ST Cells Research Articles

Transmissible gastroenteritis coronavirus induces programmed cell death in infected cells through a caspase-dependent pathway.

In this report, we show that apoptosis (or programmed cell death) is induced in different cell lines infected with a coronavirus, the porcine transmissible gastroenteritis virus (TGEV). Kinetic analysis of internucleosomal DNA cleavage by agarose gel electrophoresis and flow cytometry or cytometric monitoring of the mitochondrial transmembrane potential showed that, for ST cells infected with TGEV, the first overt signs of apoptosis appeared from 10 to 12 h postinfection on. They preceded morphological changes characteristic of cells undergoing apoptosis, as observed by light and electron microscopy. The tripeptide pan-ICE (caspase) inhibitor N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone blocked TGEV-induced apoptosis with no effect on virus production. The thiol agent pyrrolidine dithiocarbamate inhibited apoptosis, suggesting that TGEV infection may lead to apoptosis via cellular oxidative stress. The effect of TGEV infection on activation of NF-kappaB, a transcription factor known to be activated by oxidative stress, was examined. NF-kappaB DNA binding was shown to be strongly and quickly induced by TGEV infection. However, transcription factor decoy experiments showed that NF-kappaB activation is not critical for TGEV-induced apoptosis.

Adaptation and serial passage of bovine coronavirus in an established diploid swine testicular cell line and subsequent development of a modified live vaccine.

A virulent bovine coronavirus isolate (newborn calf diarrheal) was adapted and serially passaged in an established diploid swine testicular cell line (ST cells). The same cells have been used to produce modified live porcine rotavirus and coronavirus vaccines that are federally licensed and sold worldwide. Growth of the bovine coronavirus resulted in cytopathic effect characterized by cellular stranding and subsequent cell lysis. Virus yields were relatively high in the ST cells and active replication was confirmed by immune electron microscopy and immunofluorescence. Adaptation of bovine coronavirus to a diploid swine cell line has not been previously reported. Different cell culture passage levels of bovine coronavirus were evaluated by oral inoculation of clean-catch, colostrum-deprived calves. A passage level of bovine coronavirus was identified that multiplied in the calf without the clinical signs of disease associated with virulent passages. The modified live bovine coronavirus vaccine remained safe and efficacious even after 5-backpassages in calves. Further efficacy studies have shown that the modified live bovine coronavirus vaccine significantly protected calves from highly virulent challenges with either winter dysentery or newborn calf diarrheal coronavirus isolates.

Virus-receptor interactions and interspecies transfer of a mouse hepatitis virus.

Molecular mechanisms regulating virus xenotropism and cross-species transmission are poorly understood. Host range mutants (MHV-H2) of mouse hepatitis virus (MHV) strains were isolated from mixed cultures containing progressively increasing concentrations of nonpermissive Syrian baby hamster kidney (BHK) cells and decreasing concentrations of permissive murine astrocytoma (DBT) cells. MHV-H2 was polytrophic, replicating efficiently in normally nonpermissive BHK cells, Syrian and Chinese hamster (DDT-1 and CHO) cells, human adenocarcinoma (HRT), primate kidney (VERO) and in murine 17Cl-1 cell lines. Little if any virus replication was detected in feline kidney (CRFK), and porcine testicular (ST) cell lines. To study the effects of xenotrophic spread on virus receptor-interactions in the original host, murine DBT cells were pretreated with a monoclonal antibody (MAb) CC1, directed against the MHV receptor, MHVR, a biliary glycoprotein (Bgp1a). Under treatment conditions that completely ablated the replication of the parental MHV strains, CC1 antireceptor antibodies did not block MHV-H2 replication. Following expression of MHVR in normally nonpermissive ST and CRFK cells, infection with the parental MHV strains, but not MHV-H2 was observed. To characterize the molecular basis preventing the interaction between MHV-H2 and MHVR, revertants of MHV-H2 (MHV-H2R6, MHV-H2R11) were isolated following a persistent MHV-H2 infection in DBT cells. These revertant viruses efficiently recognized MHVR, however infection of murine cells was resistant to MAb CC1 blockade. In addition, MHV-H2 and the revertant viruses efficiently recognized other Bgp receptors for docking and entry. These data suggest that interspecies transfer may remodel normal virus-receptor interactions that may result in altered virulence, tropism or pathogenesis in the original host.

Cell wall proteins of in vitro cultured chili pepper lines differing in water stress tolerance

As a strategy to understand cellular mechanisms of drought tolerance, we analyzed the electrophoretic pattern of cell wall proteins from non adapted cells (ST) and a 15% polyethylenglycol (PEG)-tolerant clone (T7) of chili pepper ( Capsicum annuum). To separate proteins bound to structural polymers by non-covalent links or disulfide bonds, cell walls were treated with sodium dodecyl sulphate (SDS), urea and 2-mercaptoethanol. After treatment three major proteins with apparent molecular masses of 9, 11 and 14 kDa accumulated in higher quantities in clone T7 than in ST cells. Cell walls were also solubilized with commercial lytic enzymes, such as lyticase and cellulase, to separate proteins joined by covalent bonds. The main difference was the presence of a new band with an apparent molecular mass of 10 kDa (p10) in clone T7 but not in ST cells. This band was also evident after autodigestion of walls by hydrolytic enzymes. Since p10 was liberated by cellulase and lyticase we suggest that this protein is covalently attached to the cellulose and glucan skeleton in clone T7. The potential role of p10 on the mechanism of drought tolerance is discussed.

Cell surface glycosaminoglycans are not involved in the adherence of Helicobacter pylori to cultured Hs 198.St human gastric cells, Hs 746T human gastric adenocarcinoma cells, or HeLa cells.

Hs 198.St cells (a line derived from normal human gastric tissue), Hs 746T cells (a line derived from human gastric adenocarcinoma), and HeLa cells were used together with 3H-labelled Helicobacter pylori, strain NCTC 11637 to determine if cell surface glycosaminoglycans could act as initial receptors for adherence of the bacteria. Although as much as 40% of the 3H-labelled bacteria adhered to monolayers of the cultured cells, removal of glycosaminoglycans by prior treatment of the cells with heparitinase, heparinase, or chondroitin ABC lyase had no effect in modifying the adherence. Prior addition of heparan sulfate, heparin, or chondroitin/dermatan sulfate to bacteria had no effect on adherence, nor were bacteria released when these same glycosaminoglycans or these same enzymes were added to cultures already containing adherent bacteria. These results indicated that neither heparan sulfate nor chondroitin/dermatan sulfate are involved as receptors in the initial adherence step of H. pylori to these cultured cells.

Cellular adhesion molecules in rat adjuvant arthritis

To examine adhesion molecule expression during the progression of inflammation in a rheumatoid arthritis model of adjuvant-induced arthritis (AIA) in rats. Immunohistochemical analysis was used to determine the distribution of the following adhesion molecules: lymphocyte function-associated antigen 1 (LFA-1; CD11a/CD18), Mac-1 and p150/95 (CD11bc/CD18), intercellular adhesion molecule 1 (ICAM-1), and CD44 in tissue sections from the ankle joints of rats with AIA. Control animals and those with AIA were killed at intervals over a 54-day period after injection with mineral oil and Mycobacterium butyricum, respectively. CD44 and LFA-1 were expressed on lymphocytes, macrophages, and synovial (ST) lining cells. CD44 expression on macrophages was found to be increased compared with control animals by day 18, and was significantly increased by day 41. CD44 expression on lymphocytes significantly increased earlier, on days 11-18. Increased LFA-1 expression on macrophages occurred late, on day 41. LFA-1 expression on lymphocytes was significantly increased on days 25, 47, and 54. ST lining cells exhibited two distinct periods of increased expression, one early, on days 11-25 and one later, on days 41-54. CD11b/c was expressed on macrophages and ST lining cells, showing a significant increase on AIA rat ST lining cells compared with control animals on day 4. No differences in ICAM-1 expression on endothelial cells between rats with AIA and controls were found on any of the days examined. CD44 expression is up-regulated on macrophages and lymphocytes during the early development of AIA, while LFA-1 expression is up-regulated later in the development of AIA. The up-regulation of CD44 and LFA-1 at different times in the development of AIA suggests an important role for these adhesion molecules in establishing and sustaining an inflammatory response in the AIA joint.

Molecular Characterization of Transmissible Gastroenteritis Coronavirus Defective Interfering Genomes: Packaging and Heterogeneity

Three transmissible gastroenteritis virus (TGEV) defective RNAs were selected by serial undiluted passage of the PUR46 strain in ST cells. These RNAs of 22, 10.6, and 9.7 kb (DI-A, DI-B, and DI-C, respectively) were detected at passage 30, remained stable upon further passage in cell culture, and significantly interfered with helper mRNA synthesis. RNA analysis from purified virions showed that the three defective RNAs were efficiently packaged. Virions of different densities containing either full-length or defective RNAs were sorted in sucrose gradients, indicating that defective and full-length genomes were independently encapsidated. DI-B and DI-C RNAs were amplified by the reverse transcription-polymerase chain reaction, cloned, and sequenced. DI-B and DI-C genomes are formed by three and four discontinuous regions of the wild-type genome, respectively. DI-C contains 2144 nucleotides (nt) from the 5′-end of the genome, two fragments of 4540 and 2531 nt mostly from gene 1b, and 493 nt from the 3′ end of the genome. DI-B and DI-C RNAs include sequences with the pseudoknot motif and encoding the polymerase, metal ion binding, and helicase motifs. DI-B RNA has a structure closely related to DI-C RNA with two main differences: it maintains the entire ORF 1b and shows heterogeneity in the size of the 3′ end deletion. This heterogeneity maps at the beginning of the S gene, where other natural TGEV recombination events have been observed, suggesting that either a process of template switching occurs with high frequency at this point or that the derived genomes have a selective advantage.

An adenovirus recombinant expressing the spike glycoprotein of porcine respiratory coronavirus is immunogenic in swine.

The full-length spike (S) gene of porcine respiratory coronavirus (PRCV) was inserted into the genome of human adenovirus type 5 downstream of the early transcription region 3 promoter. The recombinant virus replicated in cultures of the swine testicle ST cell line and directed the synthesis of S antigen with a maximum yield of approximately 26 micrograms per 10(6) cells. The antigen was cell-associated except in the late phase of the infection, when a small amount (3.5 micrograms per 10(6) cells) was released. The cell-associated antigen consisted of polypeptides of molecular mass 160 kDa and 175 kDa, comigrating with the authentic precursor S' and the mature S protein of PRCV, respectively. The extracellular recombinant antigen corresponded to the 175 kDa mature protein. Some recombinant S protein was exposed on the cell surface and was recognized by neutralization-mediating anti-S monoclonal antibodies. Piglets, inoculated oronasally with the recombinant adenovirus vector developed PRCV-neutralizing serum antibodies and were partially protected against PRCV challenge, demonstrating the potential of live adenovirus as vaccine vector.

Effect of specific antibodies on the cell-associated spread of pseudorabies virus in monolayers of different cell types.

The effect of specific pseudorabies virus (PRV) antibodies on the enlargement of plaques produced by PRV were studied in monolayers of different cell types. The plaque size was used as parameter for the efficacy of the cell-associated spread (CAS) of PRV. First, the effect of anti-PRV hyperimmune serum on the plaque growth was examined in monolayers of the continuous cell lines ST, SK-6 and MDCK and monolayers of the primary cultures of porcine fibroblasts, endothelial cells and endometrial cells. A tenfold increase in the serum concentration did reduce the plaque size with 50% in both SK-cells and fibroblasts and with 40, 28 and 16% in MDCK, endothelial and endometrial cells, respectively. In ST cells, no change in size was observed with increasing antibody concentrations. Secondly, the effects of monoclonal antibodies (mAbs) directed against PRV glycoproteins gB, gC, gD and gE and polyclonal antibodies against gC were evaluated in SK-6 cells. MAbs against gB, gD and gE were able to reduce the CAS with a cumulative effect between mAbs against gD and either mAbs against gB or mAbs against gE. Monoclonal and polyclonal antibodies against gC did not change the plaque size.

Translocation of both lysosomal LDL-derived cholesterol and plasma membrane cholesterol to the endoplasmic reticulum for esterification may require common cellular factors involved in cholesterol egress from the acidic compartments (lysosomes/endosomes)

Using a stable cell line 25-RA derived from wild-type Chinese hamster ovary (CHO) cells as the parental cell, this laboratory previously reported the isolation and characterization of CHO cell mutants (cholesterol-trafficking or CT) defective in transporting LDL-derived cholesterol out of the acidic compartment(s) (lysosomes/endosomes) to the endoplasmic reticulum (ER) for esterification. In this report, we show that the CT mutation can be complemented by fusion with human cells; however, attempts to complement the CT defect through DNA transfection have resulted in a collection of stable cell lines designated as ST cells. Under cholesterol starvation condition, the ST cells exhibit an elevated rate of cholesterol ester biosynthesis (by 3- to 5-fold) compared to both the parental CHO cells and the CT cells. The phenotypes of the ST cells are stable. ST cells are thus new cell lines arisen from the CT cells. When the plasma membranes of the parental, CT, and ST cells are labelled with [ 3H]cholesterol, ST cells show rates of [ 3H]cholesterol esterification much higher than that observed in CT cells but lower than that observed in the parental CHO cells. This result shows that translocation of plasma membrane cholesterol to the ER for esterification is defective in the CT cells. This result also suggests that ST cells acquire increased cholesterol trafficking activity between the lysosome and the ER without mixing with the plasma membrane cholesterol pool. The characteristics of CT cells and ST cells reported here suggest that translocation of both lysosomal LDL-derived cholesterol and plasma membrane cholesterol to the ER for esterification may require common cellular factors involved in cholesterol egress from the acidic compartment(s) (lysosomes/endosomes).

Cellular receptors for transmissible gastroenteritis virus on porcine enterocytes.

The activity of aminopeptidase-N (APN), reported to be a major receptor for porcine transmissible gastroenteritis virus (TGEV), in enterocyte fractions harvested from the jejunal villi and crypts of newborn and weaned piglets, did not correspond with the levels of saturable virus binding previously demonstrated for the same fractions. Plasma membranes prepared from enterocytes harvested from the jejunal villi of a newborn piglet were used in the preparation of a monoclonal antibody (MAb) which blocked the binding of TGEV, but not that of the porcine respiratory coronavirus (PRCV), to ST cells. This MAb immunoprecipitated a 200 kDa non-glycosylated protein from lysates of ST cells, which was not precipitated by an anti-APN MAb. The 200 kDa protein was shown by immunostaining and fluorescence activated cell scanning to be present on ST cells and on villous enterocytes from newborn piglets, but not on MDBK cells or enterocytes from weaned piglets. APN was demonstrated by the same techniques to be present on villous enterocytes from both newborn and weaned piglets, as well as on ST cells. It was concluded that the 200 kDa protein may be a second receptor for TGEV, contributing to the high susceptibility of newborn piglets to the virus.

Molecular cloning and physical mapping of porcine adenovirus types 1 and 2.

The 25R strain of porcine adenovirus type 1 (PAV-1) and the A47 strain of PAV-2 were propagated in ST cells, and DNA was extracted from the infected cells by a modified Hirt method. The DNA of each virus was digested by each of nine restriction endonucleases, and restriction enzyme fragments representing the entire genome were cloned. The genomic size of each virus was approximately 33 kb. Physical maps for the nine restriction endonucleases were constructed from the results of double digestion and Southern blot hybridization experiments, and oriented with respect to the PAV-3 genome. PAV-1 and PAV-2 were found to be related genetically to PAV-3, and there was a closer relationship between PAV-1 and PAV-3 than between PAV-1 and PAV-2 or between PAV-2 and PAV-3.

Expression and immunogenicity of the spike glycoprotein of porcine respiratory coronavirus encoded in the E3 region of adenovirus.

The full length spike (S) gene of porcine respiratory coronavirus (PRCV) was inserted into the genome of human adenovirus type 5 downstream of the early transcription region 3 promoter. The recombinant virus replicated in cultures of the swine testicle ST cell line and directed the synthesis of S antigen to an amount of approximately 33 micrograms per 10(6) cells, as determined by ELISA. The antigen was cell-associated except in the late phase of the infection, when a low amount (4 micrograms per 10(6) cells) was released in the culture supernatant. The cell-associated antigen consisted of 2 polypeptides of 160 K and 175 K, respectively. The 160 K polypeptide comigrated with the authentic S' precursor from PRCV-infected cells. The 175 K polypeptide had the same mobility as the authentic mature S protein from PRCV-infected cells and from PRCV released in the supernatant. The extracellular recombinant antigen corresponded with the 175 K mature protein. Immunofluorescent staining gave evidence that some recombinant S protein was exposed on the cell surface; it also showed that the protein was recognized by conformation-specific anti-S monoclonal antibodies. Piglets, immunized oronasally with the recombinant adenovirus vector developed PRCV-neutralizing serum antibodies and were partially protected against PRCV-challenge.

Evidence for a putative second receptor for porcine transmissible gastroenteritis virus on the villous enterocytes of newborn pigs.

Aminopeptidase-N (APN) has been identified [B. Delmas, J. Gelfi, R. L'Haridon, L. K. Vogel, H. Sjostrom, O. Noren, and H. Laude, Nature (London) 357:417-420, 1992] as a major receptor for porcine transmissible gastroenteritis virus (TGEV). Binding of TGEV to villous enterocytes from the jejuna of newborn pigs is saturable and at a higher level than that of binding of virus to newborn cryptal enterocytes or to enterocytes from older piglets (H. M. Weingartl and J. B. Derbyshire, Vet. Microbiol. 35:23-32, 1993). The distribution of APN in enterocytes in the jejuna of neonatal and 3 week-old-piglets, as determined by the measurement of enzymatic activity and by labeling of the cells with an anti-APN monoclonal antibody, did not correspond with the reported distribution of saturable binding sites on enterocytes. Monoclonal antibodies, which were prepared against plasma membranes derived from enterocytes harvested from the upper villi of newborn pigs, blocked the replication of TGEV, but not the porcine respiratory coronavirus, in ST cells and immunoprecipitated a 200-kDa protein in ST cell lysates. This protein was demonstrated by immunohistochemistry and by fluorescence-activated cell scanning to be present on the villous enterocytes of newborn pigs but to be lacking on the cryptal enterocytes of newborn pigs and on the villous and cryptal enterocytes of 3-week-old piglets. Since this distribution of the protein corresponds to the previously demonstrated distribution of saturable binding sites, we conclude that the 200-kDa protein may be an additional receptor for TGEV which is restricted to the villous enterocytes of newborn pigs and which contributes to the age sensitivity of these animals to the virus.

Identification of a 40- to 42-kDa Attachment Polypeptide for Canine Parvovirus in A72 Cells

The attachment of canine parvovirus (CPV) to different cell lines was quantitated by a fluorescence-activated cell sorter assay. The viral attachment was observed to both permissive A72 and nonpermissive ST cells but not to nonpermissive MDBK cells. The binding of and infectivity for CPV to A72 cells was reduced upon prior treatment of cells with Vibrio cholerae neuraminidase or lectins, specific for sialic acid. Similarly, treatment of cells with any of several proteases reduced virus binding; however, phospholipase treatment had no effect indicating that one or more membrane glycoproteins were involved in virus binding. These proteins were characterized with a virus overlay protein blot assay. Virus bound to a protein with a molecular mass of 40 to 42 kDa in membranes prepared from A72 and ST cells and not from MDBK cells. The binding to this polypeptide was specific since increasing amounts of unlabeled virions competitively inhibited binding of radiolabeled virions in a dose-dependent manner. A polypeptide of similar molecular mass was immunoprecipitated from radiolabeled octyl glucoside (OG) extract of A72 cells using purified virions, virion-specific antiserum, and protein A. The binding to this polypeptide was decreased but not abolished upon prior treatment of the membrane with V. cholerae neuraminidase. CPV preferentially recognized a polypeptide of similar molecular size in the OG extract prepared from the biotinylated basolateral surface of polarized MDCK monolayer. Hence, we propose that the 40- to 42-kDa glycoprotein represents a specific attachment molecule for CPV in A72 cells.

Swine testis cells contain functional heparan sulfate but are defective in entry of herpes simplex virus

Herpes simplex virus (HSV) enters and infects most cultured cells. We have found that swine testis cells (ST) produce yields of infectious HSV-1 up to four orders of magnitude lower than those of human embryonic lung (HEL) and HEp-2 cells because of a defect in virus entry. For ST cells, virus binding is reduced, DNA from input virus cannot be detected, and virus proteins are not synthesized. Polyethylene glycol treatment of ST cells after exposure to HSV allows viral entry, protein synthesis, and productive infection. Transfection of viral genomic DNA that bypasses the normal entry process produces similar yields of infectious virus from ST, HEL, and HEp-2 cells. Therefore, all three cell lines can support the HSV replicative cycle. Biochemical analyses and inhibition of sulfation by sodium chlorate treatment show that ST cells contain amounts and types of heparan sulfate (HS) similar to those of highly susceptible cells. HSV infection of sodium chlorate-treated HEL and ST cells indicates the presence of a second, non-HS receptor(s) on susceptible HEp-2 and HEL cells that is missing, or not functional, on poorly susceptible ST cells. We conclude that ST cells are defective in HSV entry, contain functional HS, but lack a functional non-HS receptor(s) required for efficient HSV-1 entry. Further, ST cells provide a novel resource that can be used to identify, isolate, and characterize an HSV non-HS receptor(s) and its role in the entry and tropism of this important human pathogen.

Trypanosoma cruzi: Murine Virus Contaminant of the Experimental Infection

Evidence of apoptosis caused by infection with the Purdue strain of transmissible gastroenteritis virus (TGEV) was sought in vitro (in infected swine testicular [ST] cells) and in vivo (in the intestinal tissues of infected piglets). The methods used were (1) DNA electrophoresis for detection of DNA fragmentation, and (2) terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-fluorescein nick and labelling (TUNEL). DNA “laddering” was detected in TGEV-infected ST cells only. Numerous signs of apoptosis were detected in TGEV-infected ST cells by TUNEL assay, the positive (dark brown) staining reaction being present in the majority of cell nuclei, without background staining. No such staining was seen in TGEV-infected enterocytes at various times after inoculation of piglets. Thus, it would appear that apoptosis does not occur in the enterocytes of piglets infected with TGEV.

Differential Distribution of Intercellular Adhesion Molecules (ICAM–1, ICAM–2, and ICAM–3) and THE MS–1 Antigen in Normal and Diseased Human Synovia

Cellular adhesion and differentiation molecules (CAMs) may play a role in the recruitment and retention of inflammatory cells into rheumatoid arthritis synovial tissue (RA ST). In order to determine if certain CAMs are up-regulated in RA ST compared with normal ST, we studied the distribution of intercellular adhesion molecules (ICAMs) 1, 2, and 3 in ST. We also studied the MS-1 antigen since it is preferentially expressed on discontinuous endothelia, such as those found in RA ST; MS-1 is also expressed differentially upon cytokine activation of cells in vitro or in pathologic conditions in situ. Thus, we postulated a possible similarity between MS-1 and ICAM-1 expression in inflamed ST. Immunohistochemical analysis was used to determine the distribution of ICAMs and MS-1 in ST from 10 patients with RA, 10 with osteoarthritis (OA), and 4 normal individuals. ICAM-1 expression was found on significantly more RA ST endothelial cells compared with normal cells, as well as on RA ST macrophages and lining cells. ICAM-2, also found on endothelial cells, showed no differential staining pattern. ICAM-3 was present on RA ST macrophages and lining cells as well as on some RA and OA endothelial cells. The MS-1 antigen was present on most RA and OA ST endothelia, lining cells, and macrophages. ICAM-1 expression and MS-1 expression in the lining layer were positively correlated in both RA and OA. ICAM-1, while found mainly on endothelial cells, is up-regulated on RA ST macrophages and lining cells, suggesting a role for these cells in the infiltration and tissue damage seen in the RA ST: ICAM-3, which is present mainly on normal resting leukocytes but not on normal endothelium, is expressed by some diseased ST leukocytes and endothelial cells. MS-1 is also found on the RA ST specialized, fenestrated endothelium, on macrophages, and in the lining layer. These results suggest that the differential expression of ICAMs and MS-1 in RA ST compared with normal ST might play a special role in the pathogenesis of RA.

Evolution and tropism of transmissible gastroenteritis coronavirus.

Transmissible gastroenteritis coronavirus (TGEV) is an enteropathogenic coronavirus isolated for the first time in 1946. Nonenteropathogenic porcine respiratory coronaviruses (PRCVs) have been derived from TGEV. The genetic relationship among six European PRCVs and five coronaviruses of the TGEV antigenic cluster has been determined based on their RNA sequences. The S proteins of six European PRCVs have an identical deletion of 224 amino acids starting at position 21. The deleted area includes the antigenic sites C and B of TGEV S glycoprotein. Interestingly, two viruses (NEB72 and TOY56) with respiratory tropism have the S protein with a similar size to the enteric viruses. NEB72 and TOY56 viruses have 2 and 15 specific amino acid differences with the enteric viruses, respectively. Four of the residues changed are located within the deletion present in the PRCVs and may influence the enteric tropism of TGEV in vivo. A receptor binding site (RBS) used by the virus to infect ST and other cell types might be located between sites A and D of the S glycoprotein, since monoclonal antibodies (MAbs) specific for these sites inhibit the binding of the virus to ST cells. An evolutionary tree relating 13 enteric and respiratory isolates has been proposed. According to this tree, a main virus lineage evolved from a recent progenitor which was circulating around 1941. From this, secondary lineages originated PUR46, NEB72, TOY56, MIL65, BRI70, and the PRCVs, in this order. Least squares estimation of the origin of TGEV-related coronaviruses showed a significant constancy in the mutation fixation rate.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of colcemid concentration on chromosome aberration analysis in human lymphocytes.

As a part of technical improvements of chromosome aberration analysis on human peripheral lymphocytes for biological radiation dosimetry, we examined the optimal conditions for the use of colcemid in chromosome preparation in order to obtain enough number of cells at metaphase in the first cell division. When treated with colcemid at concentrations below 0.01 microgram/ml from the beginning of culture, cultures harvested at 48 hours had low mitotic indices. Colcemid treatment at 0.025 to 0.05 microgram/ml during 48 hours resulted in high mitotic indices (8 to 15%) and almost of the mitotic cells remaining in the 1st cell division, suggesting that this range of colcemid concentration was appropriate for continuous treatment with colcemid. We further examined the effect of colcemid concentration on the quantitative consistency of the yields of radiation-induced chromosome aberration. Repeated experiments showed that the yield of dicentrics and centric rings in the culture having colcemid at 0.025 microgram/ml concentration were larger than that at 0.05 microgram/ml. These data indicate the importance of assuring the accuracy of colcemid concentration in the lymphocyte culture for cytogenetic radiation dosimetry.