Biomedical subjects

J T Roehrig

Publications and source records attributed to J T Roehrig.

At least 37 records · Page 2Linked to original sources

PubMed1998

Monoclonal antibody mapping of the envelope glycoprotein of the dengue 2 virus, Jamaica.

Although dengue (DEN) virus is the etiologic agent of dengue fever, the most prevalent vector-borne viral disease in the world, precise information on the antigenic structure of the dengue virion is limited. We have prepared a set of murine monoclonal antibodies (MAbs) specific for the envelope (E) glycoprotein of DEN 2 virus and used these antibodies in a comprehensive biological and biochemical analysis to identify 16 epitopes. Following domain nomenclature developed for the related flavivirus, tick-borne encephalitis, three functional domains were identified. Five epitopes associated with domain A were arranged in three spatially independent regions. These A-domain epitopes were destroyed by reduction, and antibodies reactive with these epitopes were able to block virus hemagglutination, neutralize virus infectivity, and block virus-mediated cell membrane fusion. Domain-A epitopes were present on the full-length E glycoprotein, a 45-kDa tryptic peptide representing its first 400 amino acids (aa) and a 22-kDa tryptic peptide representing at least aa 1-120. Four epitopes mapped into domain B, as determined by their partial resistance to reduction and the localization of these epitopes on a 9-kDa tryptic or chymotryptic peptide fragment (aa 300-400). One domain-B-reactive MAb was also capable of binding to a DEN 2 synthetic peptide corresponding to aa 333-351 of the E glycoprotein, confirming the location of this domain. Domain-B epitopes elicited MAbs that were potent neutralizers of virus infectivity and blocked hemagglutination, but they did not block virus-mediated cell-membrane fusion. Domains A and B were spatially associated. As with tick-borne encephalitis virus, determination of domain C was more problematic; however, at least four epitopes had biochemical characteristics consistent with C-domain epitopes.

Animals↗

PubMed1998

Resistance to alpha/beta interferons correlates with the epizootic and virulence potential of Venezuelan equine encephalitis viruses and is determined by the 5' noncoding region and glycoproteins.

We compared the alpha/beta interferon (IFN-alpha/beta) sensitivities of the TC-83 vaccine strain and 24 enzootic and epizootic Venezuelan equine encephalitis (VEE) isolates. The IFN-resistant or -sensitive phenotype correlated well with epizootic or enzootic potential. IFN-alpha/beta resistance of Trinidad donkey (TRD) virus correlated with virulence determinants in the 5' noncoding region and glycoproteins. Infection of mice lacking a functional IFN system with the IFN-sensitive TC-83 virus resulted in disease equivalent to that produced by the virulent, IFN-resistant TRD virus, further demonstrating that IFN resistance contributes to VEE virus virulence and is a biological marker of epizootic potential.

Animals↗

PubMed1998

Venezuelan equine encephalitis febrile cases among humans in the Peruvian Amazon River region.

A survey was conducted from October 1, 1993 to June 30, 1995 to determine the arboviral etiologies of febrile illnesses in the city of Iquitos in the Amazon River Basin of Peru. The study subjects were patients who were enrolled at medical care clinics or in their homes by Peruvian Ministry of Health (MOH) workers as part of the passive and active disease surveillance program of the MOH. The clinical criterion for enrollment was the diagnosis of a suspected viral-associated, acute, undifferentiated febrile illness of < or = 5 days duration. A total of 598 patients were enrolled in the study. Demographic information, medical history, clinical data, and blood samples were obtained from each patient. The more common clinical features were fever, headache, myalgia, arthralgia, retro-ocular pain, and chills. Sera were tested for virus by the newborn mouse and cell culture assays. Viral isolates were identified initially by immunofluorescence using polyclonal antibody. An ELISA using viral-specific monoclonal antibodies and nucleotide sequence analysis were used to determine the specific variety of the viruses. In addition, thin and thick blood smears were observed for malaria parasites. Venezuelan equine encephalitis (VEE) virus subtype I, variety ID virus was isolated from 10 cases, including three cases in October, November, and December 1993, five cases in January and February 1994, and two cases in June 1995. The ELISA for IgM and IgG antibody indicated that VEE virus was the cause of an additional four confirmed and four presumptive cases, including five from January through March 1994 and three in August 1994. Sixteen cases were positive for malaria. The 18 cases of VEE occurred among military recruits (n = 7), agriculture workers (n = 3), students (n = 3), and general laborers (n = 5). These data indicated that an enzootic strain of VEE virus was the cause of at least 3% (18 of 598) of the cases of febrile illnesses studied in the city of Iquitos in the Amazon Basin region of Peru.

Adolescent↗

PubMed1998

Nucleotide sequences of the 26S mRNAs of the viruses defining the Venezuelan equine encephalitis antigenic complex.

Genetic relationships among viruses defining the Venezuelan equine encephalitis (VEE) virus antigenic complex were determined by analyzing the 3'-terminal 561 nucleotides of the nonstructural protein 4 gene and the entire 26S RNA region of the genome. New sequence information is reported for VEE 78V-3531 (VEE subtype-variety IF), Mucambo (IIIA), Tonate (IIIB), 71D-1252 (IIIC), Pixuna (IV), Cabassou (V), and AG80-663 (VI) viruses. The results reported here and by previous investigators largely support the current classification scheme of these viruses, while clearly identifying Everglades (II) as a subtype I virus. A genetic relationship between 78V-3531 (IF) and AG80-663 (VI) viruses contradicted previous serologic results. Mutations near the amino terminus of the E2 envelope proteins of Pixuna and AG80-663 viruses probably account for the previously reported low reactivity of the protective monoclonal antibody 1A2B-10 with these two viruses. Variations in the distribution of potential glycosylation sites in the E2 glycoprotein are discussed.

Amino Acid Sequence↗

PubMed1997

Construction of infectious cDNA clones for dengue 2 virus: strain 16681 and its attenuated vaccine derivative, strain PDK-53.

We identified nine nucleotide differences between the genomes of dengue-2 (DEN-2) 16681 virus and its vaccine derivative, strain PDK-53. These included a C-to-T (16681-to-PDK-53) mutation at nucleotide position 57 of the 5'-untranslated region, three silent mutations, and substitutions prM-29 Asp to Val, NS1-53 Gly to Asp, NS2A-181 Leu to Phe, NS3-250 Glu to Val, and NS4A-75 Gly to Ala. Unpassaged PDK-53 vaccine contained two genetic variants as a result of partial mutation at NS3-250. We constructed infectious cDNA clones for 16681 virus and each of the two PDK-53 variants. DEN-2 16681 clone-derived viruses were identical to the 16681 virus in plaque size and replication in LLC-MK2 cells, replication in C6/36 cells, E and prM epitopes, and neurovirulence for suckling mice. PDK-53 virus and both clone-derived PDK-53 variants were attenuated in mice. However, the variant containing NS3-250-Glu was less temperature sensitive and replicated better in C6/36 cells than did PDK-53 virus. The variant containing NS3-250-Val had smaller, more diffuse plaques, decreased replication, and increased temperature sensitivity in LLC-MK2 cells relative to PDK-53 virus. Both PDK-53 virus and the NS3-250-Val variant replicated poorly in C6/36 cells relative to 16681 virus. Unpassaged PDK-53 vaccine virus and the virus passaged once in LLC-MK2 cells had genomes of identical sequence, including the mixed NS3-250-Glu/Val locus. Although the NS3-250-Val mutation clearly affected virus replication in vitro, it was not a major determinant of attenuation for PDK-53 virus in suckling mice.

Animals↗

PubMed1997

Monoclonal antibodies capable of distinguishing epizootic from enzootic varieties of subtype 1 Venezuelan equine encephalitis viruses in a rapid indirect immunofluorescence assay.

We used previously characterized murine monoclonal antibodies to develop a panel useful in subtyping Venezuelan equine encephalitis (VEE) viruses by an indirect fluorescent antibody assay. This panel worked well with either prototype VEE viruses or a series of more recent VEE virus isolates. The panel is particularly useful for rapidly differentiating VEE viruses with epidemic-epizootic potential from other endemic varieties of this virus. Using this panel, we identified an antigenic variant of prototype VEE subtype 1E virus currently present in Mexico. This antigenic change in the E2 glycoprotein was confirmed by enzyme-linked immunosorbent assay. Because VEE virus virulence has been associated in part with the E2 glycoprotein, this observed antigenic change in the 1E virus E2 glycoprotein may explain the apparent equine virulence of this unusual VEE 1E virus.

Animals↗

PubMed1997

Venezuelan equine encephalitis and Oropouche virus infections among Peruvian army troops in the Amazon region of Peru.

An outbreak of a febrile illness characterized by headache, ocular pain, myalgia, and arthralgia occurred during June 1994 among Peruvian army troops in Northern Peru. On June 14-16, 1994, clinical data and blood samples were obtained from eight soldiers with a febrile illness, and from 26 others who had a history of febrile illness during the past three months. A follow-up blood sample was obtained 107 days later from four of the febrile and seven of the afebrile soldiers. Serum samples were tested for dengue (DEN), Oropouche (ORO), and Venezuelan equine encephalitis (VEE) IgM and IgG antibodies by an enzyme-linked immunosorbent assay (ELISA). Virus isolation was performed by inoculation of newborn mice and Vero cell cultures. Viral isolates were identified by immunofluorescence, ELISA, and nucleotide sequencing. A VEE virus infection was confirmed in three of the eight febrile soldiers, two by virus isolation, and one by serology. Antigenic analysis indicated that one of the virus isolates was similar to VEE subtype I, variety ID, viruses previously isolated in Colombia and Venezuela. Nucleotide sequence data showed that both viral isolates were identical to one another and closely related to VEE ID viruses previously isolated in Peru, Colombia, and Venezuela. Serologic results showed that two of 26 afebrile soldiers had IgM antibody to VEE and four had IgG antibody to VEE; two febrile soldiers had IgG antibody in their first serum samples. Oropouche-specific IgM antibody was detected in one of the eight febrile and five of the afebrile soldiers, and 18 of the 34 soldiers had low titers of ORO IgG antibody titers, which did not meet the diagnostic criteria for confirmed cases. All soldiers were negative for DEN IgM antibody, and 10 had flavivirus IgG antibody that reacted with DEN antigens. These data indicated that VEE ID virus was one of the causes of illness among Peruvians soldiers and that this was the first association of this VEE subtype with human disease in Peru.

Adolescent↗

PubMed1995

Murray valley encephalitis virus envelope protein antigenic variants with altered hemagglutination properties and reduced neuroinvasiveness in mice.

Neutralization escape variants of Murray Valley encephalitis virus were selected using a type-specific, neutralizing, and passively protective anti-envelope protein (E) monoclonal antibody (4B6C-2) which defines epitope E-1c. Nucleotide sequence analysis revealed single nucleotide changes in the E genes of 15 variants resulting in nonconservative amino acid substitutions in all cases. One variant had a three-nucleotide deletion in the E gene which resulted in loss of serine at residue 277. Changes were clustered into two separate regions of the E polypeptide (residues 126-128 and 274-277), indicating that E-1c is a discontinuous epitope. One variant (BHv1), altered at residue 277 (Ser-->Ile), failed to hemagglutinate across the pH range 5.5-7.5, in contrast to parental virus and the other escape variants which hemagglutinated at an optimal pH of 6.6. BHv1 was also of reduced neuroinvasiveness in 21-day-old mice following intraperitoneal inoculation compared to the other viruses. Parental virus and the neutralization escape variants grew equally well in both vertebrate and invertebrate cell cultures, indicating that the reduced neuroinvasiveness of BHv1 was not due to a major abnormality of replication.

Amino Acid Sequence↗

PubMed1995

Localization of a protective epitope on a Venezuelan equine encephalomyelitis (VEE) virus peptide that protects mice from both epizootic and enzootic VEE virus challenge and is immunogenic in horses.

In order to define more precisely the protective epitope encoded within the first 25 amino acids (aa) of the E2 glycoprotein of the Trinidad donkey strain of Venezuelan equine encephalomyelitis (VEE) virus, we examined the immunogenicity of smaller peptides within the first 19 aa. pep1-9 and pep3-10 elicited virus-reactive antibody, but failed to protect mice from virus challenge. Additionally, pep3-10 was identified by a competitive binding assay using overlapping peptide octamers as the putative binding site of the antipeptide monoclonal antibody (mAb) 1A2B-10. Since the E2 amino-terminal sequence for all VEE subtype viruses is conserved, we tested the protective capacity in mice of passively transferred mAb 1A2B-10 and found it to protect from both epizootic and enzootic VEE virus challenge. Since horses are an important natural host for VEE virus, pep1-19 was used to immunize horses and was found to be immunogenic and to elicit virus-reactive antibody.

Animals↗

PubMed1994

T-helper cell epitopes on the E-glycoprotein of dengue 2 Jamaica virus.

To identify T-helper (Th)-cell epitopes, we analyzed 25 synthetic peptides, which included most of the 495-amino-acid sequence of the envelope (E)-glycoprotein of dengue 2 virus. The peptides were analyzed in three mouse strains, BALB/c (H-2d), C57BL/6 (H-2b), and outbred NIH-Swiss, for their ability to elicit antibody or prime the Th-cell compartment following two inoculations in Freund's incomplete adjuvant. Sixteen peptides were able to elicit an antipeptide antibody response in one or more mouse strain. Eleven antipeptide serum pools were able to bind to virus in ELISA. Fifteen peptides primed one or more haplotype for an in vitro antipeptide Th-cell response as measured by blastogenesis. Th-cell activation was generally confirmed by measurable in vitro production of interleukin (IL)-2/IL-4. Nine peptides that were positive for in vitro blastogenesis, 1-2, 35, 4-6, 79, 142, 208, 06, 16, and 17, elicited virus-reactive Th-cells in vitro in H-2d mice. Two of these peptides (4-6 and 17) were able to prime virus-reactive Th-cells in H-2b mice. Nine peptides primed outbred mice in vitro for an antiviral antibody response significantly greater than that seen in animals primed with an irrelevant peptide. These results correlate with, and expand on, our previous observations based on a smaller set of synthetic peptides derived from the E-glycoprotein of Murray Valley encephalitis virus and suggest that synthetic peptides can function as E-glycoprotein Th-cell epitopes. The similarity of results between two distantly related flaviviruses suggests that E-glycoprotein Th-cell epitopes are consistent in location and activity.

Animals↗

PubMed1994

The envelope glycoproteins of dengue 1 and dengue 2 viruses grown in mosquito cells differ in their utilization of potential glycosylation sites.

We have previously isolated and characterized two dengue (DEN) 2 viruses mutant in their fusion-from-within (FFWI) phenotype in the insect cell line C6/36. Both viruses lost a potential glycosylation site (Asn-153) in the envelope (E) glycoprotein. To determine whether the change in FFWI phenotype was due to a change in E-glycoprotein glycosylation, we characterized the patterns of glycosylation on the E-glycoprotein of wild-type DEN 1 and DEN 2 viruses. The E-glycoproteins were isolated from purified virus grown in Aedes albopictus C6/36 cells, by use of high-performance size-exclusion chromatography. The tryptic maps of wild-type glycosylated and enzymatically (PNGase F) deglycosylated E-glycoproteins were compared by reverse-phase high-performance liquid chromatography. The DEN 1 virus E-glycoprotein was found to have two peaks in the tryptic map that exhibited shifts after deglycosylation, whereas the DEN 2 virus E-glycoprotein had only one. Besides the potential glycosylation site at Asn-153, both DEN 1 and DEN 2 virus E-glycoproteins have another potential site located at Asn-67. Amino-terminal sequencing of the shifted peaks revealed that DEN 2 virus E-glycoprotein is glycosylated only at Asn-67; however, DEN 1 virus E-glycoprotein is glycosylated at both Asn-67 and Asn-153. These DEN virus serotypes are thus heterogeneous in their use of glycosylation sites. We also determined by a lectin-binding assay that the attached carbohydrates for both viruses were likely to be of the high-mannose type.

Aedes↗

PubMed1994

Murine T-helper cell immune response to recombinant vaccinia-Venezuelan equine encephalitis virus.

The T-helper (Th) cell immune response following immunization of C3H (H-2k) mice with a recombinant vaccinia (VAC) virus (TC-5A) expressing the structural proteins (capsid, E1 and E2) of the attenuated vaccine strain (TC-83) of Venezuelan equine encephalitis (VEE) virus was compared with the immune response induced in mice after immunization with TC-83 virus. TC-5A virus elicited Th cells that strongly recognized both VAC and TC-83 viruses in in vitro lymphoblastogenesis tests. Th-cell activation was associated with elevated levels of interleukin-2. TC-5A virus induced long-term humoral immunity; VEE virus-binding and neutralizing antibodies were detected in mouse sera collected from mice 16 months after a single immunization.

Animals↗

PubMed1994

Analysis of a yellow fever virus isolated from a fatal case of vaccine-associated human encephalitis.

The virulence of a yellow fever (YF) virus (P-16065) isolated from a fatal case of vaccine-associated viral encephalitis was investigated. P-16065 appeared identical to its parent vaccine virus (17D-204 USA, lot 6145) when examined with monoclonal antibodies except that YF wild type-specific MAb S24 recognized P-16065 but not 17D-204 USA 6145. Thus, a mutation of at least one epitope on the envelope (E) protein had occurred. Unlike 17D-204 USA 6145 and other 17D vaccine viruses, P-16065 was neuroinvasive and virulent for mice after intranasal inoculation, and neurovirulent for monkeys after intracerebral inoculation. The E protein of P-16065 differed from 17D-204 USA by two amino acids at positions 155 and 303. Changes at amino acid position 155 are found in other YF vaccine viruses that are not neurovirulent, and it is therefore postulated that the change at position 303 is involved in the alteration of the phenotype of P-16065 and may be important for virulence of YF virus.

Aedes↗

PubMed1993

Selection and partial characterization of dengue 2 virus mutants that induce fusion at elevated pH.

Two types of dengue (DEN) 2 virus mutants were selected either by repeated exposure to acidic pH (acid mutant, AM), or by the addition of ammonium chloride to Aedes albopictus C6/36 cells prior to and during viral infection (fusion mutant, FM). Both mutants grew more slowly than the parent strain and induced smaller plaques in Vero cells. The 50% fusion from within index for both mutants occurred at least 0.65 pH units higher than with the wild-type DEN virus. A single amino acid substitution (Asn-153 to Asp) was found in the envelope (E)-glycoprotein of the AM virus. Three amino acid substitutions were detected on the E-glycoprotein of the FM virus: Ile-6 to Met, Asn-134 to Ser, and Asn-153 to Tyr. No mutations were found in the precursor to the membrane protein, prM. The DEN virus E-glycoprotein has two potential glycosylation sites: Asn-67 and Asn-153. The loss of the potential glycosylation site at Asn-153 or the change in the chemical characteristics resultant from the amino acid substitutions in both mutants implicates these regions of the E-glycoprotein in virus-mediated membrane fusion.

Acids↗

PubMed1993

Immunogens of encephalitis viruses.

The equine encephalitis viruses are members of the genus Alphavirus, in the family Togaviridae. Three main virus serogroups represented by western (WEE), eastern (EEE) and Venezuelan equine encephalitis (VEE) viruses cause epizootic and enzootic infection of horses throughout the western hemisphere. All equine encephalitis viruses are transmitted through the bite of an infected mosquito. The first equine encephalitis virus vaccines were produced by virus inactivation. Problems with inadequate inactivation, which may have caused a major epidemic/epizootic of VEE in central America and Texas in the 1970s, led to the development of a live attenuated VEE virus vaccine (TC-83) derived by cell culture passage. Inactivated vaccines are still used to prevent equine infections with WEE and EEE viruses. Alphaviruses are small single stranded, positive sense RNA viruses. The 12000 nucleotide genome is enclosed in an icosahedral nucleocapsid composed of multiple copies of the capsid (C) protein. The virion is enveloped. The membrane is modified by the insertion of heterodimers of two glycoproteins: E1 and E2. Monoclonal antibody analysis of the surface glycoproteins have provided a detailed understanding of important protective antigens. Recent studies comparing gene sequences from virulent and avirulent VEE viruses have begun to delineate mechanisms of alphavirus attenuation.

Alphavirus↗

PubMed1993

Molecular and biological characterization of a non-glycosylated isolate of St Louis encephalitis virus.

The glycosylation patterns of the envelope (E) glycoprotein of several naturally occurring strains of St Louis encephalitis (SLE) virus were investigated. SLE viruses were found that contained both glycosylated and non-glycosylated E proteins, and one isolate (Tr 9464) that lacks N-linked glycosylation sites on its E protein was identified. SLE virus monoclonal antibodies that define E protein B cell epitopes and demonstrate biological activities reacted essentially to the same extent with glycosylated and non-glycosylated virions. These results indicate that glycosylation is not essential for epitope conformation or recognition. However, failure to glycosylate the E protein was associated with possible morphogenetic differences as manifested by reduced virus yields and differences in specific infectivity.

Amino Acid Sequence↗

PubMed1993

Attenuation of Venezuelan equine encephalitis virus strain TC-83 is encoded by the 5'-noncoding region and the E2 envelope glycoprotein.

The virulent Trinidad donkey (TRD) strain of Venezuelan equine encephalitis (VEE) virus and its live attenuated vaccine derivative, TC-83 virus, have different neurovirulence characteristics. A full-length cDNA clone of the TC-83 virus genome was constructed behind the bacteriophage T7 promoter in the polylinker of plasmid pUC18. To identify the genomic determinants of TC-83 virus attenuation, TRD virus-specific sequences were inserted into the TC-83 virus clone by in vitro mutagenesis or recombination. Antigenic analysis of recombinant viruses with VEE E2- and E1-specific monoclonal antibodies gave predicted antigenic reactivities. Mouse challenge experiments indicated that genetic markers responsible for the attenuated phenotype of TC-83 virus are composed of genome nucleotide position 3 in the 5'-noncoding region and the E2 envelope glycoprotein. TC-83 virus amino acid position E2-120 appeared to be the major structural determinant of attenuation. Insertion of the TRD virus-specific 5'-noncoding region, by itself, into the TC-83 virus full-length clone did not alter the attenuated phenotype of the virus. However, the TRD virus-specific 5'-noncoding region enhanced the virulence potential of downstream TRD virus amino acid sequences.

Animals↗

PubMed1992

The hamster immune response to tick-transmitted Borrelia burgdorferi differs from the response to needle-inoculated, cultured organisms.

The human immune response to natural infection with Borrelia burgdorferi appears to differ from that seen in small mammals infected by needle inoculation. In humans, antibody to outer surface proteins A and B (OspA and OspB) is not detectable until late in infection, but small mammals inoculated with B. burgdorferi produce early antibody to OspA and OspB. To investigate this disparity we compared the immune response in hamsters to B. burgdorferi after needle inoculation with cultured organisms or infected tick homogenates with the immune response after tick transmitted (natural) infection. We determined that the antibody response to OspA and OspB after natural infection of hamsters is similar to that seen in humans, and differs from the antibody response after hamster infection by needle inoculation. High titers of antibody to OspA and OspB were undetectable even 42 wk after bite by B. burgdorferi-infected ticks. The failure to produce antibody to OspA and OspB was not dependent on challenge dose, because animals inoculated by needle with low doses (1 x 10(5) to 1 x 10(6) cells) of B. burgdorferi produced antibody to OspA and OspB. A rapid but limited anti-41-kDa response was observed. One possible new Ag, 43 kDa (p43), was identified. The antibody response to p43 was independent of the route of inoculation. Our results suggest that the hamster immune response to tick-transmitted Borrelia burgdorferi differs from the response to needle inoculated, cultured organisms.

Animals↗