psittaci strains meningopneumonitis and 6BC and insusceptible to poliovirus type 1. The cells were susceptible to vesicular stomatitis virus and herpes simplex type 1 virus. Identity of the cells was established by cytotoxicity, isozyme, and cytogenetic studies.

Animals↗