Programmed cell death-associated gene transcripts in bovine embryos exposed to bovine Herpesvirus type 5.

In vitro-produced bovine embryos become infected after exposure to bovine Herpesvirus type 5 (BoHV-5), yet no changes in developmental rates, mitochondrial activity and inhibition of apoptosis are detected in comparison to unexposed embryos. Thus, the aim of the present study was to assess the transcription of mitochondria-mediated apoptosis genes using TaqMan real-time polymerase chain reaction. Transcripts of mcl-1, caspase-2, -3, Apaf-1 and Bax genes were measured after exposure to BoHV-5 in vitro. Mitochondrial dehydrogenase activity was evaluated by MTT test and compared between groups of exposed and unexposed embryos, at day 7 of development. The rate of oocyte maturation was assessed by the extrusion of the first polar body. In summary, BoHV-5 exposed embryos retained their viability, mitochondrial dehydrogenase activity and displayed up-regulation of transcription of survival mcl-1 gene and down-regulation of Bax transcription in relation to mitochondria-mediated pathway which might improve embryo viability. These findings demonstrate that BoHV-5 exposed embryos maintain their viability and mitochondrial dehydrogenase activity with no compromise of embryos produced in vitro.

Evaluation of developmental changes in bovine in vitro produced embryos following exposure to bovine Herpesvirus type 5.

BACKGROUND: Bovine Herpesvirus type-5 (BoHV-5) is a neurovirulent α-Herpesvirus which is potentially pathogenic for cows and suspected to be associated with reproductive disorders. Interestingly, natural transmission of BoHV-5 by contaminated semen was recently described in Australia. Additionally, BoHV-5 was also isolated from the semen of a healthy bull in the same country and incriminated in a natural outbreak of reproductive disease after artificial insemination. In contrast with BoHV-1, experimental exposure of in vitro produced bovine embryos to BoHV-5 does not affect embryo viability and seems to inhibit some pathways of apoptosis. However, the mechanisms responsible for these phenomena are poorly understood. In this study, we examined mitochondrial activity, antioxidant protection, stress response and developmental rates of in vitro produced bovine embryos that were exposed and unexposed to BoHV-5. METHODS: For this purpose, bovine embryos produced in vitro were assayed for cell markers after experimental infection of oocytes (n = 30; five repetitions), in vitro fertilization and development. The indirect immunofluorescence was employed to measure the expression of superoxide dismutase 1 (SOD1), anti-oxidant like protein 1 (AOP-1), heat shock protein 70.1 (Hsp 70.1) and also viral antigens in embryos derived from BoHV-5 exposed and unexposed oocytes. The determination of gene transcripts of mitochondrial activity (SOD1), antioxidant protection (AOP-1) and stress response (Hsp70.1) were evaluated using the reverse transcriptase polymerase chain reaction (RT-PCR). MitoTracker Green FM, JC-1 and Hoechst 33342-staining were used to evaluate mitochondrial distribution, segregation patterns and embryos morphology. The intensity of labeling was graded semi-quantitatively and embryos considered intensively marked were used for statistical analysis. RESULTS: The quality of the produced embryos was not affected by exposure to BoHV-5. Of the 357 collected oocytes, 313 (+/- 6.5; 87.7%) were cleaved and 195 (+/- 3.2; 54.6%) blastocysts were produced without virus exposure. After exposure, 388 oocytes were cleaved into 328 (+/- 8.9, 84.5%), and these embryos produced 193 (+/- 3.2, 49.7%) blastocysts. Viral DNA corresponding to the US9 gene was only detected in embryos at day 7 after in vitro culture, and confirmed by indirect immunofluorescence assay (IFA). These results revealed significant differences (p < 0.05) between exposed and unexposed oocytes fertilized, as MitoTracker Green FM staining Fluorescence intensity of Jc-1 staining was significantly higher (p < 0.005) among exposed embryos (143 +/- 8.2). There was no significant difference between the ratios of Hoechst 33342-stained nuclei and total cells in good-quality blastocysts (in both the exposed and unexposed groups). Using IFA and reverse transcriptase polymerase chain reaction (RT-PCR) for the set of target transcripts (SOD1, AOP-1 and Hsp 70.1), there were differences in the mRNA and respective proteins between the control and exposed embryos. Only the exposed embryos produced anti-oxidant protein-like 1 (AOP-1). However, neither the control nor the exposed embryos produced the heat shock protein Hsp 70.1. Interestingly, both the control and the exposed embryos produced superoxide dismutase (SOD1), revealing intense mitochondrial activity. CONCLUSION: This is the first demonstration of SOD1 and AOP-1 production in bovine embryos exposed to BoHV-5. Intense mitochondrial activity was also observed during infection, and this occurred without interfering with the quality or number of produced embryos. These findings further our understanding on the ability of α-Herpesviruses to prevent apoptosis by modulating mitochondrial pathways.

Viral infections during pregnancy.

Viral infections during pregnancy have long been considered benign conditions with a few notable exceptions, such as herpes virus. The recent Ebola outbreak and other viral epidemics and pandemics show how pregnant women suffer worse outcomes (such as preterm labor and adverse fetal outcomes) than the general population and non-pregnant women. New knowledge about the ways the maternal-fetal interface and placenta interact with the maternal immune system may explain these findings. Once thought to be 'immunosuppressed', the pregnant woman actually undergoes an immunological transformation, where the immune system is necessary to promote and support the pregnancy and growing fetus. When this protection is breached, as in a viral infection, this security is weakened and infection with other microorganisms can then propagate and lead to outcomes, such as preterm labor. In this manuscript, we review the major viral infections relevant to pregnancy and offer potential mechanisms for the associated adverse pregnancy outcomes.

Pathogenicity of infectious laryngotracheitis virus as measured by chicken embryo inoculation.

A comparison was made between pathogenicities for chicken embryos of unattenuated and attenuated strains or isolates of infectious laryngotracheitis (ILT) virus. All 11-day-old chicken embryos inoculated with 10(3.0) or 10(4.0) TCID50 of unattenuated strain NS175 via allantoic cavity died within 6 days. On the contrary, no chicken embryos of the same age died when inoculated with the same amount of cell-culture-attenuated isolate C7 in a like manner. The mortality index for chicken embryos (MICE) was obtained by dividing the cumulative number of embryos dying within 7 days by the cumulative number of embryos surviving 7 days. The reliability of the MICE test was confirmed by duplicate and triplicate experiments with strain NS175 and isolate C7. MICE obtained in the experiments with 9 different strains or isolates of ILT virus ranged from 0 to 1, and the values were well correlated with the pathogenicities for chickens. The results from the present work suggest that strains or isolates with MICE less than 0.16 would have low or no pathogenicity for chickens, and those with MICE more than 0.27 would be highly pathogenic. Further studies are needed using additional isolates of ILT virus with varied pathogenicities.

EHV-1-induced abortion in mice and its relationship to stage of gestation.

The most important consequence of equine herpesvirus-1 (EHV-1) infection is abortion. The object of the present study was to characteristic further a murine EHV-1 abortion model and to make comparisons with the natural host with particular reference to the stage of gestation during which the infection occurs. BALB/c mice at different stages of pregnancy were infected intranasally with EHV-1 (strain AB4); they suffered respiratory distress, weight loss, and other constitutional signs of infection. When the virus was inoculated in the late second or early third week of gestation dead or dying fetuses were aborted, whereas infection between seven and nine days of pregnancy led to fetal death and resorption. During the process of resorption, complications were observed. Virus was frequently isolated from the placentas and occasionally from the tissues of the aborting fetuses, depending on the severity of the infection of the placentas. In some cases, therefore, the inoculation resulted in abortion although the infection was restricted to the placenta. Virus antigen was detected in the placentas, lungs and occasionally in other tissues of the aborting fetuses. The potential of this murine model for testing methods for the diagnosis and control of equine abortion is discussed.

Use of transabdominal ultrasound-guided amniocentesis for detection of equid herpesvirus 1-induced fetal infection in utero.

OBJECTIVE: To evaluate transabdominal ultrasound-guided amniocentesis for detection of equid herpes-virus 1 (EHV-1)-induced fetal infection in utero. ANIMALS: 4 Welsh Mountain mares. PROCEDURE: Pregnant mares were inoculated intranasally with EHV-1 during the ninth month of gestation. Amniocentesis was initiated on postinoculation day (PID) 12, and was performed at 2- to 3-day intervals in standing mares under deep sedation. Amniotic fluid samples were tested by virus isolation (VI), polymerase chain reaction (PCR), and immunoperoxidase cytologic examination (IC) for detection of EHV-1. RESULTS: Exposure to EHV-1 in the ninth month of gestation resulted in nasal shedding of infective virus, establishment of cell-associated viremia, and seroconversion. Equid herpesvirus 1 was detected by VI, PCR, and IC in amniotic fluid collected on PID 14 from 1 mare and on PID 16 and 17 from a second mare. Specimens of amniotic fluid from a third mare were VI negative until PID 18, when collections ceased, although this mare subsequently aborted an EHV-1-infected fetus on PID 28. The fourth mare aborted an EHV-1 infected fetus on PID 14. The 2 mares with VI-positive amniotic fluid were each carrying an EHV-1 infected fetus in utero, confirmed by examination of the uterus, placenta, and fetus, using specific immunohistochemistry and in situ hybridization. Endothelial cells in the endometrium and allantochorion were often virus-infected, with accompanying vascular lesions. The fetus had been infected via the chorionic vasculature in the first and fourth mares, and by inhalation of infected amniotic fluid in the second mare. CONCLUSION: Amniocentesis permits specific detection of EHV-1-induced fetal infection in utero. CLINICAL RELEVANCE: Amniocentesis may have a clinical role in the specific identification and isolation of mares carrying virus-infected fetuses during EHV-1-induced abortion epizootics.

[Arguments for an infectious cause of IUGR]. / Quels arguments pour déterminer l'origine infectieuse d'un retard de croissance intra-utérin?

Intra-uterine growth retardation (IUGR) is a frequent cause of consultation in antenatal care unit. The prognosis relies on the etiology: vascular, chromosomic, genetic, or infectious. Because of chronic fetal distress, hypotrophy increase morbidity, mortality and neurosensorial long term effect. Usually, infection is involved in 5 to 15% of the IUGR, mainly by Cytomegalovirus (CMV), Varicella Zoster virus, rubella, toxoplasmosis, herpes and syphilis. Maternal sera and amniotic liquid analysis make the diagnosis possible but fetal ultrasound scan is used to find other features. Most of the abnormalities are unspecific but their combination can worsen fetal prognosis. Infection should always be ruled out in the assessment of IUGR.

Developmental disorders of the mouse brain induced by murine cytomegalovirus: animal models for congenital cytomegalovirus infection.

Developmental disorders induced by congenital cytomegalovirus (CMV) infection mainly involve the central nervous system. The type and degree of the brain disorders seems to depend on infection time during gestation, virulence, route of infection and viral susceptible cells in each embryonal stage. Since transplacental transmission has been reported not to occur with murine CMV (MCMV), we developed mouse models for congenital CMV infection by surgical injection of MCMV into the mouse conceptus or embryo at different gestational stages. For the early stage, the mouse embryos were not infected with MCMV even after injecting the virus into the blastocysts, which were developed in the pseudo-pregnant mothers or cultured in vitro. Isolated whole mouse embryos of day 7.5 of gestation (E7.5), adsorbed with a high titer of MCMV and cultured for 3 days, were susceptible to MCMV infection. Therefore, the mouse embryo acquires the susceptibility around this period. Microphthalmia and cerebral atrophy were induced in mouse embryos after injection of MCMV into the conceptus on E8.5. Viral antigen-positive cells were widely distributed in the mesenchyme around the oral and nasal cavities and in the mesenchyme around the brain, especially the endothelial cells of vessels and the perivascular mesodermal cells, then infection extends to the eyes, brain or choroid plexus. This finding suggests that mesenchymal infection may be the critical step in disrupting organogenesis, resulting in brain disorders. For the late stage, mouse embryos were infected with MCMV by injecting the virus into the cerebral ventricles on E15.5. Brains of the offspring showed massive necrosis with gliomesodermal proliferation in the cerebral cortex. Viral antigen-positive cells were observed in laminar array in the lesion-free cortex and the hippocampus, suggesting that the infected cells migrate in association with the lamina formation. Immunohistochemical double-staining showed that brain cells susceptible to MCMV infection may be mainly neuronal and endothelial cells, resulting in cerebral atrophy with reduction of neuronal cells and cystic lesions, presumably due to ischemic vascular changes.

A polymerase chain reaction for detection of equine herpesvirus-1 in routine diagnostic submissions of tissues from aborted foetuses.

Equine herpesvirus 1 (EHV-1) is the causative agent of abortion, perinatal foal mortality, neurological and acute respiratory diseases in horses. Conventional laboratory diagnosis involving viral isolation from aborted foetuses is laborious and lengthy and requires processing of samples within 24 h of collection, which is problematic for samples that come from long distances. The aim of this study was to develop a polymerase chain reaction (PCR) assay useful in Argentina to detect DNA sequences of EHV-1 in different tissues from aborted equine foetuses with variable quality of preservation and without the use of conventional DNA fenolic extraction. Several DNA extraction protocols and primers were evaluated. The amplification method was standardized and its specificity was analysed using 38 foetal samples of variable quality of preservation. Of the 38 different foetal tissues, nine livers, six spleens and two lungs in good preservation and eight livers, one spleen and four lungs in a poor state of preservation were positive for PCR. EHV-1 was recovered only from the nine livers, five spleens and two lungs in good preservation. No virus was isolated from the samples that were poorly preserved. Viral isolation was confirmed by cytopathic effect and indirect immunofluorescence. The specificity of the PCR results was confirmed by the restriction endonuclease digestion of PCR products and hybridization.

Reactivation of and primary infection with human herpesvirus 8 among solid-organ transplant recipients.

Human herpesvirus 8 (HHV-8) is the causal agent of all forms of Kaposi's sarcoma, including the iatrogenic form that presents in solid-organ transplant recipients. A longitudinal study of HHV-8 seropositivity was conducted among a cohort consisting of children and adult solid-organ transplant recipients. Antibodies to HHV-8 lytic proteins were detected by an indirect immunofluorescence assay in serum samples of 100 transplant recipients. HHV-8 seropositivity increased significantly, from 5.3% before transplantation to 15.8% after transplantation (P<.01). Seropositivity was not related to the age of the patient or the type of organ transplanted. HHV-8 seroconversion occurred in both children and adult recipients. None of the seroconversion events was related to the source of the donor organ. These findings suggest that HHV-8 infection is not uncommon among both adult and children transplant recipients and that viral infection may be acquired from an outside source other than the transplanted organ.

Secuelas oculares en recién nacidos son síndrome de TORCH / Ocular sequelae in newborns with TORCH syndrome

Se evaluó la incidencia de alteraciones oculares en infantes nacidos de madres con titulación positiva contra el complejo TORCH y la infección por sífilis. Incluimos a 33 pacientes, captados en el periodo de septiembre de 1997 a junio de 1998. Se valoró la presencia de alteraciones oculares congénitas en infantes con antecedentes de infección intrauterina por el síndrome de TORCH. Se incluyeron 8 mujeres (24.2 por ciento) y 25 hombres (75.7 por ciento). Las lesione que más se encontró fue: catarata en 6 pacientes (18.1 por ciento), coloboma en 2 pacientes (6.0 por ciento), microftalmía, entropión, queratoconjuntivitis, masa retrobulbar, desprendimiento de retina, coriorretinitis y atrofia óptica en los restantes pacientes (3.0 por ciento). Las infecciones intrauterinas influyen en el dessarrollo del ojo. Estos datos sugieren que el diagnóstico temprano de tales infecciones por el complejo TORCH permite realizar el tratamiento adecuado