Use of a tetanus toxoid marker to allow differentiation of infected from vaccinated poultry without affecting the efficacy of a H5N1 avian influenza virus vaccine.

Tetanus toxoid (TT) was assessed as a positive marker for avian influenza (AI) virus vaccination in chickens, in a vaccination and challenge study. Chickens were vaccinated twice with inactivated AI H5N2 virus vaccine, and then challenged three weeks later with highly pathogenic AI H5N1 virus. Vaccinated chickens were compared with other groups that were either sham-vaccinated or vaccinated with virus with the TT marker. All sham-vaccinated chickens died by 36 hours postinfection, whereas all vaccinated chickens, with or without the TT marker, were protected from morbidity and mortality following exposure to the challenge virus. Serological testing for H5-specific antibodies identified anamnestic responses to H5 in some of the vaccinated birds, indicating active virus infection.

Development and characterization of new flavivirus-resistant mouse strains bearing Flv(r)-like and Flv(mr) alleles from wild or wild-derived mice.

A single genetic locus, flavivirus resistance (Flv), controls virus titres and severity of flavivirus infection in mouse brain. It has been mapped to mouse chromosome 5 and shown to include different allelic forms. While the majority of laboratory mouse strains are susceptible to flaviviruses and carry the Flv(s) allele, wild mice and laboratory mouse strains recently derived from them are resistant and carry flavivirus-resistance alleles including Flv(r)-like and Flv(mr) alleles. Although there is a mouse model of flavivirus resistance conferred by the Flv(r) allele, other resistance alleles have not been adequately studied due to a lack of appropriate animal models. In this paper we describe the development of new flavivirus-resistant mouse strains, C3H.M.domesticus-Flv(r) and C3H.MOLD-Flv(mr), which carry the novel resistance alleles Flv(r)-like and Flv(mr) on the genetic background of flavivirus susceptible C3H/HeJ mice. The new strains were created by 10 to 11 generations of backcrossing followed by brother-sister matings resulting in a generation of homozygous founder stocks. Genome analysis of the newly developed mouse strains has revealed chromosomal regions of approximately 9 and 11 cM, respectively, encompassing Flv on chromosome 5, which are derived from resistant donor mice. These segments are much smaller than the segment of approximately 31 cM described in the congenic resistant mouse strain C3H.PRI-Flv(r) (also known as C3H/RV). The new congenic mouse strains, which were created to carry the Flv(r)-like and Flv(mr) alleles on the standardized genetic background of susceptible mice, represent new animal models of flavivirus resistance conferred by these novel resistance alleles.

Acoustic method to quantitatively assess the position and patency of infant endotracheal tubes: preliminary results in rabbits.

In this preliminary laboratory study, an acoustical method was evaluated to quantitatively assess the position and patency of an infant-size endotracheal tube (ETT) by in vivo and in vitro measurements. The method consists of emitting an audible sound pulse into the ETT and the airways, and deriving position and patency information from the timing and characteristics of the returning echoes. The method's capacity to measure ETT changes of position in the tracheae of five anesthetized New Zealand white rabbits (weight, 4.3-4.9 kg; age, 1.5-3 years) was found to be accurate to 0.7 +/- 3.6 mm (mean +/- 95% CI) over a distance of 5 cm. The method was also shown to reliably differentiate between tracheal, bronchial, and esophageal intubations by means of an acoustically inferred diameter of the passageway just beyond the ETT tip. To assess the accuracy of estimating lumen obstruction, in vitro acoustical measurements were performed in different size ETTs (2.5, 3.0, 3.5, and 4.0 mm inner diameter), with obstructions ranging from 5-100% reduction in cross-sectional area. The system identified the sizes of these obstructions to within +/-7%. This technology has the potential for continuous, computer-based monitoring of breathing-tube function through instantaneous detection of ETT malposition or obstruction before it leads to a serious medical condition.

Molecular characterization of virus-specific RNA produced in the brains of flavivirus-susceptible and -resistant mice after challenge with Murray Valley encephalitis virus.

Natural resistance to flaviviruses in mice is controlled by a single genetic locus, FIv, on chromosome 5. Although the mechanism of this resistance is not fully understood, it is believed to operate at the level of virus replication rather than the immune response. It has been hypothesized that enhanced production of viral defective interfering (DI) particles is responsible for a substantial reduction in the titres of infectious virus in resistant mice. However, this has never been established at the molecular level since such particles have not been isolated and characterized. We have studied the products of virus replication in the brains of flavivirus-susceptible C3H/HeJ (Flv(s)) and -resistant congenic C3H/RV (Flv(r)) mice after an intracerebral challenge (i.c.) with Murray Valley encephalitis (MVE) virus and have found no evidence for the accumulation of truncated viral RNA in the brains of resistant mice. All three major viral RNA species, the replicative intermediate (RI), replicative form (RF) and virion RNA (vRNA) together with a subgenomic RNA species of 0.6 kb, which has not been previously described, were present in the brains of both mouse strains. However, the viral RF and RI RNA forms preferentially accumulated in the brains of resistant mice. Thus, we confirm that the resistance allele Flv(r) interferes with discrete steps in flavivirus replication, although the precise mechanism remains to be determined.

Low resolution mapping around the flavivirus resistance locus (Flv) on mouse chromosome 5.

Although the phenomenon of innate resistance to flaviviruses in mice was recognized many years ago, it was only recently that the genetic locus (Flv) controlling this resistance was mapped to mouse Chromosome (Chr) 5. Here we report the fine mapping of the Flv locus, using 12 microsatellite markers which have recently been developed for mouse Chr 5. The new markers were genotyped in 325 backcross mice of both (C3H/HeJ x C3H/RV)F1 x C3H/HeJ and (BALB/c x C3H/RV)F1 x BALB/c backgrounds, relative to Flv. The composite genetic map that has been constructed identifies three novel microsatellite loci, D5Mit68, D5Mit159, and D5Mit242, tightly linked to the Flv locus. One of those loci, D5Mit159, showed no recombinations with Flv in any of the backcross mice analyzed, indicating tight linkage (< 0.3 cM). The other two, D5Mit68 and D5Mit242, exhibited two and one recombinations with Flv (0.6 and 0.3 cM) respectively, defining the proximal and distal boundaries of a 0.9-cM segment around this locus. The proximal flanking marker, D5Mit68, maps to a segment on mouse Chr 5 homologous to human Chr 4. This, together with the previous data produced by our group, locates Flv to a region on mouse Chr 5 carrying segments that are conserved on either human Chr 4, 12, or 7, but present knowledge does not allow precise identification of the syntenic element.

Mapping the Flv locus controlling resistance to flaviviruses on mouse chromosome 5.

Genetically determined resistance to flaviviruses in mice is a dominant trait conferred by alleles at a single autosomal locus designated Flv, but no gene products have been associated with this locus and the mechanism of resistance is not well understood. To further characterize this model of genetic resistance, we conducted mapping studies to determine the chromosomal location of Flv. Because of evidence suggesting that the Flv locus is on chromosome 5, three-point backcross linkage analyses were used to define the location of Flv relative to previously assigned chromosome 5 markers. The results confirm the chromosome 5 location of Flv and indicate a map position between the anchor loci rd and Gus-s. The chromosomal localization of Flv is the first step in the production of a detailed linkage map of the Flv region, which may open approaches to positional cloning of the resistance gene.

An acoustical guidance and position monitoring system for endotracheal tubes.

A prototype instrument to guide the placement and continuously monitor the position of an endotracheal tube (ETT) was developed. An incident audible sound pulse is introduced into the proximal ETT and detected as it travels down the ETT via a miniature microphone located in the wall. This pulse is then emitted from the tube tip into the airways and the reflected signal from the airways is detected by the microphone. A well defined reflection arises from the point where the total cross sectional area of the airways increases rapidly, and the difference in timing between detection of the incident pulse and this reflection is used to determine ETT position or movement. This reflection is not observed if the ETT is erroneously placed in the esophagus. The amplitude and polarity of an additional reflection that occurs at the ETT tip is used to estimate the cross-sectional area of the airway in which the ETT is placed. This combined information allows discrimination between tracheal and bronchial intubation and can be used to insure an adequate fit between the ETT and trachea. The instrument has proven extremely reliable in multiple intubations in eight canines and offers the potential to noninvasively and inexpensively monitor ETT position in a continuous manner.