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John T Roehrig

Publications and source records attributed to John T Roehrig.

17 recordsLinked to original sources
PubMed2006

Temporal analyses of the neuropathogenesis of a West Nile virus infection in mice.

A West Nile virus (WNV) infection in humans can produce neurological symptoms including acute flaccid paralysis, encephalitis, meningitis and myelitis. To investigate the pathogenesis of WNV in the peripheral and the central nervous system (PNS and CNS), the authors used a murine footpad inoculation model of WNV infection. Survival curves of virus-infected animals of ages 4- to 6-weeks-old demonstrated age-dependent mortality where older animals (6-weeks-old) had a higher mortality rate compared to younger animals (4- and 5-weeks-old). The mice that survived the virus infection formed WNV-reactive antibodies, confirming viral infection and clearance. The localization of viral RNA (vRNA) and antigen in infected murine tissues was investigated using TaqMan and immunohistochemistry (IHC) respectively. During a nine day infection, vRNA levels in the spinal cord and brainstem fluctuated, suggesting early viral clearance from these tissues by days 3-4 p.i. with later re-introduction. Viral antigens detected using IHC were primarily observed in three main regions of the brain: cortex, hippocampus and brainstem. Additionally, the dorsal root ganglion neurons of the PNS stained positive for viral antigens. These data are consistent with multiple routes of neuroinvasion following a peripheral inoculation of virus and do not preclude the previous observation that virus-infected peripheral neurons can introduce virus into the CNS by a retrograde transport mechanism.

Age Factors↗
PubMed2006

Non-structural proteins of dengue 2 virus offer limited protection to interferon-deficient mice after dengue 2 virus challenge.

Chimeric (D2/WN) viruses containing the pre-membrane (prM) and envelope (E) proteins of West Nile virus (WN virus) and the capsid (C) and non-structural proteins of dengue 2 (DEN2) virus were used to evaluate the protective immunity elicited by either the flaviviral E protein or non-structural proteins. AG129 interferon-deficient mice, previously shown to be protected against lethal DEN1 or DEN2 viral infection after vaccination with a wild-type or candidate vaccine strain of DEN1 or DEN2 virus, respectively, were immunized with chimeric D2/WN virus and then challenged with DEN2 virus. D2/WN chimeric viruses were non-pathogenic in AG129 mice. These viruses elicited little anti-DEN E antibody, high levels of anti-DEN NS1 antibody and no or very low levels of DEN2 virus-neutralizing antibodies. Only 15% of D2/WN-immunized mice survived challenge with DEN2 virus. However, their mean survival time increased by 11-14 days over non-immunized controls. These results suggest that, whilst the non-structural proteins were able to enhance mean survival times of AG129 mice, this protection was not as effective as protection mediated by the E protein.

Animals↗
PubMed2006

A humanized murine monoclonal antibody protects mice either before or after challenge with virulent Venezuelan equine encephalomyelitis virus.

A humanized monoclonal antibody (mAb) has been developed and its potential to protect from or cure a Venezuelan equine encephalomyelitis virus (VEEV) infection was evaluated. The VEEV-neutralizing, protective murine mAb 3B4C-4 was humanized using combinatorial antibody libraries and phage-display technology. Humanized VEEV-binding Fabs were evaluated for virus-neutralizing capacity, then selected Fabs were converted to whole immunoglobulin (Ig) G1, and stable cell lines were generated. The humanized mAb Hy4-26C, designated Hy4 IgG, had virus-neutralizing capacity similar to that of 3B4C-4. Passive antibody protection studies with purified Hy4 IgG were performed in adult Swiss Webster mice. As little as 100 ng Hy4 IgG protected 90 % of mice challenged with 100 intraperitoneal (i.p.) mean morbidity (MD(50)) doses of virulent VEEV (Trinidad donkey) 24 h after antibody transfer; also, 500 mug Hy4 IgG protected 80 % of mice inoculated with 100 intranasal MD(50) doses of VEEV. Moreover, 10 mug passive Hy4 IgG protected 70 % of mice from a VEEV challenge dose as great as 10(7) i.p. MD(50). Hy4 IgG also protected mice from challenge with another epizootic VEEV variety, 1C (P676). Importantly, therapeutic administration of the humanized mAb to mice already infected with VEEV cured 90 % of mice treated with Hy4 IgG within 1 h of VEEV inoculation and 75 % of mice treated 24 h after virus infection.

Animals↗
PubMed2005

Dengue virus: molecular basis of cell entry and pathogenesis, 25-27 June 2003, Vienna, Austria.

Multivalent dengue vaccines now in late stage development pose unique vaccine safety challenges in that primary or secondary vaccine failures might place vaccines at risk to antibody-dependent enhanced (ADE) wild-type dengue infections. This conference was organized to address this unique vaccine safety issue. New data were presented on the structure of dengue and other flaviviruses, the cellular receptors of dengue virus for biologically relevant cells, dengue viral cell entry mechanisms and mechanisms underlying in vivo protection, neutralization and enhancement of dengue virus infection. It was concluded that a targeted research program should aim to develop an in vitro test to characterize persons immunized with dengue vaccines as completely or partially protected. Achievement of this aim will require a better understanding of the basic mechanisms by which dengue viruses recognize, attach, enter and infect relevant human cells and how antibodies protect against dengue infections.

Antibodies, Viral↗
PubMed2005

Characterization of West Nile viral replication and maturation in peripheral neurons in culture.

The North American West Nile virus (WNV), New York 1999 strain, appears to be highly neurotropic, and its neuroinvasiveness is an important aspect of human disease. The authors have developed an in vitro model to study WNV replication and protein processing in neurons. They compared WNV infection of the dorsal root ganglion (DRG) neurons (sensory neurons) and PC-12 cells (sympathetic neurons) to WNV infection of the mosquito cell line, C6/36, and Vero cells. WNV infection of both neuronal cell types and C6/36 cells was not cytopathic up to 30 days post infection, and continual viral shedding was observed during this period. However, WNV infection of Vero cells was lytic. Interestingly, WNV infection of neurons was not efficient, requiring a high multiplicity of infection of > or = 10. Indirect immunofluorescence assays using normal and confocal microscopy with flavivirus-reactive antibodies and WNV-infected neurons demonstrated viral antigen mostly associated with the plasma membrane and in the neurite processes. Treatment of WNV-infected C6/36, PC-12, or DRG cells with brefeldin A (BFA; a trans-Golgi inhibitor) or nocadazole (a beta-tubulin inhibitor) had little effect on viral maturation and secretion. Treatment of WNV-infected Vero cells with BFA resulted in a 1000-fold decrease in viral titer, but nocodazole had no effect. Our studies suggest that even though PC-12 and DRG neurons are mammalian cells, viral protein processing and maturation in these cells more closely resembles replication in C6/36 insect cells than in mammalian Vero cells.

Animals↗
PubMed2004

Contribution of disulfide bridging to epitope expression of the dengue type 2 virus envelope glycoprotein.

The individual contributions of each of the six conserved disulfide (SS) bonds in the dengue 2 virus envelope (E) glycoprotein (strain 16681) to epitope expression was determined by measuring the reactivities of a panel of well-defined monoclonal antibodies (MAbs) with LLC-MK(2) cells that had been transiently transformed with plasmid vectors expressing E proteins that were mutant in their SS bonds. Three domain I (DI) epitopes (C1, C3, and C4) were affected by elimination of any SS bond and were essentially the only epitopes affected by elimination of the amino-proximal SS1 formed between Cys 3 and Cys 30. The remaining DI epitope (C2) was sensitive to only SS3-bond (Cys 74-Cys 105) and SS6-bond (Cys 302-Cys 333) elimination. Of the four DII epitopes examined, reactivities of three anti-epitope MAbs (A1, A2, and A5) were reduced by elimination of SS2 (Cys 61-Cys 121), SS3, SS4 (Cys 94-Cys 116), SS5 (Cys 185-Cys 285), or SS6. The other DII epitope examined (A3) was sensitive only to SS2- and SS3-bond elimination. The three DIII epitopes tested (B2, B3, and B4) were most sensitive to elimination of SS6. The flavivirus group epitope (A1) was less sensitive to elimination of SS3 and SS6. This result may indicate that the region proximal to the E-protein fusion motif (amino acids 98 to 110) may have important linear components. If this observation can be confirmed, peptide mimics from this region of E protein might be able to interfere with flavivirus replication.

Antibodies, Monoclonal↗
PubMed2004

Construction of yellow fever/St. Louis encephalitis chimeric virus and the use of chimeras as a diagnostic tool.

St. Louis encephalitis (SLE) and West Nile (WN) flaviviruses are genetically closely related and cocirculate in the United States. Virus neutralization tests provide the most specific means for serodiagnosis of infections with these viruses. However, use of wild-type SLE and WN viral strains for laboratory testing is constrained by the biocontainment requirements. We constructed two highly attenuated yellow fever (YF) virus chimeras that contain the premembrane-envelope (prM-E) protein genes from the virulent MSI-7 (isolated in the United States) or the naturally attenuated CorAn9124 (Argentina) SLE strains. The YF/SLE (CorAn version) virus and the previously constructed YF/WN chimera were shown to specifically distinguish between confirmed human SLE and WN cases in a virus neutralization test using patient sera. These chimeras have the potential for use as diagnostic reagents and vaccines against SLE and WN.

Amino Acid Sequence↗
PubMed2003

Enhancing biosynthesis and secretion of premembrane and envelope proteins by the chimeric plasmid of dengue virus type 2 and Japanese encephalitis virus.

We have constructed a series of plasmids encoding premembrane (prM) and envelope (E) protein genes of dengue virus type 2 (DEN-2). These plasmids included an authentic DEN-2 prM-E construct (pCBD2-14-6), and two chimeric constructs, 90% DEN-2 E-10% Japanese encephalitis (JE) virus E (pCB9D2-1J-4-3) and 80% DEN-2 E-20% JE E (pCB8D2-2J-2-9-1). Monoclonal antibody (MAb) reactivity indicated that all three plasmids expressed authentic DEN-2 virus E protein epitopes representative of flavivirus domains 1, 2, and 3. However, only the pCB8D2-2J-2-9-1 construct secreted high levels of prM, M (membrane), and E proteins into the culture fluid of plasmid-transformed COS-1 cells. The major portion of the prM and E proteins expressed by COS-1 cells transformed by pCBD2-14-6 or pCB9D2-4-3 plasmids remained membrane-bound. The results supported the notion that an unidentified membrane retention sequence is located between E-397 and E-436 of DEN-2 virus E protein. Replacing the carboxyl-terminal 20% of DEN-2 E (397-450) with the corresponding JE sequence had no effect on anti-DEN-2 MAb reactivity, indicating that this region is antigenically inert, although it is required for antigen secretion. Plasmid pCBD2-2J-2-9-1, which expressed secreted forms of prM/M and E that have the potential to form subviral particles, was superior to other constructs in stimulating an antibody response. Ninety percent neutralization titers ranging from 1:40 to >1:1000 were observed in seven of nine serum specimens from pCB8D2-2J-2-9-1-immunized mice. Eleven of twelve 2-day-old neonatal mice, derived from a pCB8D2-2J-2-9-1 immunized female mouse, survived intraperitoneal challenge of DEN-2 New Guinea C virus.

Animals↗
PubMed2003

Antigenic structure of flavivirus proteins.

The increased activity of Dengue virus in the tropical regions of the world and the recent movement of West Nile virus from the eastern to the western hemisphere emphasize the fact that vector-borne flaviviruses are medically important emerging infectious diseases. These facts warrant continued efforts to decode all facets of flavivirus immunology. This chapter reviews current understanding of the antigenic fine structure of flaviviral structural and nonstructural (NS) proteins and their involvement in B- an T-cell host responses. The virion structural glycoprotein E elicits both virus-neutralizing antibodies and antiviral Th-cell responses. Consistent with the current hypothesis of the MHC class I pathway of protein processing, immunodominant flaviviral Tc-cell epitopes mainly reside on the NS proteins. To prepare effective and inexpensive subunit vaccines, we will need to continue to better understand these structure-function relationships of flavivirus proteins.

Animals↗
PubMed2003

Epitope-blocking enzyme-linked immunosorbent assays for the detection of serum antibodies to west nile virus in multiple avian species.

We report the development of epitope-blocking enzyme-linked immunosorbent assays (ELISAs) for the rapid detection of serum antibodies to West Nile virus (WNV) in taxonomically diverse North American avian species. A panel of flavivirus-specific monoclonal antibodies (MAbs) was tested in blocking assays with serum samples from WNV-infected chickens and crows. Selected MAbs were further tested against serum samples from birds that represented 16 species and 10 families. Serum samples were collected from birds infected with WNV or Saint Louis encephalitis virus (SLEV) and from noninfected control birds. Serum samples from SLEV-infected birds were included in these experiments because WNV and SLEV are closely related antigenically, are maintained in similar transmission cycles, and have overlapping geographic distributions. The ELISA that utilized MAb 3.1112G potentially discriminated between WNV and SLEV infections, as all serum samples from WNV-infected birds and none from SLEV-infected birds were positive in this assay. Assays with MAbs 2B2 and 6B6C-1 readily detected serum antibodies in all birds infected with WNV and SLEV, respectively, and in most birds infected with the other virus. Two other MAbs partially discriminated between infections with these two viruses. Serum samples from most WNV-infected birds but no SLEV-infected birds were positive with MAb 3.67G, while almost all serum samples from SLEV-infected birds but few from WNV-infected birds were positive with MAb 6B5A-5. The blocking assays reported here provide a rapid, reliable, and inexpensive diagnostic and surveillance technique to monitor WNV activity in multiple avian species.

Animals↗
PubMed2002

Complete genome sequences and phylogenetic analysis of West Nile virus strains isolated from the United States, Europe, and the Middle East.

The complete nucleotide sequences of eight West Nile (WN) virus strains (Egypt 1951, Romania 1996-MQ, Italy 1998-equine, New York 1999-equine, MD 2000-crow265, NJ 2000MQ5488, NY 2000-grouse3282, and NY 2000-crow3356) were determined. Phylogenetic trees were constructed from the aligned nucleotide sequences of these eight viruses along with all other previously published complete WN virus genome sequences. The phylogenetic trees revealed the presence of two genetic lineages of WN viruses. Lineage 1 WN viruses have been isolated from the northeastern United States, Europe, Israel, Africa, India, Russia, and Australia. Lineage 2 WN viruses have been isolated only in sub-Saharan Africa and Madagascar. Lineage 1 viruses can be further subdivided into three monophyletic clades.

Animals↗
PubMed2002

Use of immunoglobulin m cross-reactions in differential diagnosis of human flaviviral encephalitis infections in the United States.

To define the virus specificity of the immunoglobulin M (IgM) antibody-capture enzyme-linked immunosorbent assay (MAC-ELISA) among the medically important members of the Japanese encephalitis (JE) virus serocomplex of flaviviruses, 103 IgM-positive human serum samples from patients with confirmed West Nile (WN) virus, St. Louis encephalitis (SLE) virus, or JE virus infections were assembled and simultaneously tested against all three viral antigens in a standardized MAC-ELISA. Of the serum samples tested, 96 (93%) showed higher positive-to-negative absorbance ratios (P/Ns) with the infecting virus antigen compared to those obtained with the other two virus antigens. Of the seven specimens with higher P/Ns with heterologous virus antigens, six were from patients with SLE virus infections (the serum samples had higher levels of reactivity with WN virus antigen) and one was from a patient with a JE virus infection (this serum sample also had a higher level of reactivity with WN virus antigen). Not surprisingly, similar virus specificity was observed with WN virus-elicited IgM in cerebrospinal fluid. As shown in previous studies, a subset of these specimens was even less reactive in the MAC-ELISA with dengue virus, a member of a different flavivirus serocomplex. The degree of virus cross-reactivity did not appear to be related to days postonset, at least during the first 40 days of infection. Infections with WN virus could be correctly distinguished from infections with SLE virus on the basis of the observed anti-viral IgM cross-reactivities alone 92% of the time. Infections with SLE virus resulted in antibody that was more cross-reactive, so identification of SLE virus as the infecting agent by use of MAC-ELISA cross-reactivity alone was more problematic.

Antibodies, Viral↗
PubMed2002

Detection of West Nile virus antigen in mosquitoes and avian tissues by a monoclonal antibody-based capture enzyme immunoassay.

An antigen capture immunoassay to detect West Nile (WN) virus antigen in infected mosquitoes and avian tissues has been developed. With this assay purified WN virus was detected at a concentration of 32 pg/0.1 ml, and antigen in infected suckling mouse brain and laboratory-infected mosquito pools could be detected when the WN virus titer was 10(2.1) to 10(3.7) PFU/0.1 ml. In a blindly coded set of field-collected mosquito pools (n = 100), this assay detected WN virus antigen in 12 of 18 (66.7%) TaqMan-positive pools, whereas traditional reverse transcriptase PCR detected 10 of 18 (55.5%) positive pools. A sample set of 73 organ homogenates from naturally infected American crows was also examined by WN virus antigen capture immunoassay and TaqMan for the presence of WN virus. The antigen capture assay detected antigen in 30 of 34 (88.2%) TaqMan-positive tissues. Based upon a TaqMan-generated standard curve of infectious WN virus, the limit of detection in the antigen capture assay for avian tissue homogenates was approximately 10(3) PFU/0.1 ml. The recommended WN virus antigen capture protocol, which includes a capture assay followed by a confirmatory inhibition assay used to retest presumptive positive samples, could distinguish between the closely related WN and St. Louis encephalitis viruses in virus-infected mosquito pools and avian tissues. Therefore, this immunoassay demonstrates adequate sensitivity and specificity for surveillance of WN virus activity in mosquito vectors and avian hosts, and, in addition, it is easy to perform and relatively inexpensive compared with the TaqMan assay.

Animals↗