Quality sample collection, handling, and preservation for an effective microbial forensics program.
Explore the source record for details and available documents.
Bioterrorism↗
Biomedical subjects
Luis L Rodriguez
Publications and source records attributed to Luis L Rodriguez.
Vaccination against foot-and-mouth disease virus confers complete clinical protection in 7 days and partial protection in 4 days: Use in emergency outbreak response.
Recent outbreaks of foot-and-mouth disease virus (FMDV) demonstrate that this highly contagious viral infection of cloven hoofed animals continues to be a significant economic problem worldwide. Debate about the most effective way to respond to outbreaks of FMDV in disease free countries continues to center on the use of vaccines. In this report, we present data showing that a commercially available, standard dose vaccine formulation can fully protect cattle against direct challenge with the virus in as little as 7 days with no carrier transmission to naïve animals. Cattle challenged 4 days after vaccination have reduced disease severity, no detectable virus in blood and little virus shedding from nasal secretions. These significant effects at 4 days post vaccination, confirmed in two separate trials, support the value of using currently available vaccines as a first line of defense against foot-and-mouth disease (FMD) outbreaks.
Animals↗
A rapid, simple, and humane method for submandibular bleeding of mice using a lancet.
Methods for obtaining blood samples from mice tend to be difficult, inhumane, or both. The authors describe an inexpensive, disposable, single-use lancet for submandibular bleeding of mice that allows investigators to quickly draw 0.2-0.5 ml of blood without the use of anesthesia.
Animals↗
Recombinant vesicular stomatitis (Indiana) virus expressing New Jersey and Indiana glycoproteins induces neutralizing antibodies to each serotype in swine, a natural host.
Vesicular stomatitis virus (VSV) is the most common cause of vesicular disease outbreaks in livestock throughout the Western Hemisphere. Two major serotypes, Indiana and New Jersey, cause epidemic disease in pigs, cattle, and horses. We generated recombinant viruses derived from the Indiana serotype genome that were engineered to contain and express: (1) a single copy of the glycoprotein gene from the Indiana serotype (VSIV-GI); (2) a single copy of the glycoprotein gene from the New Jersey serotype (VSIV-GNJ); or (3) two copies of the glycoprotein gene, one from each of the two major VSV serotypes (VSIV-GNJGI) [Martinez I, Rodriguez LL, Jimenez C, Pauszek SJ, Wertz GW. Vesicular stomatitis virus glycoprotein is a determinant of pathogenesis in swine, a natural host. J Virol 2003;77(14):8039-47]. These recombinant viruses and a highly pathogenic New Jersey field isolate (VSNJV), from which the GNJ gene was derived, were inoculated into swine, a natural host, and the induction of neutralizing antibodies to both serotypes was analyzed. The neutralizing antibody response induced by VSIV-GI, VSIV-GNJ and VSNJV was serotype-specific, according to the glycoprotein expressed. VSIV-GNJGI expressed both glycoproteins stably through multiple rounds of replication in swine and induced neutralizing antibodies against both VSV serotypes, with a dominance of the Indiana serotype in the serological response. Pigs immunized with VSIV-GI or VSIV-GNJ were protected against homologous high dose virus challenge. Pigs inoculated with VSIV-GNJGI were protected against challenge with VSIV-GI but three of four animals developed lesions after challenge with the highly pathogenic New Jersey field isolate.
Animals↗
A synthetic peptide containing the consensus sequence of the G-H loop region of foot-and-mouth disease virus type-O VP1 and a promiscuous T-helper epitope induces peptide-specific antibodies but fails to protect cattle against viral challenge.
A pilot study was carried out in cattle to determine the immunogenicity of a synthetic consensus peptide comprising the G-H loop region of foot-and-mouth disease virus (FMDV) type-O VP1 and a non-VP1 T-helper (Th) epitope. Cattle vaccinated intramuscularly either once (n = 5) or twice (n = 4) with 50 microg of the peptide preparation at a 21-day interval developed antibodies to the peptide as determined by ELISA with the exception of one steer that received a single dose. However, neutralizing antibody titers against FMDV type-O were modest and all animals presented with clinical FMD signs upon challenge 21 days after the last vaccination. In contrast, four of the five animals inoculated with an inactivated FMD type-O commercially prepared vaccine developed neutralizing antibodies and were fully protected against clinical disease following virus challenge 21 days post-vaccination (dpv). Nucleotide sequence comparison of the VP1 region between the challenge virus and RT-PCR products recovered from a lesion of the peptide-vaccinated animal with the highest neutralizing antibody titer 5 days post-challenge (dpc) showed no evidence for selection of a neutralization-resistant mutant. We conclude that although the synthetic peptide induced an antibody response in cattle, it failed to confer protection against FMDV challenge.
Amino Acid Sequence↗
Vesicular stomatitis virus glycoprotein is a determinant of pathogenesis in swine, a natural host.
There are two major serotypes of vesicular stomatitis virus (VSV), Indiana (VSIV) and New Jersey (VSNJV). We recovered recombinant VSIVs from engineered cDNAs that contained either (i) one copy of the VSIV G gene (VSIV-G(I)); (ii) two copies of the G gene, one from each serotype (VSIV-G(NJ)G(I)); or (iii) a single copy of the G(NJ) gene instead of the G(I) gene (VSIV-G(NJ)). The recombinant viruses expressed the appropriate glycoproteins, incorporated them into virions, and were neutralized by antibodies specific for VSIV (VSIV-G(I)), VSNJV (VSIV-G(NJ)), or both (VSIV-G(NJ)G(I)), according to the glycoprotein(s) they expressed. All recombinant viruses grew to similar titers in cell culture. In mice, VSIV-G(NJ) and VSIV-G(NJ)G(I) were attenuated. However, in swine, a natural host for VSV, the G(NJ) glycoprotein-containing viruses caused more severe lesions and replicated to higher titers than the parental virus, VSIV-G(I). These observations implicate the glycoprotein as a determinant of VSV virulence in a natural host and emphasize the differences in VSV pathogenesis between mice and swine.
Animals↗
A single-tube multiplex reverse transcription-polymerase chain reaction for detection and differentiation of vesicular stomatitis Indiana 1 and New Jersey viruses in insects.
A multiplex single-tube reverse transcription-polymerase chain reaction (RT-PCR) has been developed for the detection and differentiation of vesicular stomatitis viruses (VSV), Indiana 1 and New Jersey, from insect samples. Using this assay, detection of either or both viruses in as little as 20 fg of total RNA from tissue culture was achieved, along with detection of vesicular stomatitis (VS) RNA from macerates containing 2 infected mosquitoes in pools of 10-30 noninfected mosquitoes. Vesicular stomatitis virus was detected by RT-PCR in all culture-positive samples, and detection as low as 4 plaque forming units per milliliter was achieved. Comparison between RT-PCR and tissue culture revealed that RT-PCR was able to detect VSV in a volume of insect macerate averaging almost 100 times less than that required for detection by tissue culture. The reported RT-PCR is a potential valuable tool for rapid and sensitive detection and differentiation of VS in insects because intense work associated with viral isolation, the cytotoxicity of insect extracts, and separate virus identification steps can be avoided. Potential application to detection and differentiation of VSV serotypes from vertebrate hosts is addressed.
Animals↗
Detection of antibodies against the four subtypes of ebola virus in sera from any species using a novel antibody-phage indicator assay.
The natural host for Ebola virus, presumed to be an animal, has not yet been identified despite an extensive search following several major outbreaks in Africa. A straightforward approach used to determine animal contact with Ebola virus is by assessing the presence of specific antibodies in serum. This approach however has been made very difficult by the absence of specific reagents required for the detection of antibodies from the majority of wild animal species. In this study, we isolated a human monoclonal antibody Fab fragment, KZ51, that reacts with an immunodominant epitope on Ebola virus nucleoprotein (NP) that is conserved on all four Ebola virus subtypes. The antibody KZ51 represents a major specificity as sera from all convalescent patients tested (10/10) and sera from guinea pigs infected with each of the four Ebola virus subtypes competed strongly with KZ51 for binding to radiation-inactivated Ebola virus. These features allowed us to develop a novel assay for the detection of seroconversion irrespective of Ebola virus subtype or animal species. In this assay, the binding of KZ51 Fab-phage particles is used as an indicator assay and the presence of specific antibodies against Ebola virus in sera is indicated by binding competition. A prominent feature of the assay is that the Fab-phage particles may be prestained with a dye so that detection of binding can be directly determined by visual inspection. The assay is designed to be both simple and economical to enable its use in the field.
Animals↗
Growth and molecular evolution of vesicular stomatitis serotype New Jersey in cells derived from its natural insect-host: evidence for natural adaptation.
In this study we evaluated the growth and molecular evolution of a natural isolate of VSV-NJ (89GAS) from sand flies in cells derived from sand flies (LL5), mosquitoes (C6/36) or hamsters (BHK21). Two VSV-NJ natural isolates of mammalian origin were used for comparison. For each virus we measured: (i) ability to grow in one-step growth curve or after serial passage on each cell type; (ii) ability to induce persistent infection, and (iii) genetic stability of the glycoprotein gene (G) after serial passage in each cell type. Sand fly virus 89GAS grew to higher titers in insect cells compared with viruses of mammalian origin and showed increasing titers with each passage only in C6/36 cells. All viruses established productive persistent infections in both mosquito and sand fly cells but only LL5 cells yielded sustained high virus titers for periods of up to 81 days. Analyses of the consensus sequences of the G gene from each virus after 0, 10 or up to 25 passages in each cell line showed nucleotide substitution rates between 1.39 x l0(-4) and 6.95 x l0(-5). The majority of these changes were non-synonymous, suggesting positive selection. We did not detect increased nucleotide substitution rates on the G gene of 89GAS after passage in cell lines of mosquito or mammalian origin, nor in viruses of mammalian origin after passage in insect cells. This indicates that although VSV G is positively selected in vitro by the insect cell environment, this does not fully explain VSV natural adaptation. This is the first evidence of naturally occurring adaptation of VSV to cells derived from its natural host.
Adaptation, Physiological↗
Full-length genome analysis of natural isolates of vesicular stomatitis virus (Indiana 1 serotype) from North, Central and South America.
Most studies on the molecular biology and functional analysis of vesicular stomatitis virus Indiana 1 serotype (VSV-IN1) are based on the only full-length genomic sequence currently deposited in GenBank. This sequence is a composite of several VSV-IN1 laboratory strains passaged extensively in tissue culture over the years and it is not certain that this sequence is representative of strains circulating in nature. We describe here the complete genomic sequence of three natural isolates, each representing a distinct genetic lineage and geographical origin: 98COE (North America), 94GUB (Central America) and 85CLB (South America). Genome structure and organization were conserved, with a 47 nucleotide 3' leader, five viral genes -- N, P, M, G and L -- and a 59 nucleotide 5' trailer. The most conserved gene was N, followed by M, L and G, with the most variable being P. Sequences containing the polyadenylation and transcription stop and start signals were completely conserved among all the viruses studied, but changes were found in the non-transcribed intergenic nucleotides, including the presence of a trinucleotide at the M-G junction of the South American lineage isolate. A 102-189 nucleotide insertion was present in the 5' non-coding region of the G gene only in the viruses within a genetic lineage from northern Central America. These full-length genomic sequences should be useful in designing diagnostic probes and in the interpretation of functional genomic analyses using reverse genetics.
Base Sequence↗