br Patients and Methods br

Patients and Methods
Results At autopsy, all 3 aborted fetuses showed a congenital cataract confirmed by gross observation. Histologically, the following findings were observed. In the liver, congestion, necrotizing and inflammatory changes accompanied with hemorrhage, apoptotic hepatocytes (so called acidophil bodies), expansion of the portal area with mild to moderate inflammatory infiltrate (rarely with piecemeal necrosis), giant cell formation of hepatocytes, mild deposition of bile pigments into the hepatocytes and vacuolar degeneration of epithelial cells in the intrahepatic bile ducts were observed (Fig. 1a–d). Interestingly, active erythrophagocytosis by Kupffer cells suggesting virus-associated hemophagocytic syndrome was seen (Fig. 1e). The kidney showed mild nephritis accompanied by partial hemorrhage (Fig. 1f). In the lung, all cases had mild pneumonia accompanied by congestion, alveolar hemorrhage and interstitial edema (Fig. 1g, h). The spleen and lymph node from one case showed hypoplasia with hemorrhage (Fig. 1i, j). The heart exhibited mild myocarditis accompanied by interstitial edema (Fig. 1k). In the central nervous system (CNS), no remarkable change suggesting viral encephalitis was found. The immunohistochemical examination revealed localization of RV capsid antigen in multiple organs from all 3 cases tested (Table 1). In the liver, which showed necrotizing and inflammatory changes, virus antigen was localized on the surface of hematopoietic mononuclear cells produced in the fetal liver (Fig. 2a, b). These mononuclear cells tested positive for cell surface marker CD34 by immunohistochemical examination, suggesting that they were derived from hematopoietic stem cells. Similar RV infected hematopoietic stem cells expressing CD34 were also observed in the systemic organs including the spleen, kidney, lungs, heart, CNS and eyes. Virus antigen was also localized in the epithelial cells of the glomerulus and proximal tubules of the kidney, calmodulin dependent protein kinase and alveolus in the lungs, myocardial cells in the heart, spleen cells, lymphoid tissue, and nerve cells in the cerebral cortex (Fig. 2c–j). Furthermore, an important finding in the eye was the presence of virus antigen in the epithelial cells of the ciliary body and the lachrymal glands (Fig. 2k, l). RV capsid antigen was prominent in the cytoplasm of all infected cells. No positive reactions using normal mouse serum as the negative control were seen. By the nested RT-PCR assay using tissues from multiple organs, positive-strand RV RNA was detectable in nasal swabs, placenta and the lens of the eye in all 3 cases tested. Furthermore, viral RNA was also detectable in all of the major organs including the liver, kidney, spleen, heart, lungs, the eye and CNS (consisting of the cerebral cortex, cerebellum and brain stem) obtained from all fetuses examined (Table 1). In addition, our examination detected negative-strand RV RNA, which indicates the replicative form of the positive-strand RNA virus in infected cells. This result showed that all samples tested were positive for the negative-strand RNA of the virus.
Discussion Clinically, the most important illness during congenital rubella infection is the development of malformations in the fetus (Banatvala and Brown, 2004; Duszak, 2009). In particular, it is known that the fetus of early pregnancy is at greater risk than at later stages. So far, many epidemiological studies on RV infection have been reported from many countries, but there are very few reports of pathological studies on rubella (Töndury and Smith, 1966; Brookhouser and Bordley, 1973; Menser and Reye, 1974). For this reason, the pathogenesis of RV infection in human fetuses remains unknown. In addition, the lack of appropriate animal models for rubella infection impedes efforts to elucidate this issue. One of the important illnesses related to CRS is cataract. In our previous study in Vietnamese patients, all of 20 fetuses/newborns with congenital RV infection presented with congenital cataract (Pham et al., 2013). In the pathological study reported here, we obtained direct evidence of viral infection in the epithelial cells of the ciliary body and the lachrymal glands in the eye. Physiological function of the ciliary body is the production of aqueous humor (Goel et al., 2010). The aqueous humor is analogous to a blood surrogate for these avascular structures and provides oxygen and nutrition to the lens, removes excretory products of metabolism, transports neurotransmitters, stabilizes the ocular structure and contributes to the regulation of the homeostasis of these ocular tissues. This cycle is called the aqueous circulation (Goel et al., 2010). If this physiological function by the ciliary body is inhibited by some etiology such as virus infection, the aqueous circulation will not function smoothly. As a result, the lens will be subjected to physiological dysfunction resulting in a disorder such as a cataract. Since the ciliary body has such an important physiological function, we suggest strongly that virus infection of the ciliary body might play an important role in cataractogenesis.