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Biomedical subjects

N Nathanson

Publications and source records attributed to N Nathanson.

At least 37 records · Page 2Linked to original sources
PubMed1994

Replication in cultured C2C12 muscle cells correlates with the neuroinvasiveness of California serogroup bunyaviruses.

The neuroinvasiveness of California serogroup bunyaviruses is determined by the ability of the virus to replicate in striated muscle after peripheral inoculation of mice. Neuroinvasiveness was mapped to the medium (M) RNA segment of the virus, which encodes the viral glycoproteins, when reassortants were made between La Crosse/original virus, a neuroinvasive isolate, and Tahyna-181/57 virus, a nonneuroinvasive clone. We have tested the murine muscle cell line C2C12 as a surrogate for myotropism and have found that there is a slight, but reproducible difference in the replication of virus clones bearing the M RNA segment of La Crosse/original virus compared to clones bearing the M RNA segment of Tahyna-181/57 virus, as determined by viral titer, antigen expression, and plaque formation.

Animals↗
PubMed1994

Human immunodeficiency virus type 1 causes productive infection of macrophages in primary placental cell cultures.

To characterize the role of the placenta in vertical transmission of human immunodeficiency virus type 1 (HIV-1), the susceptibility of primary human placental cultures and of transformed trophoblast cell lines to infection by several HIV-1 isolates was examined. Placental cultures supported the replication of all strains tested, including lymphocyte-, macrophage-, and amphotropic isolates. All viruses replicated to modest levels, with production of both viral antigen and infectious virus in the culture supernatants. Placental cells demonstrated a pattern of permissiveness for HIV-1 isolates distinct from that seen with lymphocytes, blood-derived macrophages, or T cell lines. Immunofluorescent staining showed that 5%-10% of the cultured placental cells expressed viral antigens, and double labeling revealed that the HIV-positive cells were macrophages not trophoblasts. None of the trophoblast cell line (JEG-3, Jar, BeWo, HP-W1) could be infected by HIV. These results support the hypothesis that infection of the placenta could play a role in maternofetal transmission of HIV-1 and suggest that the placental macrophage is likely to be the primary cell type responsible.

Cell Line, Transformed↗
PubMed1993

Infection of colonic epithelial cell lines by type 1 human immunodeficiency virus is associated with cell surface expression of galactosylceramide, a potential alternative gp120 receptor.

The gastrointestinal tract plays a major role in the pathogenesis and pathophysiology of infection by the type 1 human immunodeficiency virus (HIV-1). It is a potential route for viral entry and it is the site of a number of complications, including both opportunistic infections and a primary HIV-induced enteropathy. Correspondingly, both in vivo and in vitro studies have demonstrated HIV infection of gastrointestinal cells of lymphoid and epithelial origin. HT-29, a human colonic epithelial cell line that is infectable with many HIV-1 strains, does not express CD4 protein or mRNA. Recent studies showed that antibodies recognizing a neutral glycolipid related to galactosylceramide (GalCer) in HT-29 cells inhibited HIV-1 infection of this cell line, extending previous findings in neural cells. In the current studies, we further analyzed the neutral glycolipids of HT-29 cells and showed that they contained authentic GalCer and that recombinant gp120 bound to this glycolipid. Moreover, by analyzing GalCer expression in clones derived from HT-29 and Caco-2 (another human colonic cell line), we observed that the level of expression of this glycolipid was associated with the sensitivity to HIV-1 infection. Subclones of Caco-2 did not express GalCer and were not infectable with any of three HIV-1 strains. These results strengthen the possibility that GalCer is an alternative receptor in CD4- cell lines. Furthermore, since GalCer is a major glycolipid in epithelial cells of the small intestine and colon, these results provide a structural basis for the binding of HIV-1 by gastrointestinal epithelial cells and the entry of the virus into those cells.

Adenocarcinoma↗
PubMed1993

Expression of the La Crosse M segment proteins in a recombinant vaccinia expression system mediates pH-dependent cellular fusion.

To study the expression of La Crosse virus (LAC) glycoproteins, G1 and G2, we constructed a cDNA copy of the open reading frame (ORF) of the middle RNA segment and expressed it in a recombinant vaccinia virus (VV.ORF). Cells infected with VV.ORF expressed G1 and G2 at the cell surface and formed syncytia with a pH profile similar to that of LAC. These experiments provide a system of studying the biological functions of the LAC glycoproteins, including processing, targeting, fusion, receptor binding, and antigenicity.

Animals↗
PubMed1993

HIV-1 pseudotype virus containing a Cocal virus genome and an HIV envelope: construction, assay and use.

A method is described for the production and assay of pseudotype viruses between human immunodeficiency virus type 1 (HIV-1) and Cocal virus (COV), containing an HIV-1 envelope and a COV genome (COV(HIV)). COV(HIV) pseudotype virus is a useful tool for the investigation of a variety of questions regarding HIV entry into susceptible cells, including steps in virus binding, fusion, and internalization, and the role of molecules which inhibit entry. COV, a rhabdovirus closely related to vesicular stomatitis virus (VSV), replicated and caused cytopathic effect in primary cultures of human peripheral blood lymphocytes (PBLs) and monocyte-derived macrophages (MDM), and in human cell lines of lymphocytoid or monocytoid origin, making it an ideal candidate for pseudotype production. 174XCEM cells, which were permissive for selected macrophage-tropic strains as well as most lymphocyte-tropic strains of HIV-1, were used to produce stocks of putative pseudotype virus. To neutralize parental COV in these stocks, a rabbit antiserum was produced which had a neutralization index of > 10(7) at a dilution of 1:100. Using these methods, pseudotype viruses were produced with a titer of about 10(4) PFU per ml; these same stocks contained HIV-1 at a titer of about 10(5) TCD50 per ml and COV at a titer of about 10(8) PFU per ml. CD4-expressing HeLa cells were used to assay pseudotype stocks made with lymphocyte-tropic strains of HIV-1. The authenticity of the pseudotype stocks was validated by several controls, including their failure to register on congenic CD4-negative HeLa cells and their inhibition by monoclonal anti-CD4 antibodies such as Leu 3a.

Animals↗
PubMed1993

The evolution of virus diseases: their emergence, epidemicity, and control.

The evolution of virus diseases, both their emergence and disappearance, involves complex interactions between the agent, the host, and the environment. These themes are illustrated by three examples, poliomyelitis of humans, bovine spongiform encephalopathy of cattle, and AIDS of humans. Emergence may be due to evolution of the virus genome, such as probably occurred in parvovirus infection of dogs and human immunodeficiency virus infection of humans. However, emergence of some new viral diseases can be traced to host or environmental factors with no change in the agent. Poliomyelitis, an enteric infection, probably emerged as an epidemic disease due to improvements in personal hygiene and public sanitation which led to a delay in the occurrence of initial infections from the perinatal period (when maternal antibody protected against paralysis) to later childhood when passive immunity had waned. Bovine spongiform encephalopathy is a common source epidemic which was transmitted through nutritional supplements which became contaminated due to a change in the method of production of bone meal supplements in rendering plants. The reduction of disappearance of virus diseases usually involves human intervention, as exemplified by immunization for smallpox and other virus diseases of humans and animals. Naturally occurring immunity may lead to fadeout of a virus as seen with measles in isolated island populations. Evolution of a virus can also result in waning of a disease as seen with myxomatosis among rabbits in Australia. The evolution of virus diseases is a provocative scientific topic and carries lessons relevant to the control of important diseases of humans, animals, and plants.

Acquired Immunodeficiency Syndrome↗
PubMed1993

Replication of a macrophage-tropic strain of human immunodeficiency virus type 1 (HIV-1) in a hybrid cell line, CEMx174, suggests that cellular accessory molecules are required for HIV-1 entry.

To investigate the mechanism underlying one aspect of the cellular tropism of human immunodeficiency virus type 1 (HIV-1), we used a macrophage-tropic isolate, 89.6, and screened its ability to infect a number of continuous cell lines. HIV-1 (89.6) was able to replicate robustly in a T-cell/B-cell hybrid line, CEMx174, while it replicated modestly or not at all in either of its parents, one of which is the CD4-positive line CEM.3. Analysis by transfection of a molecular clone, a virus uptake assay, and polymerase chain reaction all provided strong evidence that the block to HIV-1(89.6) replication in the CEM.3 line lies at the level of cellular entry. These results were complemented by preparing a CD4-expressing derivative of the B-cell parent, 721.174, and demonstrating that it is permissive for productive HIV-1(89.6) replication. Given these experimental findings, we speculate that there exist cellular accessory factors which facilitate virus entry and infection in CD4-positive cells. Furthermore, these cellular accessory factors may be quite virus strain specific, since not all macrophage-tropic strains of HIV-1 were able to replicate in the CEMx174 hybrid cell line. This experimental model provides a system for the identification of one or more of these putative cellular accessory factors.

B-Lymphocytes↗
PubMed1993

Polygenic control of neuroinvasiveness in California serogroup bunyaviruses.

The pathogenesis of the California serogroup bunyaviruses includes both extraneural and intraneural replicative phases that can be separated experimentally. The present study dissects the viral genetic determinants of extraneural replication. We have previously described two attenuated reassortant clones of California serogroup bunyaviruses which exhibit reduced neuroinvasiveness after subcutaneous inoculation into suckling mice. Clone B1-1a bears an attenuated middle RNA segment (neuroinvasiveness phenotype v alpha v), and clone B.5 bears an attenuated large RNA segment (neuroinvasiveness phenotype alpha vv). We prepared reassortant viruses between these two strains and found that the two attenuated gene segments acted independently and additively, since reassortants bearing two attenuated RNA segments were more attenuated than the parental clones. Reassortants bearing no attenuated RNA segments were much more neuroinvasive than either parental clone, indicating that a neuroinvasive strain can be derived from two attenuated clones. Pathogenesis studies demonstrated that after injection of 10(3) PFU, the attenuated reassortant clones did not replicate in peripheral tissue, failed to reach the brain, and did not cause disease. At a dose of 10(6) PFU, attenuated clones failed to replicate to a significant level in peripheral tissue and produced only a minimal passive plasma viremia during the first 24 h but nevertheless reached high titers in the brain and killed mice. Because of this result, we investigated the possibility that neuroinvasion occurs via retrograde axonal transport, by determining whether sciatic nerve sectioning could protect against virus infection after hind leg footpad inoculation. We found that nerve sectioning had no effect on lethality, ruling out this mode of entry and suggesting that passive viremia is likely to be sufficient for invasion of the central nervous system.

Animals↗
PubMed1993

Molecular determinants of the virulence and infectivity of California serogroup bunyaviruses.

California bunyaviruses cause encephalitis in mammalian hosts after peripheral infection. The virulence of these viruses is determined by their ability to replicate sequentially in striated muscle, cause viremia, and invade and replicate in the central nervous system. These viruses are also able to infect vector mosquitoes following ingestion of a blood meal containing virus. Bunyaviruses are negative stranded RNA viruses with a trisegmented genome, and the large, medium, and small RNA segments encode the polymerase, the glycoproteins, and the nucleoprotein, respectively. Reassortants between virulent and avirulent virus clones have been used to map virulence determinants in mice as well as determinants of infectivity in mosquitoes. Attenuation in mice and infectivity in mosquitoes of some virus clones maps to the medium RNA segment, implying that the virus glycoproteins, which are involved in virus entry, play a role in virulence. Attenuation in mice and mosquito infectivity of other clones maps to the large RNA segment, suggesting that cell-specific differences in the function of the viral polymerase can also determine virulence and host range.

Animals↗
PubMed1992

Neurovirulence tests of type 3 oral poliovirus vaccine manufactured by Lederle Laboratories, 1964-1988.

Oral poliovirus vaccine (OPV) is tested for safety by evaluation of neurovirulence in rhesus and cynomolgus macaques. After intraspinal or intrathalamic injection of varying doses of vaccine, monkeys are followed for 17-21 days, killed, and a histopathological evaluation is made of the severity of poliomyelitis lesions in the spinal cord and brainstem. Each production lot of vaccine is compared with a type 1 OPV reference virus tested by the same method. Records of neurovirulence tests on production lots of type 3 OPV manufactured by Lederle Laboratories, during the period 1964-1988, have recently become available, together with the corresponding tests on type 1 reference vaccine. The cumulative data were collated, using a system under which each monkey was given a single grade according to the severity and spread of neuropathological poliomyelitis lesions. These raw data were assembled into frequency distributions ('neurovirulence profiles'), and used to compare type 3 OPV with the reference vaccine. These comparisons included monkeys injected by intraspinal injection (three vaccine dose levels) and intrathalamic injection (one vaccine dose level), and comprised independent tests conducted by the Food and Drug Administration and by the vaccine manufacturer. A total of 13 different comparisons were made, each one consisting of a pair of profiles, on type 3 OPV and reference vaccine, respectively. In total, these comparisons represented tests on more than 12,000 monkeys. Based on these neurovirulence profiles, the type 3 OPV appeared to be no more virulent than the reference vaccine.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1992

An infectious molecular clone of an unusual macrophage-tropic and highly cytopathic strain of human immunodeficiency virus type 1.

We isolated and molecularly cloned a human immunodeficiency virus type 1 (HIV-1) strain (89.6) which is unusual because it is both macrophage-tropic and extremely cytopathic in lymphocytes. Moreover, this is the first well-characterized infectious molecularly cloned macrophage-tropic HIV-1 strain derived from peripheral blood. HIV-1 89.6 differs markedly from other macrophage-tropic isolates within the envelope V3 region, which is important in determining cell tropism and cytopathicity. HIV-1 89.6 may thus represent a transitional isolate between noncytopathic macrophage-tropic viruses and cytopathic lymphocyte-tropic viruses.

Amino Acid Sequence↗
PubMed1991

Reassortment of La Crosse and Tahyna bunyaviruses in Aedes triseriatus mosquitoes.

Experiments were conducted to determine if La Crosse (LAC) and Tahyna (TAH) viruses reassort in Aedes triseriatus mosquitoes and to determine the genotypic frequencies of viruses selected by in vivo vector interactions. A molecular hybridization technique was used to analyze progeny viruses. Probes specific for the La Crosse L, M and S segments (pLAC4.16: LAC L RNA; pLAC4.27: LAC M RNA; pLAC4C-26: LAC S RNA) were used to determine the parental origin of the progeny RNA segments. Following infection with a mixture of LAC and TAH viruses, mosquitoes were held for 23 days extrinsic incubation, then assayed for reassortment. Individual progeny viruses were isolated by plaque assay and propagated in BHK-21 cells. Cytoplasmic RNA was extracted from the cells, blotted in triplicate to Nytran, and each blot was hybridized with 32P-labelled pLAC4.16, pLAC4.27 or pLAC4C-26 to determine the parental origin of each RNA segment. High frequency reassortment occurred in these mosquitoes. All of the expected genotypes resulting from a cross of LAC and TAH were obtained from these mosquitoes. Genotypic frequencies of 708 virus isolates from 39 mosquitoes were: LLL, 150 (21%); LLT, 71 (10%); LTL, 39 (5.5%); LTT, 109 (15%); TTT, 259 (36%); TTL, 16 (2.2%); TLT, 55 (7.8%); TLL, 9 (1.2%).

Aedes↗
PubMed1991

Neuroattenuation of an avirulent bunyavirus variant maps to the L RNA segment.

The derivation and characterization of a neuroattenuated reassortant clone (RFC 25/B.5) of California serogroup bunyavirus was described previously (M. J. Endres, A. Valsamakis, F. Gonzalez-Scarano, and N. Nathanson, J. Virol. 64:1927-1933, 1990). To map the RNA segment responsible for this attenuation, a panel of reassortants was constructed between the attenuated clone B.5 (genotype TLL) and a virulent clone (B1-1a) of reciprocal genotype (LTT). Parent viruses and clones representing all of the six possible reassortants were examined for neurovirulence by intracerebral injection in adult mice. Reassortants bearing the large RNA segment from the virulent parent were almost as virulent as the virulent parent virus, while reassortants bearing the large RNA segment from the avirulent parent virus exhibited low or intermediate virulence. These results indicate that the large RNA segment is the major determinant of neuroattenuation of clone B.5. In addition to its neuroattenuation, clone B.5 was temperature sensitive and exhibited an altered plaque morphology. These phenotypes also segregated with the large RNA segment. The importance of the large RNA segment (which encodes the viral polymerase) in neurovirulence contrasts with prior studies which indicate that the ability to cause lethal encephalitis after peripheral injection of suckling mice (neuroinvasiveness) is primarily determined by the middle-sized RNA segment, which encodes the viral glycoproteins.

Animals↗
PubMed1990

Neuroattenuated bunyavirus variant: derivation, characterization, and revertant clones.

A neuroattenuated variant bunyavirus, designated RFC/25B.5 (B.5), was selected by serial passage of a reassortant clone (RFC virus) of a California serogroup virus in BHK-21 cells, followed by plaque purification of that passaged stock. Based on its virulence index (ratio of PFU/50% lethal dose), clone B5 was over 40,000-fold less virulent than its unpassaged RFC parent after intracerebral (i.c.) inoculation into adult mice. Clone B.5 also exhibited markedly reduced neuroinvasiveness after subcutaneous injection into neonatal mice, although it retained its ability to replicate and kill suckling mice after i.c. injection. A murine neuroblastoma line (NA cells) can be used as an in vitro surrogate for the adult mouse brain, since clone B.5 replicated to at least 1,000-fold-lower titers in NA cells than did several neurovirulent California serogroup viruses. Clone B.5 replicated in BHK-21 cells at 37 degrees C to titers similar to those achieved by other California serogroup viruses but was temperature sensitive (ts) since its replication was markedly restricted at 38.9 degrees C. Ten ts revertant clones of B.5 virus were selected at 38.9 degrees C, and all of them lost their ts phenotype and regained the ability to replicate to high titer in NA cells and to kill adult mice after i.c. injection. Clone B.5 is the first described California serogroup virus which is truly attenuated after i.c. inoculation, and its availability will permit genetic analysis of bunyavirus neurovirulence.

Animals↗
PubMed1990

Macrophage-tropic strains of human immunodeficiency virus type 1 utilize the CD4 receptor.

To characterize the role of CD4 in human immunodeficiency virus type 1 (HIV-1) infection of macrophages, we examined the expression of CD4 by primary human monocyte-derived macrophages and studied the effect of recombinant soluble CD4 and anti-CD4 monoclonal antibodies on HIV-1 infection of these cells. Immunofluorescence and Western blot (immunoblot) studies demonstrated that both monocytes and macrophages display low levels of surface CD4, which is identical in mobility to CD4 in lymphocytes. Recombinant soluble CD4 and the anti-CD4 monoclonal antibody Leu3a blocked infection of macrophages by three different macrophage-tropic HIV isolates, and the cytopathic effects of HIV-1 infection were similarly prevented. Dose-response experiments using a prototype isolate which replicates in both macrophages and T lymphocytes showed that recombinant soluble CD4 inhibited infection of macrophages more efficiently than in lymphocytes. These results indicate that CD4 is the dominant entry pathway for HIV-1 infection of macrophages. In addition, recombinant soluble CD4 effectively blocks HIV-1 infection by a variety of macrophage-tropic strains and thus has the potential for therapeutic use in macrophage-dependent pathogenesis in HIV disease.

Antibodies, Monoclonal↗
PubMed1989

Infection of monocyte-derived macrophages with human immunodeficiency virus type 1 (HIV-1). Monocyte-tropic and lymphocyte-tropic strains of HIV-1 show distinctive patterns of replication in a panel of cell types.

To characterize the host range of different strains of HIV-1, we have used four types of cells, primary monocyte-derived macrophages (MDM), primary PBL, a promonocyte cell line (U937), and a CD4+ T cell line (SUP-T1). These cells were infected with three prototype strains of HIV-1, a putative lymphocyte-tropic strain (IIIB), and two putative monocyte-tropic strains (SF162 and DV). Infections were monitored by assays for infectious virus, for cell-free and cell-associated viral antigen (p24), and for the proportion of cells infected by immunohistochemical staining. It was concluded that: (a) the use of four different cell types provides a useful biological matrix for distinguishing the tropism of different strains of HIV-1; this matrix yields more information than the infection of any single cell type. (b) A monocyte-tropic strain of HIV-1, such as strain SF162, shows a reciprocal host range when compared with a lymphocyte-tropic strain such as IIIB; strain SF162 replicates well in primary MDM but not in U937 or SUP-T1 cells, while strain IIIB replicates well in both U937 and SUP-T1 cells but not in MDM. (c) Both lymphocyte-tropic and monocyte-tropic strains of HIV-1 replicate well in PBL. (d) The promonocyte cell line, U937, and the T cell line, SUP-T1, differ markedly from primary cells, such as MDM and PBL, in their ability to support the replication of different strains of HIV-1; these cell lines cannot be used as surrogates for primary cells in host range studies of HIV-1 strains.

Antibodies, Monoclonal↗