Effect of pregnancy on stimulation of alphavirus immunity in mice

The state of pregnancy was associated with a marked enhancement of both proliferative and cytotoxic T cell antiviral immune responses after infection of mice with avirulent Semliki Forest virus. Strong responses were obtained from the spleen and the para-aortic lymph nodes that drain the uterus. This immune enhancement seemed to be in response to the increased antigenic challenge that originated from infected placental and fetal tissue and was released during the process of abortion. Immune enhancement was not observed in pregnant mice similarly infected with Ross River virus, which also established an intrauterine infection but does not cause abortion.     

Pregnancy and viral infections: Mechanisms of fetal damage,diagnosis and prevention of neonatal adverse outcomes from cytomegalovirus to SARS-CoV-2 and Zika virus

Some maternal infections, contracted before or during pregnancy, can be transmitted to the fetus, during gestation (congenital infection), during labor and childbirth (perinatal infection) and through breastfeeding (postnatal infection). The agents responsible for these infections can be viruses, bacteria, protozoa, fungi. Among the viruses most frequently responsible for congenital infections are Cytomegalovirus (CMV), Herpes simplex 1–2, Herpes virus 6, Varicella zoster. Moreover Hepatitis B and C virus, HIV, Parvovirus B19 and non-polio Enteroviruses when contracted during pregnancy may involve the fetus or newborn at birth. Recently, new viruses have emerged, SARS-Cov-2 and Zika virus, of which we do not yet fully know the characteristics and pathogenic power when contracted during pregnancy.Viral infections in pregnancy can damage the fetus (spontaneous abortion, fetal death, intrauterine growth retardation) or the newborn (congenital anomalies, organ diseases with sequelae of different severity). Some risk factors specifically influence the incidence of transmission to the fetus: the timing of the infection in pregnancy, the order of the infection, primary or reinfection or chronic, the duration of membrane rupture, type of delivery, socio-economic conditions and breastfeeding. Frequently infected neonates, symptomatic at birth, have worse outcomes than asymptomatic. Many asymptomatic babies develop long term neurosensory outcomes.The way in which the virus interacts with the maternal immune system, the maternal-fetal interface and the placenta explain these results and also the differences that are observed from time to time in the fetal?neonatal outcomes of maternal infections. The maternal immune system undergoes functional adaptation during pregnancy, once thought as physiological immunosuppression. This adaptation, crucial for generating a balance between maternal immunity and fetus, is necessary to promote and support the pregnancy itself and the growth of the fetus. When this adaptation is upset by the viral infection, the balance is broken, and the infection can spread and lead to the adverse outcomes previously described. In this review we will describe the main viral harmful infections in pregnancy and the potential mechanisms of the damages on the fetus and newborn.     

Effect of infection with the ts22 mutant of Semliki Forest virus on development of the central nervous system in the fetal mouse.

The A7 strain of Semliki Forest virus induces rapid fetal death in pregnant mice, whereas the ts22 mutant derived from it is teratogenic for a proportion of fetuses. Both A7 and ts22 induce viremia and infect the central nervous systems and fetuses of pregnant mice. Using immunogold-silver staining, a cDNA probe for a Semliki Forest virus nonstructural sequence, and a riboprobe derived from the same sequence, we showed that the skin and musculoskeletal systems of fetuses from mothers infected with ts22 were often heavily infected but the central nervous systems were not labeled before day 17 of pregnancy. Damage to the neural tube, including open-neural-tube defects, was detected in fetuses following infection of the mother at days 8 and 10 of pregnancy with both A7 and ts22. For ts22, neural tube damage induced by fetal infection before day 17 of pregnancy appeared to be indirect and caused by virus infection of mesenchymal cells surrounding the developing neural tube.     

The placenta as a site of cytomegalovirus infection in guinea pigs.

The development of cytomegalovirus (CMV) infection in the placenta was studied in Hartley guinea pigs inoculated at midgestation, and its role in determining the outcome of fetal CMV infection was assessed. A hematogenous spread of CMV from the mother to the placenta occurred early during the course of the infection. However, the virus remained present in placental tissues long after CMV had been cleared from maternal blood (i.e., 3 and 4 weeks postinoculation). At that time, the virus was able to replicate in placental tissues in the presence of specific maternal antibodies. Viral nucleocapsids were seen within nuclei of trophoblastic cells, and virions were present surrounding infected cells. In addition, typical CMV-induced histopathological lesions bearing CMV antigens were consistently localized at the transitional zone between the capillarized labyrinth and the noncapillarized interlobium. Whenever CMV infection of the fetus occurred, virus was isolated from the associated placenta. Among placental-fetal units with CMV-infected placentas, only 27% of the fetuses were found to be infected. In addition, there was a delay in the establishment of the infection in the fetus in relation to the placenta, although frequencies of virus isolation in placental and fetal tissues peaked at 3 weeks after CMV inoculation. These results suggest that during primary CMV infection of pregnant guinea pigs, the placenta not only serves as a reservoir for CMV but also acts to limit transmission of the virus to the fetus.     

Concentrations of progesterone in arterial and venous plasma of fetal pigs and their dams in late gestation

Blood samples were drawn from uterine arteries and veins of pregnant gilts and from the umbilical artery and vein of each of their fetuses during laparotomy at Day 80. Concentrations of progesterone (P) were greater in fetal than maternal plasma. Uptake of P from the placenta by the fetal blood was evident but was not equivalent to the maternal uterine arterial-venous difference in P concentration. No correlation between plasma P and fetal weight was noted. Concentrations of P in both umbilical vessels of female fetuses were higher than in male fetuses. These data indicate that fetal sex affects the rate of transport and/or synthesis of P in the utero/placental compartment and/or the rate of metabolism of P in the fetus. The relative importance of de novo synthesis and transplacental transport of P in establishing concentrations of P in fetal blood remains to be elucidated.     

Development of a chronically catheterized maternal-fetal macaque model to study in utero mother-to-fetus HIV transmission: A preliminary report

Abstract: The lack of a representative animal model that permits frequent in utero fetal blood sampling is a major limiting factor for the study of maternal-fetal HIV transmission. Therefore, we have developed a maternal-fetal virus infection model using chronically catheterized macaques to simultaneously study the time-course of viral infection in the mother and the response of the fetus to maternal HIV infection. Pregnant macaques were infected with 10³ infectious units of HIV-2₂₈₇; every 3 days blood samples from both the mother and the fetus as well as amniotic fluid samples were collected. We found a varying degree of peak and time-to-peak virus load, virus-infected PBMCs, and free virus (determined by QC-RNA-PCR method) in maternal blood. Two of the three mothers with more than 10⁸ copies of viral RNA/ml of plasma at peak viremia transmitted the virus to their fetuses at about 14 days post-infection. As observed with HIV-2₂₈₇ infected mothers, virus-infected fetuses also produced a rapid rate of CD4⁺ cell decline in utero.     

Ultrastructural evidence of transplacental transport of immunoglobulin G in bitches

In dogs, passive immunity is conferred to fetuses and neonates by the transfer of maternal immunoglobulin G through the placenta during the last trimester of pregnancy and via the mammary gland after parturition, respectively. However, morphological evidence of transplacental transport is still lacking. The aim of the present study was to localize maternal immunoglobulin G in the labyrinthine zone and in the haemophagous zone of the canine placenta by means of immunohistochemistry and immunocytochemistry. In the labyrinthine zone, immunoglobulin G was detected in all the layers of the materno-fetal barrier including the fetal capillaries. Immunoreactivity was particularly prominent in maternal basement membrane material as well as in the syncytiotrophoblast. However, this evidence of transplacental transport of immunoglobulin G originated from a limited number of unevenly distributed maternal vessels only. In the cytotrophoblast of the haemophagous zone, immunoglobulin G was localized to phagolysosomes at various stages but was never detected within fetal vessels. The results indicate that maternal immunoglobulin G is degraded in cytotrophoblast cells of the hemophagous zone and, therefore, that transplacental transport is restricted to a subpopulation of maternal vessels in the labyrinthine zone.     

Fetal immunization of baboons induces a fetal-specific antibody response

Neonates face a high risk of infection because of the immaturity of their immune systems. Although the transplacental transfer of maternal antibodies to the fetus may convey improved postnatal immunity, this transfer occurs late in gestation and may fail to prevent in utero infection. Both fetal immunization and in utero exposure to antigen can result in a state of immunologic tolerance in the neonate. Tolerance induction of fetal and premature infant lymphocytes has become a paradigm for neonatal responsiveness. However, fetal IgM responses have been demonstrated to maternal immunization with tetanus toxoid and to congenital infections such as rubella, toxoplasma, cytomegalovirus and human immunodeficiency virus. Moreover, 1-week-old infants can respond to standard pediatric vaccination, and neonates immunized with polysaccharide antigens do not develop immunologic tolerance. Here, direct immunization of the baboon fetus with recombinant hepatitis B surface antigen produced a specific fetal IgG antibody response. No specific maternal antibody response was detected, eliminating the possibility of vertical antibody transmission to the fetus. Some infants also responded to later vaccinations with hepatitis B surface antigen, indicating that no immunological tolerance was induced by prior fetal immunization. These results characterize the ability of the fetal immune system to respond to in utero vaccination. We demonstrate that active fetal immunization can serve as a safe and efficient vaccination strategy for the fetus and neonate.     

Adrenal glands are essential for activation of glucogenesis during undernutrition in fetal sheep near term

In adults, the adrenal glands are essential for the metabolic response to stress, but little is known about their role in fetal metabolism. This study examined the effects of adrenalectomizing fetal sheep on glucose and oxygen metabolism in utero in fed conditions and after maternal fasting for 48 h near term. Fetal adrenalectomy (AX) had little effect on the rates of glucose and oxygen metabolism by the fetus or uteroplacental tissues in fed conditions. Endogenous glucose production was negligible in both AX and intact, sham-operated fetuses in fed conditions. Maternal fasting reduced fetal glucose levels and umbilical glucose uptake in both groups of fetuses to a similar extent but activated glucose production only in the intact fetuses. The lack of fasting-induced glucogenesis in AX fetuses was accompanied by falls in fetal glucose utilization and oxygen consumption not seen in intact controls. The circulating concentrations of cortisol and total catecholamines, and the hepatic glycogen content and activities of key gluconeogenic enzymes, were also less in AX than intact fetuses in fasted animals. Insulin concentrations were also lower in AX than intact fetuses in both nutritional states. Maternal glucose utilization and its distribution between the fetal, uteroplacental, and nonuterine maternal tissues were unaffected by fetal AX in both nutritional states. Ovine fetal adrenal glands, therefore, have little effect on basal rates of fetal glucose and oxygen metabolism but are essential for activating fetal glucogenesis in response to maternal fasting. They may also be involved in regulating insulin sensitivity in utero.     

Sexually dimorphic effects of maternal asthma during pregnancy on placental glucocorticoid metabolism and fetal growth

Human pregnancy is associated with sexually dimorphic differences in mortality and morbidity of the fetus with the male fetus experiencing the poorest outcome following complications such as pre-eclampsia, pre-term delivery and infection. The physiological mechanisms that confer these differences have not been well characterised in the human. Work conducted on the effect of maternal asthma during pregnancy, combining data collected from the mother, placenta and fetus has found some significant sex-related mechanistic differences associated with fetal growth in both normal pregnancies and pregnancies complicated by asthma. Specifically, sexually dimorphic differences have been found in placental glucocorticoid metabolism in male and female fetuses of normal pregnancies. In response to the presence of maternal asthma, only the female fetus alters placental glucocorticoid metabolism resulting in decreased growth. The male fetus does not alter placental function or growth in response to maternal asthma. As a result of the alterations in glucocorticoid metabolism in the female, downstream changes occur in pathways regulated by glucocorticoids. These data suggest that the female fetus adjusts placental function and reduces growth to compensate for maternal disease. However, the male fetus continues to grow in response to maternal asthma with no changes in placental function. This response by the male fetus may partially contribute to the increased risk of morbidity and mortality in this sex.     

A novel approach to intrauterine viral inoculation of swine using PCV type 2 as a model

Experimentally intrauterine (IU) viral inoculation has been commonly used to circumvent maternal interference with transplacental infection of fetuses and to assess the effect of viral infection on fetal development or reproductive parameters. However, IU inoculation requires surgical procedures such as laparatomy and surgical incision of the uterus. Post-surgical complications, that frequently result in abortion or fetal death, have been a major disadvantage. An animal trial was conducted to evaluate the non-surgical procedure of ultrasound needle-guided transabdominal injection for IU inoculation of porcine circovirus type 2 (PCV2) since this virus has been reported to cause reproductive failure in pigs. Two groups of seven pregnant sows at mid- and late-gestation, respectively, were inoculated with PCV2 using an ultrasound needle-guided technique that delivered PCV2 directly into one of the fluid-filled fetal compartments. The effect of transabdominal in utero virus challenge on fetuses and sows was assessed until term. While five of six sham-inoculated control sows had no or minimal adverse affects from in utero injection, 10 of 14 virus-inoculated sows had dead and/or stillborn piglets and PCV2 infection was evident by polymerase chain reaction and/or immunohistochemistry. These results supported previous field and experimental observations that PCV2 may cause reproductive failure. In conclusion, ultrasound needle-guided transabdominal injection was a safe and efficient method for IU inoculation of virus in pigs.     

Differential transmission of parvovirus B19 in a twin gestation: a case report.

Maternal infection with parvovirus B19 during pregnancy can cause aplastic anemia in the fetus. Severe anemia may lead to nonimmune hydrops or fetal demise. In the case reported, the demise of one twin was diagnosed by ultrasonography in an asymptomatic 21-year-old para 1-0-2-1 African American at the gestational age of 25 weeks. The deceased twin (A) was grossly hydropic with anasarca, ascites, pleural and pericardial effusions, and a thickened placenta. Parvovirus B19 DNA was found in the amniotic fluid of Twin A using the polymerase chain-reaction technique. Serial scans of Twin B showed normal growth and no evidence of hydrops. The pregnancy was managed expectantly until 29 weeks when delivery was indicated by maternal disseminated intravascular coagulation. Maternal IgM antiparvovirus B19 antibodies were detected at the time of delivery. Antiparvovirus B19 IgM antibodies were not present in Twin B. These serologic studies suggest a recent acute maternal infection and refute such an infection in Twin B. We present a case of differential transmission of parvovirus B19 in a twin pregnancy with in utero death of the infected twin and subsequent maternal disseminated intravascular coagulation.     

Nrk, an X-linked protein kinase in the germinal center kinase family, is required for placental development and fetoplacental induction of labor

The complete mechanism of labor induction in eutherian mammals remains unclear. Although important roles for the fetus and placenta in triggering labor have been proposed, no gene has been shown to be required in the fetus/placenta for labor induction. Here we show that Nrk, an X-linked gene encoding a Ser/Thr kinase of the germinal center kinase family, is essential in the fetus/placenta for labor in mice. Nrk was specifically expressed in the spongiotrophoblast layer, a fetus-derived region of the placenta, and Nrk disruption caused dysregulated overgrowth of the layer. Due to preferential inactivation of the paternally derived X chromosome in placenta, Nrk heterozygous mutant placentas exhibited a similar defect to that in Nrk-null tissues when the wild-type allele was paternally derived. However, the phenotype was weaker than in Nrk-null placentas due to leaky Nrk expression from the inactivated X chromosome. Crossing of Nrk-null females to wild-type and Nrk-null males, as well as uterine transfer of Nrk-null fetuses to wild-type females, revealed that pregnant mice exhibit a severe defect in delivery when all fetuses/placentas are Nrk-null. In addition, Nrk was not expressed in female reproductive tissues such as the uterus and ovary, as well as the fetal amnion and yolk sac, in pregnant mice. Progesterone and estrogen levels in the maternal circulation and placenta, which control the timing of labor, were unaffected upon Nrk disruption. We thus provide evidence for a novel labor-inducing fetoplacental signal that depends on the X chromosome and possibly arises from the placenta.     

Aura virus structure suggests that the T=4 organization is a fundamental property of viral structural proteins

Aura and Sindbis viruses are closely related alphaviruses. Unlike other alphaviruses, Aura virus efficiently encapsidates both genomic RNA (11.8 kb) and subgenomic RNA (4.2 kb) to form virus particles. Previous studies on negatively stained Aura virus particles predicted that there were two major size classes with potential T=3 and T=4 capsid structures. We have used cryoelectron microscopy and three-dimensional image reconstruction techniques to examine the native morphology of different classes of Aura virus particles produced in BHK cells. Purified particles separated into two components in a sucrose gradient. Reconstructions of particles in the top and bottom components were computed to resolutions of 17 and 21 A, respectively, and compared with reconstructions of Sindbis virus and Ross River virus particles. Aura virus particles of both top and bottom components have similar, T=4 structures that resemble those of other alphaviruses. The morphology of Aura virus glycoprotein spikes closely resembles that of Sindbis virus spikes and is detectably different from that of Ross River virus spikes. Thus, some aspects of the surface structure of members of the Sindbis virus lineage have been conserved, but other aspects have diverged from the Semliki Forest/Ross River virus lineage.     

Multiple binding sites for cellular proteins in the 3' end of Sindbis alphavirus minus-sense RNA.

The 3' end of Sindbis virus minus-sense RNA was tested for its ability to bind proteins in mosquito cell extracts, using labeled riboprobes that represented different parts of this region. We found four domains in the first 250 nucleotides that could bind the same 50- and 52-kDa proteins, three with high affinity and one with low affinity, whereas tested domains outside this region did not bind these proteins. The first binding domain was found in the first 60 nucleotides, which represents the complement of the 5'-nontranslated region, the second in the next 60 nucleotides, the third in the following 60 nucleotides, and the fourth between nucleotides 194 and 249 (all numbering is 3' to 5'). The relative binding constants, Kr, of the first, second, and fourth sites were similar, whereas that of domain 2 was fivefold less. Deletion mapping of the first domain showed that the first 10 nucleotides were critical for binding. Deletion of nucleotides 2 to 4, deletion or replacement of nucleotide 5, or deletion of the first 15 nucleotides was deleterious for binding, deletion of nucleotides 10 to 15, 26 to 40, or 41 to 55 had little effect on the binding, and deletion of nucleotides 15 to to 25 increased the binding affinity. We also found that the corresponding riboprobes derived from two other alphaviruses, Ross River virus and Semliki Forest virus, and from rubella virus were also able to interact with the 50- and 52-kDa proteins. The Kr value for the Semliki Forest virus probe was similar to that for the Sindbis virus probe, while that for the Ross River virus probe was four times greater. The rubella virus probe was bound only weakly, consistent with the fact that mosquito cells are not permissive for rubella virus replication. We suggest that the binding of the 50- and 52-kDa proteins to the 3' end of alphavirus minus-sense RNA represents an important step in the initiation of RNA replication.     

Superinfection exclusion of alphaviruses in three mosquito cell lines persistently infected with Sindbis virus.

Three Aedes albopictus (mosquito) cell lines persistently infected with Sindbis virus excluded the replication of both homologous (various strains of Sindbis) and heterologous (Aura, Semliki Forest, and Ross River) alphaviruses. In contrast, an unrelated flavivirus, yellow fever virus, replicated equally well in uninfected and persistently infected cells of each line. Sindbis virus and Semliki Forest virus are among the most distantly related alphaviruses, and our results thus indicate that mosquito cells persistently infected with Sindbis virus are broadly able to exclude other alphaviruses but that exclusion is restricted to members of the alphavirus genus. Superinfection exclusion occurred to the same extent in three biologically distinct cell clones, indicating that the expression of superinfection exclusion is conserved among A. albopictus cell types. Superinfection of persistently infected C7-10 cells, which show a severe cytopathic effect during primary Sindbis virus infection, by homologous virus does not produce cytopathology, consistent with the idea that cytopathology requires significant levels of viral replication. A possible model for the molecular basis of superinfection exclusion, which suggests a central role for the alphavirus trans-acting protease that processes the nonstructural proteins, is discussed in light of these results.     

Venezuelan equine encephalomyelitis virus structure and its divergence from old world alphaviruses

Although alphaviruses have been extensively studied as model systems for the structural organization of enveloped viruses, no structures exist for the phylogenetically distinct eastern equine encephalomyelitis (EEE)-Venezuelan equine encephalomyelitis (VEE) lineage of New World alphaviruses. Here we report the 25-A structure of VEE virus, obtained from electron cryomicroscopy and image reconstruction. The envelope spike glycoproteins of VEE virus have a T=4 icosahedral arrangement, similar to that observed in Old World Sindbis, Semliki Forest, and Ross River alphaviruses. However, VEE virus has pronounced differences in its nucleocapsid structure relative to nucleocapsid structures repeatedly observed in Old World alphaviruses.     

Cardiac ultrasonography in structural abnormalities and arrhythmias. Recognition and treatment.

Fetal cardiac ultrasonography has become an important tool in the evaluation of fetuses at risk for cardiac anomalies. It can both guide prenatal treatment and assist the management and timing of delivery. We recommend that a fetal echocardiogram be done when there is a family history of congenital heart disease; maternal disease that may affect the fetus; a history of maternal drug use, either therapeutic or illegal; evidence of other fetal abnormalities; or evidence of fetal hydrops. The optimal timing of evaluation is 18 to 22 weeks'' gestation. An entire range of structural cardiac defects can be visualized prenatally, including atrioventricular septal defect, ventricular septal defect, cardiomyopathy, ventricular outlet obstruction, and complex cardiac defects. The outcome for a fetus with a recognized abnormality is unfavourable, with less than 50% surviving the neonatal period. Fetal cardiac arrhythmias are also a common occurrence, 15% in the series described here. Premature atrial or ventricular contractions are most commonly seen and usually require no treatment. Supraventricular tachycardia can result in hydrops and require in utero treatment to prevent fetal demise. Complete heart block, particularly in association with structural heart disease, has a poor prognosis for fetal survival.