Flow cytometric probing of mitochondrial function in equine peripheral blood mononuclear cells

BACKGROUND: The morphopathological picture of a subset of equine myopathies is compatible with a primary mitochondrial disease, but functional confirmation in vivo is still pending. The cationic dye JC-1 exhibits potential-dependent accumulation in mitochondria that is detectable by a fluorescence shift from green to orange. As a consequence, mitochondrial membrane potential can be optically measured by the orange/green fluorescence intensity ratio. A flow cytometric standardized analytic procedure of the mitochondrial function of equine peripheral blood mononuclear cells is proposed along with a critical appraisal of the crucial questions of technical aspects, reproducibility, effect of time elapsed between blood sampling and laboratory processing and reference values. RESULTS: The JC-1-associated fluorescence orange and green values and their ratio were proved to be stable over time, independent of age and sex and hypersensitive to intoxication with a mitochondrial potential dissipator. Unless time elapsed between blood sampling and laboratory processing does not exceed 5 hours, the values retrieved remain stable. Reference values for clinically normal horses are given. CONCLUSION: Whenever a quantitative measurement of mitochondrial function in a horse is desired, blood samples should be taken in sodium citrate tubes and kept at room temperature for a maximum of 5 hours before the laboratory procedure detailed here is started. The hope is that this new test may help in confirming, studying and preventing equine myopathies that are currently imputed to mitochondrial dysfunction

Characterization of the resistance of SJL/J mice to pneumonia virus of mice, a model for infantile bronchiolitis due to a respiratory syncytial virus

Respiratory syncytial virus (RSV), a prominent cause of airway morbidity in children, maintains an excessive hospitalization rate despite decades of research. Host factors are assumed to influence the disease severity. As a first step toward identifying the underlying resistance mechanisms, we recently showed that inbred mouse strains differ dramatically as regards their susceptibility to pneumonia virus of mice (PVM), the murine counterpart of RSV. PVM infection in mice has been shown to faithfully mimic the severe RSV disease in human infants. This study aimed at dissecting the remarkable PVM-resistance shown by the SJL/J strain. To characterize its genetic component, we assessed clinical, physiopathological, and virological resistance/susceptibility traits in large first (F1) and second (F2) generations obtained by crossing the SJL/J (resistant) and 129/Sv (susceptible) strains. Then, to acquire conclusive in vivo evidence in support of the hypothesis that certain radiosensitive hematopoietic cells might play a significant role in PVM-resistance, we monitored the same resistance/susceptibility traits in mock- and γ-irradiated SJL/J mice. Segregation analysis showed that (i) PVM-resistance is polygenic, (ii) the resistance alleles are recessive, and (iii) all resistance-encoding alleles are concentrated in SJL/J. Furthermore, there was no alteration of SJL/J PVM resistance after immunosuppression by γ-irradiation, which suggests that adaptive immunity is not involved. We conclude that host resistance to pneumoviruses should be amenable to genetic dissection in this mouse model and that radioresistant lung epithelial cells and/or alveolar macrophages may control the clinical severity of pneumovirus-associated lung disease

Natural intrauterine infection with Schmallenberg virus in malformed newborn calves

We comprehensively surveyed morphologic alterations in calves naturally infected in utero by Schmallenberg virus (SBV) and born deformed. SBV-specific RNA was distributed unevenly in different tissues. Implications for diagnosic procedures are highlighted

New Insights into the Susceptibility of Immunocompetent Mice to Usutu Virus.

Usutu virus (USUV) is a mosquito-borne flavivirus that shares many similarities with the closely related West Nile virus (WNV) in terms of ecology and clinical manifestations. Initially distributed in Africa, USUV emerged in Italy in 1996 and managed to co-circulate with WNV in many European countries in a similar mosquito-bird life cycle. The rapid geographic spread of USUV, the seasonal mass mortalities it causes in the European avifauna, and the increasing number of infections with neurological disease both in healthy and immunocompromised humans has stimulated interest in infection studies to delineate USUV pathogenesis. Here, we assessed the pathogenicity of two USUV isolates from a recent Belgian outbreak in immunocompetent mice. The intradermal injection of USUV gave rise to disorientation and paraplegia and was associated with neuronal death in the brain and spinal cord in a single mouse. Intranasal inoculation of USUV could also establish the infection; viral RNA was detected in the brain 15 days post-infection. Overall, this pilot study probes the suitability of this murine model for the study of USUV neuroinvasiveness and the possibility of direct transmission in mammals

Genital re-excretion of Murid gammaherpesvirus 4 following intranasal infection

Gammaherpesviruses are the archetypes of persistent viruses that have been identified in a range of animals from mice to man. As the human gammaviruses have no well-established in vivo infection model, related animal gammaherpesviruses are an important source of information. We are studying Murid herpesvirus 4 (MuHV-4) in inbred laboratory mouse strains which are commonly accepted as a good model for studying gammaherpesviruses in vivo. To date, it has however never been possible to monitor viral reexcretion and virus transmission in this species. In order to identify potential re-excretion sites, intranasally infected mice were followed through global luciferase imaging for up to six months after infection. Surprisingly, we detected transient viral replication in mice genital tract at various times after latency establishment. Ex vivo imaging, quantitative PCR and immunohistochemistry revealed that virus genomes were present in high quantity in the vaginal tissue and that viral replication occurred mainly at the vaginal external border. Moreover, we highlighted the presence of free infectious viruses in the vaginal cavity at the moment of the observation of viral replication. As this ephemeral viral reexcretion could reveal a link with reproductive cycle, we compared reexcretion in normal and ovariectomized mice. Interestingly, no viral reactivation was observed in absence of hormonal cycle. In conclusion, we experimentally indentified for the first time a reexcretion site for MuHV-4 in mice that had been intranasaly infected. In the future, these results could help us to better understand the biology of gammaherpesviruses but should also allow us to develop strategies that could prevent the spread of these viruses in natural populations

Morbidity and respiratory pattern values.

<p>(A) Evolution of daily body weight within each generation from PVM inoculation to 7 days later (% of the pre-inoculation value, mean ± SEM). Means significantly different from the corresponding pre-inoculation values (on day 0) are indicated with a (p<0.05), b (p<0.01), or c (p<0.001). Significant day-to-day changes after day 5 pi are indicated also (bottom-right). (B) Respiratory dysfunctions were measured using a double chamber plethysmograph. Before and at selected time points after intranasal inoculation of PVM, minute ventilation (MV), expiratory balance (EB), respiratory rate (RR) and tidal volume (TV) were determined within each generation (mean ± SEM). Means significantly different from the corresponding pre-inoculation values (on day 0) are indicated with a (p<0.05), b (p<0.01), or c (p<0.001). Significant day-to-day changes after day 5 pi are indicated also (bottom-right).</p

Observed means with standard deviations, and predicted means of most pertinent PVM-susceptibility traits.

<p>LVL^d7, day 7 pi lung viral load (log₁₀ PFU/g); ΔBW^d0–d7, body weight loss as measured 7 days pi (%); ΔEB^d6–d7, change in expiratory balance between days 6 and 7 pi (%).</p><p>P1, PVM-resistant parental strain (SJL/J) and P2, PVM-susceptible parental strain (129/Sv). Predicted means were calculated by the joint scaling test. Obs., observed.</p