Pathogenesis of in utero infections with abortogenic and non-abortogenic alphaviruses in mice.

The initial stages of infection of pregnant mice at gestation day 11 with either the T48 strain of Ross River virus or avirulent Semliki Forest virus are similar. With both infections, a hematogenous spread of virus to the placenta occurs. The viruses subsequently replicate to high titer in all placentas and are able to persist in the presence of specific maternal antiviral antibodies. There is a delay of at least 1 to 2 days between the initial detection of virus in the placenta and the onset of fetal infection. With Semliki Forest virus, abortion occurred in all mothers and appeared to be preceded by infection of all fetuses. However, when Semliki Forest virus was given at other stages of pregnancy, abortion was less common, and in all non-aborted pregnancies at least one uninfected fetus was observed. This situation was similar to that with Ross River virus, in which abortion was not observed and fetal infection and death were only seen in a proportion of fetuses. Within each pregnancy, the outcome of the two in utero infections appeared to result from similar mechanisms, with the fate of an individual fetus depending upon the timing of the passive transfer of anti-viral immunoglobulin G from the mother relative to the timing of fetal infection by virus from the placenta. Although the passive maternal immunoglobulin G protected susceptible fetuses against infection, antibody did not mediate in utero recovery of infected fetuses or clear placental infection.     

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.     

Identification of the target cells and sequence of infection during experimental infection of ovine fetuses with Cache Valley virus

Cache Valley virus-induced malformations have been previously reproduced in ovine fetuses; however, no studies have established the course of infection of cells and tissues with Cache Valley virus. To address these questions, ovine fetuses at 35 days of gestation were inoculated in utero with Cache Valley virus and euthanized at 7, 10, 14, 21, and 28 days postinfection. On postmortem examination, arthrogryposis and oligohydramnios were observed in some infected fetuses. Morphological studies showed necrosis in the central nervous system and skeletal muscle of infected fetuses evaluated after 7 to 14 days postinfection, and hydrocephalus, micromyelia, and muscular loss were observed in infected fetuses after 21 to 28 days postinfection. Using immunohistochemistry and in situ hybridization, intense Cache Valley virus antigen and RNA staining was detected in the brain, spinal cord, skeletal muscle, and, to a lesser degree, in fetal membranes and other tissues of infected fetuses. Viral antigen and RNA staining decreased in targeted and infected tissues with the progression of the infection.     

Semliki Forest virus infects mouse brain endothelial cells and causes blood-brain barrier damage.

Induction of experimental allergic encephalomyelitis is facilitated in a genetically resistant BALB/c mouse strain by a nonpathogenic strain of a neurotropic alphavirus, Semliki Forest virus (SFV-A7). One possible explanation for this enhancement is virus infection of endothelial cells (EC), causing increased permeability of the blood-brain barrier. We have now sought evidence for virus infection of EC in vivo by immunocytochemistry and in situ hybridization. SFV-A7 antigens and RNA were detected in vascular EC and perivascular neurons in cerebellar and spinal cord white matter. Expression of viral antigens was followed by fibrinogen leakage from the blood vessels into brain parenchyma. This was shown by immunoperoxidase staining detecting fibrinogen extravascularly in central nervous system sections of infected mice. Simultaneously, expression of ICAM-1 (intercellular adhesion molecule 1) was induced on brain EC. SFV-A7 replicated in mouse brain microvascular EC in vitro and caused lysis of the cells. SFV-A7 did not induce ICAM-1 expression of mouse brain microvascular EC in vitro, while ICAM-1 was readily induced by gamma interferon and interleukin 1 beta. The observed increase of ICAM-1 expression on EC is immune mediated and not a direct effect of the virus infection. We conclude that SFV-A7 infection causes cerebral microvascular damage which contributes to the facilitation of experimental allergic encephalomyelitis in BALB/c mice.     

Cytotoxic macrophages: a rapid nonspecific response to viral infection.

In vitro and in vivo assays have been developed to study the relative contributions of various types of immune cytolysis in the destruction of infected cells after Semliki Forest virus infection of BALB/c mice. Highly cytotoxic activated macrophages, not specific for the infecting virus, appear on day 1, peak on day 2 to 3, and disappear within a week. Specifically sensitized T cells appear around day 3, peak on day 6, and disappear within a month. Cytotoxic antibody appears on day 4 and reaches high titers by day 8. Immune spleen cells greatly reduce the yield of virus from cultured cells. Infected cells rapidly disappear after transfer to infected animals.     

Influence of dexamethasone on growth and development of rat fetuses: changes in nucleic acids and protein content

Pregnant rats were treated with dexamethasone in drinking water (10 micrograms/ml) from the 15th to the 22nd day of pregnancy. Dexamethasone significantly reduced the weight of rat fetuses and concentration of DNA, RNA and proteins in fetal adrenal glands, liver, placenta, brain, kidneys, heart, lung, testes and pituitary from the 17th to the 22nd day of pregnancy. These data show that dexamethasone given to pregnant rat may lead to potentially deleterious effects on fetal rat development.     

Influence of the infection with lipid-containing viruses on the metabolism and pools of phospholipid precursors in animal cells

The influence of infection with three different lipid-containing RNA viruses, Newcastle disease virus, fowl plague virus, and Semliki Forest virus on the phosphatidylcholine precursors of chick embryo cells and of baby hamster kidney (BHK) cells has been measured. In chick embryo cells infection with Newcastle disease virus does not influence the energy charge, or the distribution and absolute pool sizes of the precursors or the choline phosphotransferase activity. In chick embryo cells infected with fowl plague virus the CDP-choline pool increases because of an inhibition of the choline phosphotransferase activity. The phosphorylcholine and CTP pools are smaller in infected cells when compared with mock-infected ones, although the energy charge is not influenced by infection. In chick embryo cells as well as in BHK cells the energy charge is diminished by infection with Semliki Forest virus. Therefore the CTP and phosphorylcholine pools are decreased. The CDP-choline pool in chick embryo cells becomes extremely small after infection with Semliki Forest virus because of a significant stimulation of the choline phosphotransferase. In BHK cells infected with Semliki Forest virus the opposite effect is observed. There are also severe effects on the uptake of the labeled precursors by infection. One and the same virus (Semliki Forest virus) has two completely different effects on the phosphatidylcholine precursors when infecting two different cell types. If one and the same cell type (chick embryo cells) is infected with three different lipid-containing RNA viruses also completely different effects on the phosphatidylcholine precursors were observed. Thus, each virus develops its own strategy to influence the lipid metabolism of the host cell, depending also on the choice of the host. This explains the many disturbing contradictory results described in the literature about the influence of lipid-containing viruses on the lipid metabolism of the host.     

Synthesis and Broad-Spectrum Antiviral Activity in Mice of Certain Alkyl,Alkenyl and Ribofuranosyl Derivatives of 7-Deazaguanine

Abstract

A series of alkyl, alkenyl and β-D-ribofuranosyl derivatives of 7-deazaguanine were synthesized and their ability to inhibit certain RNA virus infections was assessed in mice. 8-Chloro-7-deazaguanosine (6) protected the majority of mice infected intraperitoneally (i.p.) with benzi, encephalomyocarditis, San Angelo, or Semliki Forest virus at doses as low as 50 mg/kg/day. It was also orally active.     

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.     

Functional expression and characterization of odorant receptors using the Semliki Forest virus system

The human olfactory system can recognize and discriminate a large number of different odorant molecules. The detection of chemically distinct odorants starts with the binding of an odorant ligand to a specific receptor protein on the olfactory neuron cell surface. To address the problem of olfactory perception at a molecular level, we have expressed and characterized different olfactory receptors with several expression systems. Here we provide the first documentation of functional expression of odorant receptors using the Semliki Forest virus system. The human odorant OR 17-40 receptor and the rat 17 receptor were functionally expressed in vertebrate kidney cells (HEK293) using recombinant Semliki Forest viruses. Receptors were expressed as a fusion protein with the N-terminal membrane import sequence of the guinea pig serotonin receptor. Experiments employing the Ca2+-sensitive dye fura-2 revealed a fast, transient increase in the [Ca2+]i after application of the specific agonists helional and octanal to HEK293 cells infected with viruses containing RNA for the human odorant OR 17-40 receptor and the rat 17 receptor, respectively.     

Light microscopic and ultrastructural observations in advanced stages of induced exencephaly and spinal bifida.

This investigation was performed to demonstrate the morphologic basis of the elevation of fetal proteins in the amniotic fluid of fetuses with neural tube defects. Pregnant rats were treated with hypervitaminosis. A to induce exencephaly or with trypan blue to produce spina bifida aperta. The malformations were studied on days 15-20. On day 15 of gestation, edema developed in the primitive nervous tissue. This was followed by the appearance of quickly expanding hemorrhages throughout the ventricular and intermediate zones. Some capillaries did not rupture but collapsed and showed degenerative changes of the endothelium, probably due to lack of blood perfusion. The ventricular layer in exencephaly and spina bifida aperta was exposed to the amniotic cavity due to non closure of the neural tube. On day 17, this superficial lining of the primitive nervous tissue was disrupted by the expanding hemorrhages and subsequent necrosis. As a result vast amounts of fetal blood and cell debris were extruded into the amniotic fluid. During days 18 to 20, the degeneration of the nervous tissue proceeded rapidly. This process showed the same features in the ventricular cells, the primitive neurons and the neurons. Initially it was characterized by condensation of the nuclear chromatin and the cytoplasm, irregular outlines and breakdown of the plasma membrane. Only part of the cell debris was phagocytozed by macrophages. It is concluded that the leakage of fetal serum and cell debris causes the elevation of fetal protein levels in the amniotic fluid of fetuses with open neural tube defects.     

CD4-independent infection of human neural cells by human immunodeficiency virus type 1.

A number of studies have indicated that central nervous system-derived cells can be infected with human immunodeficiency virus type 1 (HIV-1). To determine whether CD4, the receptor for HIV-1 in lymphoid cells, was responsible for infection of neural cells, we characterized infectable human central nervous system tumor lines and primary fetal neural cells and did not detect either CD4 protein or mRNA. We then attempted to block infection with anti-CD4 antibodies known to block infection of lymphoid cells; we noted no effect on any of these cultured cells. The results indicate that CD4 is not the receptor for HIV-1 infection of the glioblastoma line U373-MG, medulloblastoma line MED 217, or primary human fetal neural cells.     

Fetal mortality and malformations associated with experimental infections of western equine encephalomyelitis vaccine virus in rhesus monkeys (Macaca mulatta).

Pregnant Rhesus monkeys were infected via installation of Western Equine Encephalomyelitis (WEE) vaccine virus into the amniotic sacs at 50 and 80 days gestation to determine if the resulting infections would produce fetal mortality or fetal malformations, particularly within the central nervous system. Of those receiving virus at 50 days gestation, 13 of 18 fetuses were aborted or dead in utero at time of Caesarean section; 2 of 18 were malformed (hydrocephalus and polyarthrosis); and 3 of 18 were anatomically normal. Of those receiving virus at 80 days gestation four of eight fetuses were aborted or dead in utero at time of Caesarean section, one of eight was malformed (hydrocephalus) and three of eight were anatomically normal. Three of three controls receiving neutralized virus at each gestational age were anatomically normal. Fetal WEE vaccine virus infection significantly increased fetal mortality and resulted in a significant incidence of fetal malformations.     

Temperature sensitive mutant of Herpes simplex virus type 1. II. Neurovirulence and latency]

The course of acute infection of mice with ts mutant or the native strain DNA and the antigens of HSV in brain nerve cells were determined. Virus DNA was detected in brains of all mice in both animal groups while the virus antigens--only in cells of mice infected with the native strain. It can be suggested, therefore, that the ability of ts mutant to replicate in central nervous system of the infected mice is lacking or much lower. The detection of virus nucleic acid 3-5 months after virus infection might indicate a possibility of establishing latent infection. However, ts mutant showed a significantly lower possibility of latency induction, as compared with highly virulent strains. It was found that the mutant ability to induce latent infection was markedly increased when mice were treated with both ts mutant and Depo-Medrol as immunosuppressive agent. This finding shows both a possibility of increase of frequency of latent infections in the state of immunosuppression, and of activation of the latent infection (recurrence of acute form of infection).     

A Single Amino Acid Change in the Nuclear Localization Sequence of the nsP2 Protein Affects the Neurovirulence of Semliki Forest Virus

The replicase protein nsP2 of Semliki Forest virus (SFV) has a ⁶⁴⁸RRR nuclear localization signal and is transported to the nucleus. SFV-RDR has a single amino acid change which disrupts this sequence and nsP2 nuclear transport. In BHK cells, SFV4 and SFV-RDR replicate to high titers, but SFV-RDR is less virulent in mice. We compared the replication of SFV4 and SFV-RDR in adult mouse brain. Both SFV4 and SFV-RDR were neuroinvasive following intraperitoneal inoculation. SFV4 spread rapidly throughout the brain, whereas SFV-RDR infection was confined to small foci of cells. Both viruses infected neurons and oligodendrocytes. Both viruses induced apoptosis in cultured BHK cells but not in the cells of the adult mouse brain. SFV-RDR infection of mice lacking alpha/beta interferon receptors resulted in widespread virus distribution in the brain. Thus, a component of the viral replicase plays an important role in the neuropathogenesis of SFV.     

Effects of Malaria (Plasmodium berghei) on the Maternal-Fetal Relationship in Mice

Plasmodium berghei infection was more severe in pregnant than in nonpregnant mice. Infection initiated on gestation day 7 resulted in rapidly increasing parasitemia and deaths of all pregnant mice within 12 days, while some nonpregnant mice survived until day 21 postinfection. When mice were infected on gestation day 12 or 14, a proportion of mice died before parturition; but some animals survived to deliver living pups. Reduced birthweights and increased spleen weight to body weight ratios were seen in pups from infected mice as compared with pups from uninfected animals. Histopathological abnormalities of placentae from infected animals included degeneration of the normal labyrinthine architecture and thickening of the trophobast separating maternal and fetal blood vessels.     

Transplacental transmission of Toxoplasma gondii in minipigs infected with strains of different virulence

Infections with the zoonotic protozoan Toxoplasma gondii during pregnancy can result in severe fetal infections. To investigate the use of pigs as animal models for congenital toxoplasmosis, tachyzoites of 5 T. gondii strains, with low to intermediate virulence in mice, were intravenously inoculated into pregnant minipig gilts. Two strains caused abortions of uninfected fetuses following severe disease of the mothers. One strain caused no disease in the gilts but slightly elevated anti-T. gondii antibodies in 2 of 9 fetuses. One strain produced clinical disease with 4 mummified fetuses and 2 full-term, congenitally infected piglets in 1 gilt and no clinical disease but elevated specific fetal antibodies in both piglets of the other gilt. Infection with the fifth strain (SVS-O14), which was considered apathogenic to both pigs and mice based on the clinical course of this and previous experiments, resulted in significant numbers of congenitally infected piglets, as indicated by production of anti-T. gondii antibodies in all 12 fetuses; the parasite was identified in 3 of these fetuses. This pattern of infection indicates that pigs infected with SVS-O14 (or a similar strain) are relevant animal models for studies of transplacental transmission and pathogenesis of congenital toxoplasmosis.     

Role of primary and secondary maternal viremia in transplacental guinea pig cytomegalovirus transfer.

The modulation of the outcome of intrauterine guinea pig cytomegalovirus (GPCMV) infection by maternal viremia was investigated in the guinea pig model. Virus assay and in situ hybridization were used to study GPCMV infection of maternal blood, placentas, and fetuses following inoculation of pregnant guinea pigs by the subcutaneous, intracardiac, or intranasal route. Animals were inoculated in early gestation and were evaluated every 7 to 10 days throughout pregnancy. Although placental and fetal infections occurred in all groups examined, transfer of GPCMV to placentas and fetuses was most efficient in mothers inoculated subcutaneously. Primary viremia was followed by virus clearance from blood and by an episode of secondary viremia in the three groups of mothers examined. Placental and fetal infections in animals infected subcutaneously or intracardially were first detected at the time of primary viremia, persisted throughout gestation, and increased during secondary viremia. In contrast, placental and fetal infections in animals inoculated intranasally were demonstrated primarily during secondary viremia. Fetal infection was detected in all mothers with detectable primary and secondary viremia but in only 33% of mothers that experienced only primary viremia. These results suggest that secondary maternal viremia is associated with increased placental and fetal GPCMV infections.     

Zinc supplementation during pregnancy protects against lipopolysaccharide-induced fetal growth restriction and demise through its anti-inflammatory effect

LPS is associated with adverse developmental outcomes, including preterm delivery, fetal death, teratogenicity, and intrauterine growth restriction (IUGR). Previous reports showed that zinc protected against LPS-induced teratogenicity. In the current study, we investigated the effects of zinc supplementation during pregnancy on LPS-induced preterm delivery, fetal death and IUGR. All pregnant mice except controls were i.p. injected with LPS (75 μg/kg) daily from gestational day (GD) 15 to GD17. Some pregnant mice were administered zinc sulfate through drinking water (75 mg elemental Zn per liter) throughout the pregnancy. As expected, an i.p. injection with LPS daily from GD15 to GD17 resulted in 36.4% (4/11) of dams delivered before GD18. In dams that completed the pregnancy, 63.2% of fetuses were dead. Moreover, LPS significantly reduced fetal weight and crown-rump length. Of interest, zinc supplementation during pregnancy protected mice from LPS-induced preterm delivery and fetal death. In addition, zinc supplementation significantly alleviated LPS-induced IUGR and skeletal development retardation. Further experiments showed that zinc supplementation significantly attenuated LPS-induced expression of placental inflammatory cytokines and cyclooxygenase-2. Zinc supplementation also significantly attenuated LPS-induced activation of NF-κB and MAPK signaling in mononuclear sinusoidal trophoblast giant cells of the labyrinth zone. It inhibited LPS-induced placental AKT phosphorylation as well. In conclusion, zinc supplementation during pregnancy protects against LPS-induced fetal growth restriction and demise through its anti-inflammatory effect.