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1.
J Comp Pathol ; 128(4): 289-92, 2003 May.
Article in English | MEDLINE | ID: mdl-12834613

ABSTRACT

The white-throated woodrat is a principal host of Whitewater Arroyo (WWA) virus, an arenavirus, in the western United States. The purpose of the present study was to investigate the pathology of WWA infection in this species. Twenty-one animals (eight newborn, seven juvenile, and six adult) were inoculated with WWA virus and killed at varying intervals after inoculation. The most striking histological findings were lymphocytic meningitis and perivascular lymphocytic cuffing in the brains of the animals killed on day 85, 113 or 121. Arenaviral antigen was detected immunohistochemically in the brain of each affected animal, suggesting that the inflammatory lesions in the brain were caused by WWA virus. Comparisons of the results of tests for infectious virus and antigen in brain and other solid tissues indicated that immunohistochemistry may be a useful method for detection of WWA viral antigen in post-mortem specimens.


Subject(s)
Arenaviridae Infections/pathology , Arenavirus , Rodent Diseases/pathology , Sigmodontinae , Animals , Animals, Newborn , Antigens, Viral/immunology , Arenaviridae Infections/immunology , Arenaviridae Infections/virology , Arenavirus/immunology , Arenavirus/isolation & purification , Cerebral Cortex/pathology , Cerebral Cortex/virology , Female , Immunohistochemistry , Kidney/pathology , Male , Meningitis/pathology , Meningitis/virology , Neurons/immunology , Neurons/pathology , Neurons/virology , Rats , Rodent Diseases/immunology , Rodent Diseases/virology , Time Factors
2.
J Virol ; 76(3): 1154-62, 2002 Feb.
Article in English | MEDLINE | ID: mdl-11773391

ABSTRACT

Pichinde virus is an arenavirus that infects guinea pigs and serves as an animal model for human Lassa fever. An attenuated Pichinde virus variant (P2) and a virulent variant (P18) are being used to delineate pathogenic mechanisms that culminate in shock. In guinea pigs, the infection has been shown to begin in peritoneal macrophages following intraperitoneal inoculation and then spreads to the spleen and other reticuloendothelial organs. We show here that infection of the murine monocytic cell line P388D1 with either Pichinde virus variant resulted in the induction of inflammatory cytokines and effectors, including interleukin-6 and tumor necrosis factor alpha. Since these genes are regulated in part by the cellular transcription factors NF-kappaB and RBP-Jkappa, we compared the activities of NF-kappaB and RBP-Jkappa in P388D1 cells following infection with Pichinde virus. The attenuated P2 virus inhibited NF-kappaB activation and caused a shift in the size of the RBP-Jkappa complex. The virulent P18 virus showed less inhibition of NF-kappaB and failed to alter the size of the RBP-Jkappa complex. Peritoneal cells from P2-infected guinea pigs showed induction of NF-kappaB RelA/p50 heterodimer and p50/p50 homodimer and manifested an increase in the size of RBP-Jkappa. By contrast, P18 induced large amounts of the NF-kappaB p50/p50 dimer but failed to induce RelA/p50 or to cause an increase in the RBP-Jkappa size. Taken together, these changes suggest that the attenuated viral strain induces an "activation" of macrophages, while the virulent form of the virus does not.


Subject(s)
Arenaviridae Infections/immunology , DNA-Binding Proteins/biosynthesis , Macrophages, Peritoneal/immunology , Macrophages/immunology , NF-kappa B/biosynthesis , Nuclear Proteins , Pichinde virus/immunology , Animals , Arenaviridae Infections/virology , Cell Line , Cell Nucleus/metabolism , Guinea Pigs , Immunoglobulin J Recombination Signal Sequence-Binding Protein , Interleukin-6/biosynthesis , Macrophages/virology , Macrophages, Peritoneal/virology , Male , Mice , NF-kappa B/genetics , Peritoneum/cytology , Peritoneum/virology , Pichinde virus/physiology , Tumor Necrosis Factor-alpha/biosynthesis
3.
Virology ; 290(1): 30-8, 2001 Nov 10.
Article in English | MEDLINE | ID: mdl-11883003

ABSTRACT

The new world arenavirus Pichinde (PIC) is the basis of an accepted small animal model for human Lassa fever. PIC (Munchique strain) variant P2 is attenuated in guinea pigs, whereas variant P18 is extremely virulent. Previous sequence analysis of the S segments of these two viruses indicated a small number of possible virulence markers in the glycoprotein precursor (GPC) and nucleoprotein (NP) genes. In order to determine the role of these S segment genes in guinea pig virulence in this system, we have generated reassortant viruses. When tested in outbred guinea pigs, the reassortant containing the S segment from the virulent parent P18 (S18L2) caused significantly higher morbidity than the reciprocal reassortant. This increased morbidity was associated with higher viral titers in serum and spleen. However, the S18L2 reassortant was not as fully virulent in this system as the P18 parent, indicating a role for L segment genes in virulence.


Subject(s)
Arenaviridae Infections/virology , Pichinde virus/genetics , Reassortant Viruses/genetics , Animals , Arenaviridae Infections/physiopathology , Base Sequence , Chlorocebus aethiops , DNA, Viral , Disease Models, Animal , Genetic Variation , Guinea Pigs , Male , Molecular Sequence Data , Pichinde virus/pathogenicity , Reassortant Viruses/pathogenicity , Recombination, Genetic , Sequence Homology, Nucleic Acid , Vero Cells
4.
Gastroenterology ; 119(6): 1537-47, 2000 Dec.
Article in English | MEDLINE | ID: mdl-11113075

ABSTRACT

BACKGROUND & AIMS: Enteropathy is a frequent complication of diclofenac and other nonsteroidal anti-inflammatory drugs, yet little is known about the underlying mechanism. One possibility is that reactive metabolites of diclofenac form adducts with enterocyte macromolecules, as previously shown for liver. We addressed this possibility by using immunohistochemistry to detect diclofenac adducts. METHODS: Rats were treated orally with diclofenac (10-100 mg/kg) and killed after 1-24 hours, and their gastrointestinal (GI) tracts were evaluated for ulcer number and area. Adduct distribution and intensity were assessed by immunohistochemistry by using a technique to simultaneously process and stain multiple intestinal rings. RESULTS: Drug treatment led to dose-dependent formation of both adducts and ulcers only in small intestine and only in animals with intact enterohepatic circulation. Adducts formed within enterocytes by 1 hour, translocated to the brush border, preceded ulceration and vascular protein leakage, and were intense at sites of ulceration. Adducts and ulcers exhibited a parallel distribution within intestinal quintiles: 3rd > 5th >> 1st. CONCLUSIONS: Diclofenac treatment resulted in the formation of drug adducts in enterocytes. Because this molecular change occurred before ulceration, was dose dependent, and exhibited concordant distribution with extent of ulceration, the results suggest a causal role for drug adduct formation in diclofenac enteropathy.


Subject(s)
Anti-Inflammatory Agents, Non-Steroidal/adverse effects , Anti-Inflammatory Agents, Non-Steroidal/metabolism , Diclofenac/adverse effects , Diclofenac/metabolism , Enterocytes/metabolism , Intestinal Diseases/chemically induced , Ulcer/chemically induced , Animals , Bile/metabolism , Dose-Response Relationship, Drug , Intestinal Diseases/pathology , Male , Rats , Rats, Sprague-Dawley , Time Factors , Tissue Distribution , Ulcer/pathology
5.
Virology ; 270(1): 111-23, 2000 Apr 25.
Article in English | MEDLINE | ID: mdl-10772984

ABSTRACT

The early stages of Venezuelan equine encephalitis virus (VEE) pathogenesis in the mouse model have been examined using a genetic approach. Disease progression of a molecularly cloned single-site mutant was compared with that of the parental virus to determine the step in the VEE pathogenetic sequence at which the mutant was blocked. Assuming that such a block constitutes a genetic screen, isolates from different tissues thought to be distal to the block in the VEE pathogenetic sequence were analyzed to determine the pathogenetic step at which revertants of the mutant were selected. Directed mutation and analysis of reversion in vivo provide two powerful genetic tools for the dissection of the wild-type VEE pathogenetic sequence. Virus from the parental virulent clone, V3000, first replicated in the draining lymph node after subcutaneous inoculation in the left rear footpad. Movement of a cloned avirulent mutant, V3010 (E2 76 Glu to Lys), to the draining lymph node was impaired, replication in the node was delayed, and spread beyond the draining lymph node was sporadic. Serum, contralateral lymph node, spleen, and brain isolates from V3010 inoculated animals were invariably revertant with respect to sequence at E2 76 and/or virulence in mice. Revertants isolated from serum and contralateral lymph node retained the V3010 E2 Lys 76 mutation but also contained a second-site mutation, Glu to Lys at E2 116. Modification of the V3010 clone by addition of the second-site mutation at E2 116 produced a virus that bypassed the V3010 block at the draining lymph node but that did not possess full wild-type capacity for replication in the central nervous system or for induction of mortality. A control construct containing only the E2 116 reverting mutation on the V3000 background was identical to V3000 in terms of early pathogenetic steps and virulence. Therefore, analysis of mutant replication and reversion in vivo suggested (1) that the earliest steps in VEE pathogenesis are transit to the draining lymph node and replication at that site, (2) that the mutation in V3010 impairs transit to the draining lymph node and blocks dissemination to other tissues, and (3) that reversion can overcome the block without restoring full virulence.


Subject(s)
Encephalitis Virus, Venezuelan Equine/genetics , Encephalitis Virus, Venezuelan Equine/pathogenicity , Encephalomyelitis, Venezuelan Equine/virology , Point Mutation/genetics , Suppression, Genetic/genetics , Animals , Brain/virology , Cell Line , Cloning, Molecular , Disease Progression , Encephalitis Virus, Venezuelan Equine/physiology , Encephalomyelitis, Venezuelan Equine/mortality , Female , Lymph Nodes/virology , Mice , Phenotype , RNA, Viral/genetics , RNA, Viral/metabolism , Spleen/virology , Structure-Activity Relationship , Vaccines, Attenuated/genetics , Viral Envelope Proteins/genetics , Viral Envelope Proteins/physiology , Viral Vaccines/genetics , Viremia , Virulence/genetics , Virus Replication
6.
Virology ; 263(2): 339-48, 1999 Oct 25.
Article in English | MEDLINE | ID: mdl-10544107

ABSTRACT

The age-related acquisition of resistance to fatal Sindbis virus infection was examined using a molecularly cloned laboratory strain of the AR339 isolate designated TRSB. TRSB caused 100% mortality in mice up to 5 days of age. Resistance to fatal infection developed abruptly between 5 and 9 days of age. Lethal Sindbis virus infection of mice inoculated at 4 days of age was characterized by high levels of virus replication, induction of high levels of interferon-alpha/beta and TNF-alpha and severe thymic involution indicative of a systemic stress response. These changes correlated with predominantly noninflammatory lesions. In contrast, TRSB infection of older mice was characterized by survival, more limited virus replication, reduced cytokine induction, and the development of inflammatory responses leading to encephalitis, myositis, and myocarditis. Previous studies utilized infections of neonatal mice with TRSB and an attenuated mutant of TRSB to compare fatal and nonfatal Sindbis infection (Trgovcich et al., 1996. Virology 224, 73-83). The experiments reported here utilize mouse age at the time of infection to create conditions for examination of fatal and nonfatal TRSB infections. Both experiments suggest that fatal infection is associated with a shock-like syndrome and little or no inflammatory pathology, while survival is correlated with greatly reduced cytokine levels and inflammatory lesions.


Subject(s)
Aging , Alphavirus Infections/physiopathology , Interferons/metabolism , Sindbis Virus/physiology , Stress, Physiological/pathology , Tumor Necrosis Factor-alpha/metabolism , Alphavirus Infections/mortality , Alphavirus Infections/pathology , Alphavirus Infections/virology , Animals , Brain/pathology , Brain/virology , Cell Line , Cricetinae , Disease Models, Animal , Disease Progression , Disease Susceptibility , Encephalitis, Viral/immunology , Encephalitis, Viral/pathology , Encephalitis, Viral/virology , Inflammation/pathology , Inflammation/virology , Mice , Mice, Inbred Strains , Sindbis Virus/genetics , Sindbis Virus/immunology , Sindbis Virus/pathogenicity , Stress, Physiological/virology , Survival Rate , Thymus Gland/pathology , Thymus Gland/virology , Virulence , Virus Replication
7.
J Forensic Sci ; 44(5): 1082-5, 1999 Sep.
Article in English | MEDLINE | ID: mdl-10486964

ABSTRACT

Accidental hypothermia has been described in the forensic literature but reports of occurrence in hospitalized patients are rare. Associated anatomic lesions include acute hemorrhagic pancreatitis and characteristic acute gastric ulcers termed Wischnewski ulcers. We report here two patients with cirrhosis and ascites; one also had hepatocellular carcinoma. Portal vein thrombosis, acute hemorrhagic pancreatitis and Wischnewski ulcers were present in both. The clinical records documented hypothermia that progressed over several days. Temperature nadirs of 31.0 degrees C (87.8 degrees F) and 32.2 degrees C (90.0 degrees F) were recorded in each patient, respectively, one day before death, although each transiently reached temperatures that did not register on standard monitoring devices. This is the first report that chronicles antemortem body temperatures in hypothermic patients with Wischnewski ulcers and pancreatitis at autopsy. Also, the association of these findings with portal vein thrombosis and cirrhosis has not been previously described. We discuss this constellation of findings with regard to possible mechanistic interrelations.


Subject(s)
Hypothermia/complications , Liver Cirrhosis/complications , Pancreatitis/etiology , Portal Vein , Stomach Ulcer/etiology , Venous Thrombosis/complications , Acute Disease , Body Temperature , Carcinoma, Hepatocellular/complications , Carcinoma, Hepatocellular/pathology , Fatal Outcome , Female , Forensic Medicine , Hospitalization , Humans , Hypothermia/pathology , Liver Cirrhosis/pathology , Liver Neoplasms/complications , Liver Neoplasms/pathology , Male , Middle Aged , Pancreatitis/pathology , Stomach Ulcer/pathology , Venous Thrombosis/pathology
8.
Am J Trop Med Hyg ; 61(2): 220-5, 1999 Aug.
Article in English | MEDLINE | ID: mdl-10463670

ABSTRACT

The established animal model for Lassa fever is based on the new world arenavirus Pichinde (PIC). Natural isolates of PIC virus are attenuated in guinea pigs, but serial guinea pig passage renders them extremely virulent in that host. We have compared the nucleotide sequences of the small RNA segments of two attenuated, low-passage variants of the PIC virus Munchique strain (CoAn 4763) and two virulent, high-passage derivatives. Missense mutations in the glycoprotein precursor (GPC) gene at codons GPC-119, GPC-140, and GPC-164 and the nucleoprotein gene (NP) codons NP-35 and NP-374 were most closely associated with virulence. Codon GPC-140 is predicted to represent a region of peak hydrophilicity of the glycoprotein 1 (GP1); it is conceivable that mutations at this site could influence virulence by altering B cell epitopes or virus attachment protein conformation.


Subject(s)
Hemorrhagic Fever, American/virology , Pichinde virus/genetics , RNA/genetics , Animals , Base Sequence , Codon , Genetic Variation , Guinea Pigs , Lethal Dose 50 , Male , Models, Genetic , Mutation, Missense , Phenotype , Pichinde virus/classification , Pichinde virus/isolation & purification , Pichinde virus/pathogenicity , Reverse Transcriptase Polymerase Chain Reaction , Sequence Analysis, RNA
9.
Med Mycol ; 36(5): 263-7, 1998 Oct.
Article in English | MEDLINE | ID: mdl-10075494

ABSTRACT

Fungal-specific primers targeted for highly conserved genomic nucleic acid sequences were used in a polymerase chain reaction (PCR) to amplify DNA from lobomycosis lesions in a bottlenose dolphin. Sequence alignments of this DNA possessed high homology to fungal ribosomal DNA sequences found in the genus Cladosporium. When used for in situ hybridization, the riboprobe transcribed from a cloned PCR-generated fragment bound to Loboa loboi cells. These results support the hypothesis that L. loboi in dolphin tissue is a fungus.


Subject(s)
DNA, Ribosomal/isolation & purification , Dermatomycoses/veterinary , Dolphins/microbiology , Entomophthorales , Skin/microbiology , Zygomycosis/veterinary , Animals , Base Sequence , Cladosporium/genetics , DNA, Fungal/genetics , DNA, Fungal/isolation & purification , DNA, Ribosomal/genetics , Dermatomycoses/microbiology , Molecular Sequence Data , Sequence Alignment , Sequence Homology, Nucleic Acid , Zygomycosis/microbiology
10.
Virology ; 227(1): 234-8, 1997 Jan 06.
Article in English | MEDLINE | ID: mdl-9007079

ABSTRACT

Neonatal mice were infected with virus derived from a molecular clone of a laboratory strain of Sindbis virus, TRSB. The resulting acute fatal infection was typified by few if any of the classic hallmarks of encephalitis, very high levels of interferon-alpha/beta (IFNalphabeta), and lesions in the thymus and hematopoietic tissues usually associated with a severe stress response. Infection with an attenuated mutant of TRSB, which harbors a single amino acid change in the E2 surface glycoprotein (TRSBr114), was characterized by encephalitis, reduced mortality, low levels of IFNalphabeta, and no thymic pathology (J. Trgovcich, J. F. Aronson, and R. E. Johnston, 1996, Virology 224, 73-83). Here we report that infection of neonatal mice with TRSB, but not TRSBr114, resulted in induction of high levels of tumor necrosis factor-alpha as well as high and sustained levels of adrenalcorticotropin-releasing hormone and corticosterone. This syndrome of potentially toxic cytokine and stress hormone induction correlates with lethal Sindbis virus infection and constitutes a previously unrecognized aspect of Sindbis virus pathogenesis in mice.


Subject(s)
Alphavirus Infections/physiopathology , Sindbis Virus , Stress, Physiological/virology , Adrenocorticotropic Hormone/biosynthesis , Alphavirus Infections/immunology , Alphavirus Infections/mortality , Alphavirus Infections/virology , Animals , Animals, Newborn , Brain/pathology , Corticosterone/biosynthesis , Mice , Point Mutation , Shock, Septic/immunology , Shock, Septic/physiopathology , Shock, Septic/virology , Sindbis Virus/genetics , Stress, Physiological/immunology , Thymus Gland/pathology , Tumor Necrosis Factor-alpha/biosynthesis
11.
Virology ; 224(1): 73-83, 1996 Oct 01.
Article in English | MEDLINE | ID: mdl-8862401

ABSTRACT

A comparative pathogenesis study was performed in neonatal mice using a molecularly cloned laboratory variant of Sindbis strain AR339, designated TRSB, and a single-site attenuated mutant of TRSB derived by site-directed mutagenesis of the E2 glycoprotein from Ser to Arg at residue 114 (TRSBr114). TRSB caused 100% mortality with an average survival time of 3.0 +/- 0.7 days, whereas mice inoculated with TRSBr114 exhibited an attenuated disease course with 46% mortality and an extended average survival time of 7.5 +/- 3.4 days for those animals that died. Reduced virulence of TRSBr114 was characterized by delayed appearance of detectable virus, relative to TRSB, and by lower peak virus titers in both sera and brains of infected mice. TRSB infection induced very high peak serum titers of interferon alpha/beta (215,000 units/ml compared to 2100 units/ml for TRSBr114). In situ hybridization analysis demonstrated replication of TRSB in brain, but only minimal histopathological changes and no evidence of encephalitis were observed. However, extensive extraneural lesions and viral replication were found in skin, connective tissue, and muscle. Moreover, dramatic involution of the thymus and loss of hematopoietic tissues were observed in the absence of virus replication at these sites, suggesting the involvement of a systemic physiological stress response in TRSB infection. TRSBr114 infection did not cause thymic lesions. Otherwise, the attenuated mutant demonstrated a similar pattern of tissue and organ involvement, but lesions and positive in situ hybridization signal were much more limited in scope and intensity compared to TRSB. TRSBr114-infected mice developed myositis and encephalomyelitis approximately 6 days postinfection. Therefore, TRSB-infected animals may succumb to an early syndrome associated with the stress response, preventing their survival for a time sufficient for the development of encephalitis. Alternatively, a systemic stress response, as evidenced by thymic involution, may result in immunosuppression, thus contributing to the absence of encephalitis. In any event, the attenuating mutation in the E2 glycoprotein significantly altered the course of Sindbis-induced disease by limiting virus replication and associated damage early in infection. Mutant-infected animals survived beyond Day 4 and progressed to a classical encephalomyelitis from which about half recovered.


Subject(s)
Alphavirus Infections/virology , Encephalitis, Viral/virology , Sindbis Virus/pathogenicity , Viral Envelope Proteins/genetics , 3T3 Cells , Alphavirus Infections/pathology , Animals , Animals, Newborn , Cell Line , Cricetinae , In Situ Hybridization , Interferon-alpha , Interferon-beta , Mice , Mutagenesis, Site-Directed , Sindbis Virus/genetics , Sindbis Virus/physiology , Structure-Activity Relationship , Virulence/genetics , Virus Replication/genetics
12.
Am J Trop Med Hyg ; 52(3): 262-9, 1995 Mar.
Article in English | MEDLINE | ID: mdl-7694969

ABSTRACT

Pichinde virus (PIC) is a reticuloendothelial arenavirus of the New World tropics. A guinea pig passage-adapted strain of this virus (adPIC) is uniformly lethal for inbred guinea pigs, while the related, prototype strain (PIC3739) has attenuated virulence. The abilities of adPIC and PIC3739 to induce tumor necrosis factor (TNF) in vivo and in cultured macrophages were compared. Infection with adPIC, but not PIC3739, was associated with detectable serum TNF that peaked in week 2 of infection. Tumor necrosis factor was found in the spleens of adPIC- and PIC3739-infected animals in week 1 of infection; TNF alpha mRNA levels in spleens and livers of adPIC infected animals increased and remained high throughout infection, whereas PIC3739-infected organs showed down regulation of TNF alpha mRNA late in infection. Peritoneal macrophages explanted from adPIC-infected animals showed enhanced lipopolysaccharide-inducible TNF production. Altered regulation of TNF production may play a role in the pathogenesis of guinea pig arenavirus disease.


Subject(s)
Disease Models, Animal , Hemorrhagic Fever, American/etiology , Pichinde virus/pathogenicity , Tumor Necrosis Factor-alpha/analysis , Animals , Blotting, Northern , Cells, Cultured , Guinea Pigs , Hemorrhagic Fever, American/virology , Liver/metabolism , Macrophages/metabolism , Macrophages, Peritoneal/metabolism , Male , RNA, Messenger/analysis , Spleen/cytology , Spleen/metabolism , Tumor Necrosis Factor-alpha/biosynthesis , Tumor Necrosis Factor-alpha/genetics
13.
Virology ; 206(2): 994-1006, 1995 Feb 01.
Article in English | MEDLINE | ID: mdl-7856110

ABSTRACT

The pathogenesis of Venezuelan equine encephalitis virus (VEE) was examined in the mouse model using V3000, a virus derived from a molecular clone of the Trinidad donkey strain of VEE. These results were compared in parallel experiments with avirulent mutants of VEE derived by site-directed mutagenesis of the clone. Adult mice, inoculated subcutaneously in their left rear footpad with V3000, were followed in a time course study for 6 days in which 15 organs were tested for histopathological changes, for the presence of viral antigen by immunohistochemical staining, for the presence of viral nucleic acid by in situ hybridization analysis, and for content of viable virus. Virus was detected in the footpad inoculation site, but until 12 hr postinoculation (pi), the level of virus did not suggest early viral replication. By 4 hr pi, however, replication of V3000 was evident in the draining popliteal lymph node. At this early time point, no virus could be isolated from any other organ examined. At 12 hr, a significant serum viremia was observed, and virus was detected at a low level in a number of well vascularized organs, including spleen, heart, lung, liver, kidney, and adrenal gland. By 18 hr, high virus titers were present in serum and all the lymphoid organs examined, and these tissues appeared to be the major peripheral sites of V3000 replication. Virus in serum and peripheral organs was cleared by 3-4 days pi. In a second phase of the infection, V3000 invaded the central nervous system (CNS), replicated predominantly in neurons, and persisted in the brain until death by encephalitis. Pathologic findings as well as the results of immunocytochemical and in situ hybridization examination were generally coordinate with virus titration. A site-directed mutant of V3000, V3010, contained a mutation in the gene for the E2 glycoprotein at codon 76 (Glu to Lys) which rendered it avirulent after footpad inoculation. Detection of V3010 replication in the draining lymph node was sporadic and was sometimes delayed to as long as 3 days pi. Infrequent and/or delayed virus spread to other sites also was observed. Analogous experiments were performed with other mutants which were avirulent by the footpad inoculation route: V3014, a mutant differing from V3000 at three loci (E2 Lys 209, E1 Thr 272, and E2 Asn 239), as well as single-site mutants V3032 (E2 Lys 209) and V3034 (E1 Thr 272).(ABSTRACT TRUNCATED AT 400 WORDS)


Subject(s)
Encephalitis Virus, Venezuelan Equine/pathogenicity , Encephalomyelitis, Venezuelan Equine/physiopathology , Encephalomyelitis, Venezuelan Equine/virology , Virus Replication , Animals , Brain/pathology , Brain/virology , Cell Line , Cloning, Molecular , Cricetinae , Death , Drug Design , Encephalitis Virus, Venezuelan Equine/genetics , Encephalitis Virus, Venezuelan Equine/physiology , Encephalomyelitis, Venezuelan Equine/pathology , Female , Horses , In Situ Hybridization , Islets of Langerhans/pathology , Islets of Langerhans/virology , Kidney , Lymph Nodes/pathology , Lymph Nodes/virology , Mice , Mice, Inbred Strains , Mutagenesis, Site-Directed , Necrosis , Neurons/pathology , Neurons/virology , Organ Specificity , Pancreas/pathology , Pancreas/virology , RNA, Viral/analysis , Trinidad and Tobago , Viral Vaccines , Virulence
14.
Am J Pathol ; 145(1): 228-35, 1994 Jul.
Article in English | MEDLINE | ID: mdl-8030751

ABSTRACT

A guinea pig passage-adapted strain of the arena-virus Pichinde (adPIC) is highly virulent in inbred guinea pigs, whereas the related strain PIC3739 is attenuated. Both viruses were macrophage tropic and infected peritoneal, splenic, liver, and alveolar macrophages during experimental Pichinde virus infection. Infection with the virulent strain was associated with unlimited viral replication in the face of exaggerated delayed-type hypersensitivity response, manifested by the macrophage disappearance reaction. Histopathological lesions unique to adPIC-infected guinea pigs included intestinal villus blunting with mucosal infiltration by pyknotic debris-laden macrophages and apoptosis of crypt epithelial cells. Splenic red pulp necrosis was also significantly associated with adPIC infection but not PIC3739 infection. These findings may provide clues to the pathogenesis of a group of poorly understood human viral hemorrhagic fevers.


Subject(s)
Hemorrhagic Fever, American/microbiology , Hemorrhagic Fever, American/pathology , Pichinde virus/pathogenicity , Animals , Disease Models, Animal , Guinea Pigs , Hemorrhagic Fever, American/mortality , Macrophages/microbiology , Macrophages/pathology , Necrosis , Species Specificity , Spleen/pathology
15.
Mutat Res ; 192(1): 79-82, 1987 Sep.
Article in English | MEDLINE | ID: mdl-3657838

ABSTRACT

In this note we distinguish between multiple mutations affecting a given locus which are generated at separate error-prone lesions and multiple independent mutations generated at a single error-prone lesion. We describe a basis for determining the probability with which the latter class of mutations occurs based on the mutant fraction in the progeny and determine an average probability of 0.6 mutations/replication/mutagenic site for those EMS-modified sites which are mutagenic for G6PDH activity in CHO cells.


Subject(s)
DNA Damage , Ethyl Methanesulfonate/toxicity , Mutation/drug effects , Animals , Cricetinae , DNA/drug effects , DNA Repair , DNA Replication , Probability
16.
Mutat Res ; 177(2): 277-81, 1987 Apr.
Article in English | MEDLINE | ID: mdl-3561427

ABSTRACT

Treatment of G1-synchronized mammalian cells with mutagenic agents which act on one strand of the DNA at a given site would be expected to produce colonies containing both mutant and wild-type cells (mosaic). We have observed that in addition to mosaic colonies, G1-synchronized Chinese hamster ovary cells which had been treated with the single-strand mutagen ethyl methanesulfonate (EMS), produced colonies in which all the cells lacked glucose-6-phosphate dehydrogenase activity. These completely mutant (pure) colonies could be derived from a potentially mosaic colony by the "death" of the wild-type cell after the first cell division, leaving only the glucose-6-phosphate dehydrogenase (G6PD)-deficient cell to grow into a colony (lethal sectoring). Using time-lapse photography to analyze cell lineages after EMS treatment, we find that cell lysis, cell release, cell migration, or proliferative failure of one lineage at the 2-cell stage accounts for only 20-25% of the pure mutant colonies observed. This result suggests that in the Chinese hamster cell there exists a mutational mechanism which fixes the mutation in both strands of the DNA before the next replication cycle following EMS treatment.


Subject(s)
Ethyl Methanesulfonate/toxicity , Mutation , Animals , Cell Cycle/drug effects , Cell Division/drug effects , Cell Line , Cell Survival/drug effects , Clone Cells , Cricetinae , Cricetulus , Female , Ovary
17.
Dev Biol ; 101(1): 1-7, 1984 Jan.
Article in English | MEDLINE | ID: mdl-6692968

ABSTRACT

Birefringence is shown to be a property of lamellar bodies in living lung type II cells and is used to demonstrate the appearance of differentiated type II cells in cultures of fetal rat lung explanted as early as Day 13. The culture medium was F-12 K without added serum, hormones, or growth factors. This system is considered to be potentially useful in analyzing factors and cell interactions involved in determination and cytodifferentiation of the type II cell from lung bud epithelium.


Subject(s)
Lung/cytology , Animals , Cell Differentiation/drug effects , Cell Division/radiation effects , Cells, Cultured , Epithelial Cells , Hydrocortisone/pharmacology , Lung/embryology , Lung/radiation effects , Microscopy, Polarization , Phosphatidylcholines/metabolism , Rats
18.
In Vitro ; 19(8): 642-50, 1983 Aug.
Article in English | MEDLINE | ID: mdl-6193052

ABSTRACT

Human retinal pigment epithelium (RPE)-derived cell lines were established from RPE-covered choroid tissue fragments, which had been generated by culture on nontissue culture plastic. Two phenotypes were apparent in a given line: (a) a compact cell which formed domes and ultimately melanosomes before being sloughed; and (b) a squamous cell which was often elongated and which bound antibody to human keratins. This latter cell did not become black or form domes. The average number of cell doublings for the 13 lines tested was between 15 and 40 when cultured in a modified Eagle's minimum essential medium containing 10% fetal bovine serum. Cell lines newly established from material that had been in culture for more than 6 months had normal mitotic chromosomes and still developed areas with strongly pigmented cells when refed. Normal human epithelial cell lines of this kind may be useful in studies of cell aging and defining change associated with the development of neural cells from ectoderm.


Subject(s)
Cell Line , Pigment Epithelium of Eye/cytology , Cell Division , Cell Survival , Cell Transformation, Viral , Choroid , Humans , Karyotyping , Keratins/analysis , Melanocytes/ultrastructure , Pigment Epithelium of Eye/analysis , Simian virus 40/physiology , Trypsin
19.
Mech Ageing Dev ; 21(3-4): 229-44, 1983.
Article in English | MEDLINE | ID: mdl-6887978

ABSTRACT

The loosely associated lung mesenchymal cell found between the ducts of the 3-to 4-month fetus is considered to be the probable progenitor of lung fibroblast cultures and of alveolar interstitial cells. A possible stage- and tissue-specific property of this cell type, the reduction of cortisone to cortisol, is defined and its activity studied in a variety of cell lines. Fibroblast lines derived from both fetal and adult lung had about ten times the cortisone-reducing activity of corresponding skin fibroblast lines. This relatively high activity was also characteristic of cell lines established by cloning the initial fetal lung digest. Lines established from developmentally related esophagus and from trachea had less activity than corresponding lung fibroblasts. A high level of cortisone-reducing activity was maintained in four serially passaged lung fibroblast lines for at least 85% of the proliferative life span and a second cell property, the enhancement of this activity by pretreatment with cortisol, was also maintained in three of the four lines.


Subject(s)
Cortisone/metabolism , Hydrocortisone/metabolism , Lung/physiology , Cell Count , Cell Division , Cell Line , Fetal Organ Maturity , Fibroblasts/cytology , Fibroblasts/physiology , Humans , Lung/cytology , Lung/embryology , Microscopy, Electron , Skin
20.
In Vitro ; 17(1): 61-70, 1981 Jan.
Article in English | MEDLINE | ID: mdl-7216240

ABSTRACT

Cells obtained by tryptic digestion of the surface of intact adult and fetal Fischer 344 rat lungs were plated on glass fragments. Epithelial cell lines were readily established by selecting fragments with 2 to 10 cells 2 days after plating and growing them in F12 K media containing 10% fetal bovine serum (FBS). These cell lines and new lines that can be easily obtained a provide a reliable source of diploid, density-inhibited epithelial cells. These cells of mesothelial origin may serve as models for the study of mesothelial cells in situ.


Subject(s)
Pleura/cytology , Animals , Cell Division , Cells, Cultured , Chromosome Mapping , Epithelium/ultrastructure , Microscopy, Electron, Scanning , Rats , Trypsin
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