Congenital eye malformations associated with extensive periocular neural crest apoptosis after influenza B virus infection during early embryogenesis.

PURPOSE: Congenital eye malformations are a leading cause of blindness in children. Influenza virus infections prevail worldwide and have been implicated in congenital defects. Infections acquired during gestation may disrupt eye morphogenesis. We investigated the effects of influenza B virus infection on eye malformations during early embryogenesis. METHODS: Chick embryos were exposed to influenza B virus at Hamburger-Hamilton stage 9. Maternal infection was conducted by exposing pregnant ICR mice to influenza B virus at the embryonic gestation stage E 5.0. After infection, virus RNA distribution was detected by in situ hybridization at various developmental stages. The distribution of periocular neural crest cells and the extent of apoptosis were examined by immunohistochemical staining, in correlation with eye malformations. RESULTS: Microphthalmos and anophthalmos, together with neural tube defects, were found in the chick and mouse embryos following the infections. The viral RNA was detected in the head neuroepithelium, optic vesicle, periocular mesenchyme, and the forming ventricles of the developing brain. Immunohistochemical staining revealed aberrant neural crest distribution and extensive apoptosis in the head surface ectoderm, periocular mesenchyme, and optic vesicle in the chick embryos. Furthermore, transplacental infection was confirmed by the presence of viral RNA in the mouse fetuses, with eye and neural tube defects similar to those found in the chick embryos after experimental infections. CONCLUSIONS: Influenza B virus may act as a teratogen to cause aberrant periocular neural crest cell contribution to eye development and extensive apoptosis, resulting in congenital eye malformations.

Influenza-B-virus-induced eye and brain malformations during early chick embryogenesis and localization of the viral RNA in specific areas.

Influenza is prevalent worldwide, and the teratogenic effects of influenza infection have been suspected to occur within the developing central nervous system. We herein report the sequelae of influenza B viral infection during early chick embryogenesis. Chick embryos at Hamburger-Hamilton stage 9 were infected by an in ovo injection under the blastoderm of influenza B virus (B/Taiwan/25/99). At 48 h after infection, gross malformations of the eye and brain, ranging from 25 to 58% of 168 infected embryos, were observed, in contrast to 3-6% among 71 mock-infected controls (p < 0.0001 for both eye and brain malformations). Histological analyses showed extensive tissue degeneration and aggregates of cells in the head mesenchyme, suggesting cell death and heterotopia. Influenza B viral RNA was directly localized by in situ hybridization with probes specific for the HA segment. Viral RNA was extensively detected in the head surface ectoderm and in the lung bud. In the developing brain, viral RNA was specifically located in the anterior neural retina, habenular area, mid-thalamus, and rhombencephalon. Our data show that influenza B virus can be a teratogenic agent in neural and nonneural embryonic tissues, raising concern for transplacental infection during early pregnancy.

Prenatal viral infection leads to pyramidal cell atrophy and macrocephaly in adulthood: implications for genesis of autism and schizophrenia.

We investigated the role of maternal exposure to human influenza virus (H1N1) in C57BL/6 mice on Day 9 of pregnancy on pyramidal and nonpyramidal cell density, pyramidal nuclear area, and overall brain size in Day 0 neonates and 14-week-old progeny and compared them to sham-infected cohorts. Pyramidal cell density increased significantly (p < 0.0038) by 170% in Day 0 infected mice vs. controls. Nonpyramidal cell density decreased by 33% in Day 0 infected progeny vs. controls albeit, nonsignificantly. Pyramidal cell nuclear size decreased significantly (p < 0.0465) by 29% in exposed newborn mice vs. controls. Fourteen-week-old exposed mice continued to show significant increases in both pyramidal and nonpyramidal cell density values vs. controls respectively (p < 0.0085 E1 (exposed group 1), p < 0.0279 E2 (exposed group 2) pyramidal cell density; p < 0.0092 E1, p < 0.0252 E2, nonpyramidal cell density). By the same token, pyramidal cell nuclear size exhibited 37-43% reductions when compared to control values; these were statistically significant vs. controls (p < 0.04 E1, p < 0.0259 E2). Brain and ventricular area measurements in adult exposed mice also showed significant increases and decreases respectively vs. controls. Ventricular brain ratios exhibited 38-50% decreases in exposed mice vs. controls. While the rate of pyramidal cell proliferation per unit area decreased from birth to adulthood in both control and exposed groups, nonpyramidal cell growth rate increased only in the exposed adult mice. These data show for the first time that prenatal exposure of pregnant mice on Day 9 of pregnancy to a sublethal intranasal administration of influenza virus has both short-term and long-lasting deleterious effects on developing brain structure in the progeny as evident by altered pyramidal and nonpyramidal cell density values; atrophy of pyramidal cells despite normal cell proliferation rate and final enlargement of brain. Moreover, abnormal corticogenesis is associated with development of abnormal behavior in the exposed adult mice.

Human influenza viral infection in utero increases nNOS expression in hippocampi of neonatal mice.

We investigated the role of maternal exposure to human influenza virus (HI) in C57BL/6 mice on day 9 of pregnancy on hippocampal expression of nNOS in day 0 neonates and compared that to sham-infected pups. Qualitative analysis using polyclonal antibody to nNOS showed overall increases in immunoreactivity (IR) in hippocampal and dentate layers of day 0 infected neonates when compared to sham-infected animals. These increases in nNOS immunoreactivity were pronounced in hippocampal plate, intermediate, molecular, subplate, and dentate areas. Quantitative analysis of specific immunogold silver-enhanced nNOS IR via densitometry showed nNOS IR increases of 26-71.6% in all layers, i.e., hippocampal plate (35.1%), dentate area (71.6%), molecular area (43.75%), subplate (45.7%), and intermediate zone (26%) in infected neonatal brains vs. controls. The changes in levels of nNOS expression in hippocampi of neonates born to mothers exposed to HI virus during the second trimester of pregnancy may reflect the potential for glutamatergic excitotoxicity via activation of NMDA receptors in the developing brains of these neonatal mice.

Induction of chick embryo feather malformations by an influenza C virus.

The effect of influenza C virus, strain JJ/50, on the development of chicken embryos infected at 10 or 12 days was documented by microscopic techniques, as well as by gross observations of embryos or chicks at hatching. The infected, newly hatched chicks displayed marked abnormalities in their feathering. Such abnormalities were observed neither in mock-infected embryos nor in embryos injected with virus which had been previously treated with specific influenza C virus antibody. At a microscopic level, the abnormalities apparently are a result of hypertrophy and/or hyperplasia of the developing barb and barbule cells. Further, the additional development of integumental necrotic foci was correlated with the development of relatively high viral titers (greater than 256) as measured by hemagglutination (HA). Embryos infected after 12 instead of 10 days incubation showed normal feathering at hatching. Infection at 12 days, however, was correlated with the development of relatively low viral titers (HA = 4) and limited degeneration of the respiratory epithelium. The relationship of teratogenic effects to the site of viral replication in rapidly differentiating tissue is discussed.

The effects of maternal influenzal viraemia in late gestation on the conceptus of the pregnant ferret.

Pregnant ferrets were inoculated intra-cardially on day 30 of gestation with influenza virus. The animals were sacrificed on days 5 to 11 after inoculation and the products of conception including the uterus were examined virologically and histopathologically. The results indicate that the initial site of infection of the conceptus is the haemophagous organ and that spread occurs from this site to the endometrium, placental labyrinth and fetus. Lesions in the fetus are confined to the liver and respiratory tract. In the liver they may represent either a viral hepatitis or a secondary response to placental damage resulting in the stimulation of erythropoiesis. In the respiratory tract they first occur in the nasal sinuses and upper airways suggesting that infection is via the amniotic fluid rather than via the blood stream. The relevance of these findings to human pregnancy is discussed.