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Biomedical subjects

N L Davis

Publications and source records attributed to N L Davis.

At least 19 recordsLinked to original sources
PubMed2004

Future vaccines against emerging encephalitides.

The vaccine strategies available for control of emerging encephalitides range in a continuum from traditional approaches to those utilizing new technologies. In this report, we explore the use of live attenuated vaccines where the attenuating mutations have been selected in a rational way to improve attenuation without sacrificing effectiveness. A strategy for paired lethal and resuscitating mutations is presented that will greatly reduce the possibility of reversion to virulence. Finally, we describe an example of a vaccine vector system that could be rapidly adapted for use against these virus diseases as they emerge.

Animals↗
PubMed2001

Immunopathogenesis and immune modulation of Venezuelan equine encephalitis virus-induced disease in the mouse.

The course of Venezuelan equine encephalitis (VEE) disease in immunodeficient and immunologically normal mice was compared to define the role of the immune system in this disease process. Immunocompetent mice infected with VEE exhibited a biphasic illness characterized by an early self-limiting lymphoid phase and a fatal CNS phase. The lymphoid phase of the illness was characterized by extensive viral replication within spleen, thymus, Peyer's patches, and lymph nodes, was accompanied by a high-titered serum viremia, and resolved with the production of VEE-specific IgM class antibody at 72 h postinfection (p.i.). Immunocompetent animals survived an average of 6.8 +/- 1.2 days before succumbing to fulminant encephalitis. In contrast, SCID mice infected with VEE showed a persistent replication of virus throughout all organs tested beginning at 24 h p.i. VEE-infected SCID mice exhibited a severe spongiform encephalopathy with 100% mortality and an average survival time of 8.9 +/- 0.9 days. These studies indicated that the characteristic organ tropism of VEE in the mouse is due in large part to an early anti-viral state, the establishment of which is dependent upon the presence of an intact immune system. Finally, the CNS pathology in a VEE-infected mouse had a significant immunologic component. However, in contrast to other neurovirulent alphaviruses, VEE was directly cytopathic for the cells of the CNS, even in the absence of an immune response.

Animals↗
PubMed2001

Role of alpha/beta interferon in Venezuelan equine encephalitis virus pathogenesis: effect of an attenuating mutation in the 5' untranslated region.

Venezuelan equine encephalitis virus (VEE) is an important equine and human pathogen of the Americas. In the adult mouse model, cDNA-derived, virulent V3000 inoculated subcutaneously (s.c.) causes high-titer peripheral replication followed by neuroinvasion and lethal encephalitis. A single change (G to A) at nucleotide 3 (nt 3) of the 5' untranslated region (UTR) of the V3000 genome resulted in a virus (V3043) that was avirulent in mice. The mechanism of attenuation by the V3043 mutation was studied in vivo and in vitro. Kinetic studies of virus spread in adult mice following s.c. inoculation showed that V3043 replication was reduced in peripheral organs compared to that of V3000, titers in serum also were lower, and V3043 was cleared more rapidly from the periphery than V3000. Because clearance of V3043 from serum began 1 to 2 days prior to clearance of V3000, we examined the involvement of alpha/beta interferon (IFN-alpha/beta) activity in VEE pathogenesis. In IFN-alpha/betaR(-/-) mice, the course of the wild-type disease was extremely rapid, with all animals dying within 48 h (average survival time of 30 h compared to 7.7 days in the wild-type mice). The mutant V3043 was as virulent as the wild type (100% mortality, average survival time of 30 h). Virus titers in serum, peripheral organs, and the brain were similar in V3000- and V3043-infected IFN-alpha/betaR(-/-) mice at all time points up until the death of the animals. Consistent with the in vivo data, the mutant virus exhibited reduced growth in vitro in several cell types except in cells that lacked a functional IFN-alpha/beta pathway. In cells derived from IFN-alpha/betaR(-/-) mice, the mutant virus showed no growth disadvantage compared to the wild-type virus, suggesting that IFN-alpha/beta plays a major role in the attenuation of V3043 compared to V3000. There were no differences in the induction of IFN-alpha/beta between V3000 and V3043, but the mutant virus was more sensitive than V3000 to the antiviral actions of IFN-alpha/beta in two separate in vitro assays, suggesting that the increased sensitivity to IFN-alpha/beta plays a major role in the in vivo attenuation of V3043.

5' Untranslated Regions↗
PubMed2001

Hearing preservation in acoustic neuroma surgery.

This article reviews the Montreal experience of hearing preservation in acoustic neuroma surgery. The medical records since 1995 of 36 patients who underwent acoustic neuroma extirpation with the intent to preserve hearing were examined. Intraoperative monitoring was conducted using auditory brainstem response measurement with electrocochleography via a transtympanic electrode. The role of intraoperative monitoring in guiding surgical technique and its correlation with postoperative hearing outcome are discussed. A review of the literature regarding hearing preservation in acoustic neuroma surgery is included.

Adult↗
PubMed2000

Influenza virus (A/HK/156/97) hemagglutinin expressed by an alphavirus replicon system protects chickens against lethal infection with Hong Kong-origin H5N1 viruses.

Venezuelan equine encephalitis virus replicon particles (VRP) containing the gene expressing hemagglutinin (HA) from the human Hong Kong Influenza A isolate (A/HK/156/97) were evaluated as vaccines in chicken embryos and young chicks. Expressed HA was readily detected in bird-tissue staining with anti-H5 HA antibody and in chicken cells infected with the replicon preparations following immunoprecipitation with monoclonal antibody. Birds challenged with a dose of the lethal parent virus were protected to different extents depending on the age of the bird. In ovo and 1-day-old inoculated animals that received no boost with the VRP were partially protected; birds 2 weeks of age were completely protected with a single dose of VRP.

Age Factors↗
PubMed2000

A single-site mutant and revertants arising in vivo define early steps in the pathogenesis of Venezuelan equine encephalitis virus.

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.

Animals↗
PubMed2000

Vaccination of macaques against pathogenic simian immunodeficiency virus with Venezuelan equine encephalitis virus replicon particles.

Vaccine vectors derived from Venezuelan equine encephalitis virus (VEE) that expressed simian immunodeficiency virus (SIV) immunogens were tested in rhesus macaques as part of the effort to design a safe and effective vaccine for human immunodeficiency virus. Immunization with VEE replicon particles induced both humoral and cellular immune responses. Four of four vaccinated animals were protected against disease for at least 16 months following intravenous challenge with a pathogenic SIV swarm, while two of four controls required euthanasia at 10 and 11 weeks. Vaccination reduced the mean peak viral load 100-fold. The plasma viral load was reduced to below the limit of detection (1,500 genome copies/ml) in one vaccinated animal between 6 and 16 weeks postchallenge and in another from week 6 through the last sampling time (40 weeks postchallenge). The extent of reduction in challenge virus replication was directly correlated with the strength of the immune response induced by the vectors, which suggests that vaccination was effective.

Animals↗
PubMed2000

Expression of the two major envelope proteins of equine arteritis virus as a heterodimer is necessary for induction of neutralizing antibodies in mice immunized with recombinant Venezuelan equine encephalitis virus replicon particles.

RNA replicon particles derived from a vaccine strain of Venezuelan equine encephalitis virus (VEE) were used as a vector for expression of the major envelope proteins (G(L) and M) of equine arteritis virus (EAV), both individually and in heterodimer form (G(L)/M). Open reading frame 5 (ORF5) encodes the G(L) protein, which expresses the known neutralizing determinants of EAV (U. B. R. Balasuriya, J. F. Patton, P. V. Rossitto, P. J. Timoney, W. H. McCollum, and N. J. MacLachlan, Virology 232:114-128, 1997). ORF5 and ORF6 (which encodes the M protein) of EAV were cloned into two different VEE replicon vectors that contained either one or two 26S subgenomic mRNA promoters. These replicon RNAs were packaged into VEE replicon particles by VEE capsid protein and glycoproteins supplied in trans in cells that were coelectroporated with replicon and helper RNAs. The immunogenicity of individual replicon particle preparations (pVR21-G(L), pVR21-M, and pVR100-G(L)/M) in BALB/c mice was determined. All mice developed antibodies against the recombinant proteins with which they were immunized, but only the mice inoculated with replicon particles expressing the G(L)/M heterodimer developed antibodies that neutralize EAV. The data further confirmed that authentic posttranslational modification and conformational maturation of the recombinant G(L) protein occur only in the presence of the M protein and that this interaction is necessary for induction of neutralizing antibodies.

Animals↗
PubMed1999

Venezuelan equine encephalitis virus vectors expressing HIV-1 proteins: vector design strategies for improved vaccine efficacy.

A live virus vaccine vector has been constructed from a molecularly cloned attenuated strain of Venezuelan equine encephalitis virus (VEE). High levels of foreign protein expression are regulated by an additional copy of the 26 S viral subgenomic RNA promoter. The position of this additional promoter and foreign gene in the VEE genome was predicted to have a major influence on expression level of the heterologous protein. Two sites in the genome were tested to determine the optimal site for expression of the matrix/capsid (MA/CA) coding region of human immunodeficiency virus (HIV-1). One vector contained the additional promoter and the MA/CA genes immediately downstream of the VEE E1 gene at the 3' end of the genome. In the second vector, the additional promoter was introduced immediately upstream from the authentic 26 S subgenomic promoter. Significantly higher levels of MA/CA were expressed from the downstream vector compared to the upstream vector. However, the stability of expression for both vectors was similar following passage in baby hamster kidney cells (BHK) cells. In BALB/c mice, the two vectors elicited similar levels of cellular immune responses to MA/CA as determined by bulk cytotoxic T-lymphocyte assays and precursor frequency analysis, but the humoral response induced by the downstream vector was significantly stronger. At 11 months post boosting with the downstream vector, serum antibody levels against HIV MA/CA were undiminished, and MA/CA specific CTLp were detectable in all mice tested. These findings suggest that VEE vectors can be optimized to elicit strong, balanced and long-lived immune responses to foreign viral proteins.

AIDS Vaccines↗
PubMed1999

Intratympanic cholesteatoma.

Cholesteatomas occurring behind an intact TM are relatively unusual, and intratympanic keratomas are even less common. This article presents the case of a Baffin Zone Inuit gentleman with an intratympanic cholesteatoma confined to the membranous portion of his TM. Although it is generally accepted that many native populations, including the Inuit, demonstrate an elevated preponderance of aural disease, cholesteatoma formation, particularly within the TM, in an individual from this area of the high Arctic is extremely rare.

Adult↗
PubMed1997

Replicon-helper systems from attenuated Venezuelan equine encephalitis virus: expression of heterologous genes in vitro and immunization against heterologous pathogens in vivo.

A replicon vaccine vector system was developed from an attenuated strain of Venezuelan equine encephalitis virus (VEE). The replicon RNA consists of the cis-acting 5' and 3' ends of the VEE genome, the complete nonstructural protein gene region, and the subgenomic 26S promoter. The genes encoding the VEE structural proteins were replaced with the influenza virus hemagglutinin (HA) or the Lassa virus nucleocapsid (N) gene, and upon transfection into eukaryotic cells by electroporation, these replicon RNAs directed the efficient, high-level synthesis of the HA or N proteins. For packaging of replicon RNAs into VEE replicon particles (VRP), the VEE capsid and glycoproteins were supplied in trans by expression from helper RNA(s) coelectroporated with the replicon. A number of different helper constructs, expressing the VEE structural proteins from a single or two separate helper RNAs, were derived from attenuated VEE strains Regeneration of infectious virus was not detected when replicons were packaged using a bipartite helper system encoding the VEE capsid protein and glycoproteins on two separate RNAs. Subcutaneous immunization of BALB/c mice with VRP expressing the influenza HA or Lassa virus N gene (HA-VRP or N-VRP, respectively) induced antibody responses to the expressed protein. After two inoculations of HA-VRP, complete protection against intranasal challenge with influenza was observed. Furthermore, sequential immunization of mice with two inoculations of N-VRP prior to two inoculations of HA-VRP induced an immune response to both HA and N equivalent to immunization with either VRP construct alone. Protection against influenza challenge was unaffected by previous N-VRP immunization. Therefore, the VEE replicon system was characterized by high-level expression of heterologous genes in cultured cells, little or no regeneration of plaque-forming virus particles, the capability for sequential immunization to multiple pathogens in the same host, and induction of protective immunity against a mucosal pathogen.

Animals↗
PubMed1997

Mucosal immunity induced by parenteral immunization with a live attenuated Venezuelan equine encephalitis virus vaccine candidate.

Induction of a mucosal immune response is generally thought to require introduction of an immunogen directly onto the mucosal surface. It has been observed, however, that live, attenuated mutants of the alphavirus, Venezuelan equine encephalitis virus (VEE), induce protection from virulent challenge at the respiratory mucosa even after parenteral inoculation. In this report, we propose a mechanism by which subcutaneous immunization with a molecularly cloned, attenuated double mutant of VEE is able to stimulate the production of mucosal anti-VEE IgA. Our results showed that the immunizing virus spread to, and replicated within, lymphoid tissues throughout the mouse. Several tissues known to be inductive sites of the mucosal immune system were found to be positive for the presence of VEE RNA by 48 hr postimmunization. Moreover, this mucosal lymphotropism resulted in the production of virus-specific IgA antibody detectable in vaginal secretions of immunized mice.

Administration, Intranasal↗
PubMed1997

The development of a novel intraperitoneal tumor-seeding prophylactic.

BACKGROUND: Peritoneal tumor dissemination and implantation is a complication of both open and laparoscopic oncologic surgery. This study evaluates the efficacy of paclitaxel-loaded poly(L-lactic acid) microspheres as prophylaxis against intraabdominal tumor seeding. METHODS: 2 x 10(6) 9L glioblastoma cells were introduced into the abdominal cavity of Wistar rats. Fifteen minutes later, the peritoneal cavity was washed with the experimental solutions, and 2 weeks later the presence of tumor implantation was determined. After defining the optimum dose of paclitaxel PLA microspheres in a dose-ranging study, the microsphere formulation was then compared with conventional paclitaxel in four experimental groups (n = 5) as follows: 100 mg of 30% paclitaxel-loaded microspheres; 100 mg PLA microspheres; paclitaxel 4.1 mg; and controls receiving no intraabdominal therapy. RESULTS: Although carcinomatosis developed in all control animals, none in the paclitaxel-loaded microsphere group had biopsy proven cancer. The conventional paclitaxel group (3) showed significant toxicity; only 1 animal survived and had positive histology. CONCLUSIONS: In this animal model of peritoneal carcinomatosis, the paclitaxel-loaded microsphere formulation was more effective than conventional paclitaxel in preventing tumor seeding.

Animals↗
PubMed1997

Humoral, mucosal, and cellular immunity in response to a human immunodeficiency virus type 1 immunogen expressed by a Venezuelan equine encephalitis virus vaccine vector.

A molecularly cloned attenuated strain of Venezuelan equine encephalitis virus (VEE) has been genetically configured as a replication-competent vaccine vector for the expression of heterologous viral proteins (N. L. Davis, K. W. Brown, and R. E. Johnston, J. Virol. 70:3781-3787, 1996). The matrix/capsid (MA/CA) coding domain of human immunodeficiency virus type 1 (HIV-1) was cloned into the VEE vector to determine the ability of a VEE vector to stimulate an anti-HIV immune response in mice. The VEE-MA/CA vector replicated rapidly in the cytoplasm of baby hamster kidney (BHK) cells and expressed large quantities of antigenically identifiable MA/CA protein. When injected subcutaneously into BALB/c mice, the vector invaded and replicated in the draining lymphoid tissues, expressing HIV-1 MA/CA at a site of potent immune activity. Anti-MA/CA immunoglobulin G (IgG) and IgA antibodies were present in serum of all immunized mice, and titers increased after a second booster inoculation. IgA antibodies specific for MA/CA were detected in vaginal washes of mice that received two subcutaneous immunizations. Cytotoxic T-lymphocyte responses specific for MA/CA were detected following immunization with the MA/CA-expressing VEE vector. These findings demonstrate the ability of a VEE-based vaccine vector system to stimulate a comprehensive humoral and cellular immune response. The multifaceted nature of this response makes VEE an attractive vaccine for immunization against virus infections such as HIV-1, for which the correlates of protective immunity remain unclear, but may include multiple components of the immune system.

AIDS Vaccines↗
PubMed1996

Complete nucleotide sequence and full-length cDNA clone of S.A.AR86 a South African alphavirus related to Sindbis.

S.A.AR86 and Girdwood S.A., two South African Sindbis-like arboviruses, are closely related antigenically to the Swedish isolate, Ockelbo82 [Lundström, J. O., Vene, S., Saluzzo, J. F., and Niklasson, B. (1993) Am. J. Trop. Med. Hyg. 49(5), 531-537]. Each of these viruses is associated with human disease, and Girdwood S.A. was isolated from a human case. In addition, S.A.AR86 is unique among Sindbis-like viruses in that adult mice remain sensitive to lethal infection with S.A.AR86. The complete genomic sequences of S.A.AR86 and Girdwood S.A. were determined. The S.A.AR86 RNA genome contained 11,663 nucleotides, excluding the 5' CAP structure and 3' poly(A) tail. In comparison to the consensus sequence of the prototype Egyptian Sindbis strain AR339, S.A.AR86 differed at 5.57% of the nucleotides, including a 54-nucleotide deletion, two insertions of 6 nucleotides each, and a 3-nucleotide insertion in the 3' terminal one-third of the S.A.AR86 nsP3 gene. S.A.AR86 is one of only three alphaviruses sequenced to date that does not have an opal termination codon between the nsP3 and the nsP4 genes. These genes are separated by a cysteine codon in the S.A.AR86 genome. The genome of Girdwood S.A. was 11,717 nucleotides in length, excluding the 5' CAP and 3' poly(A) tail. Girdwood S.A. contained an opal termination codon between nsP3 and nsP4 and did not have the large 54-nucleotide deletion in nsP3, although Girdwood S.A. did contain the remaining insertions and deletions characteristic of S.A.AR86. S.A.AR86 was more closely related to Girdwood S.A. than to the Egyptian isolate, and the South African isolates as a group were more closely related to the Swedish isolate. Comparison of the S.A.AR86 sequence to that of Ockelbo82, Girdwood S.A., and Sindbis virus AR339 revealed several codons where S.A.AR86 differed from the conserved amino acid encoded by the other three viruses. These changes may be related to the ability of S.A.AR86 to initiate a lethal central nervous system infection in adult mice. To fulfill a prerequisite for testing this hypothesis, a full-length cDNA clone of S.A.AR86 was constructed from which infectious genomic RNA replicas could be derived. The sequence of this clone differed from the S.A.AR86 genomic RNA sequence at four translationally silent positions, and virus derived from the clone reproduced the adult mouse neurovirulence phenotype of its biological progenitor.

Alphavirus↗
PubMed1996

A modified method of carcinogenesis induction in the DMBA hamster cheek pouch model of squamous neoplasia.

BACKGROUND: The classic hamster cheek pouch model of squamous cell neoplasia requires triweekly application of 9,10-dimethyl-1,2-benzanthracene (DMBA) on tissues for 20 weeks. This study describes a new methodology for induction of neoplasia using a sustained-release delivery of carcinogen that is less labor intensive with decreased risk to personnel. METHODS: Cotton sutures were impregnated with DMBA and coated with silicone elastomer. These sutures were then placed in the cheek pouch of animals, were harvested at weekly intervals, and residual DMBA was measured confirming sustained release. Carcinogenesis was compared in Syrian hamsters grouped into classic triweekly painted model (n = 10), sustained-release DMBA (n = 19), sustained-release with weekly painting (n = 17), and control (n = 10). RESULTS: The sustained-release implants resulted in a 90% yield of squamous neoplasia at 20 weeks, similar to the classic painted model but with less handling of the carcinogen and the animals. CONCLUSIONS: This model is safer for personnel, time efficient, and effective for inducing carcinogenesis.

9,10-Dimethyl-1,2-benzanthracene↗
PubMed1996

Deduced consensus sequence of Sindbis virus strain AR339: mutations contained in laboratory strains which affect cell culture and in vivo phenotypes.

The consensus sequence of the Sindbis virus AR339 isolate, the prototype alphavirus, has been deduced. THe results presented here suggest (i) that a substantial proportion of the sequence divergence evident between the consensus sequence and sequences of laboratory strains of AR339 has resulted from selection for efficient growth in cell culture, (ii) that many of these changes affect the virulence of the virus in animal models, and (iii) that such modified genetic backgrounds present in laboratory strains can exert a significant influence on genetic studies of virus pathogenesis and host range. A laboratory strain of Sindbis virus AR339 was sequenced and cloned as a cDNA (pTRSB) from which infectious virus (TRSB) could be derived. The consensus sequence was deduced from the complete sequences of pTRSB and HRsp (E. G. Strauss, C. M. Rice, and J. H. Strauss, Virology 133:92-110, 1984), from partial sequences of the glycoprotein genes of three other AR339 laboratory strains, and by comparison with the sequences of the glycoprotein genes of three other AR339 sequence. HRsp differed form the consensus sequence by eight coding changes, and TRSB differed by three coding changes. In the 5' untranslated region, HRsp differed from the consensus sequence at nucleotide (nt) 5. These differences were likely the result of cell culture passage of the original AR339 isolate. At three of the difference loci (one in TRSB and two in HRsp), selection of cell-culture-adaptive mutations was documented with Sindbis virus or other alphaviruses. Selection in cell culture often results in attenuation of virulence in animals. Considering the TRSB and HRsp sequences together, one noncoding difference from the consensus (an A-for-G substitution in the 5' untranslated region at nt 5) and six coding differences in the glycoprotein genes (at E2 amino acids 1, 3, 70, and 172 and at E1 amino acids 72 and 237) were at loci which, either individually or in combination, significantly affected alphavirus virulence in mice. Although the levels of virulence of isogenic strains containing either nt 5 A or nt 5 G did not differ significantly in neonatal mice, the presence of nt 5 A greatly enhanced the effect of a second attenuating mutation in the E2 gene. These results suggest that minimal differences in the "wild type" genetic background into which an additional mutation is introduced can have a dramatic effect on apparent virulence and pathogenesis phenotypes. A cDNA clone of the consensus AR339 sequence, a sequence devoid of occult attenuating mutations introduced by cell culture passage, will allow the molecular genetic examination of cell culture and in vivo phenotypes of a virus which may best reflect the sequence of Sindbis virus AR339 at the time of its isolation.

Alphavirus Infections↗