Electron microscopic observation on a non-occluded baculo-like virus in shrimps

Summary.  A severe disease of farm-raised shrimp, Penaeus chinensis has spread throughout Chinese coasts since 1993. Recently, a baculo-like virus has been diagnosed as the causative agent for this epidemic disease. The electron microscopic observation of the virus is described. Thin sections of hepatopancreatic and hypodermic tissue of diseased P. chinensis showed many rod-shaped, enveloped, baculo-like virions in hypertrophied nuclei of infected cells. The virion was filled with a highly electron dense core. No occlusion bodies have been found. Negative stained intact virions, purified from infected tissues by centrifugation on sucrose discontinuous gradients, demonstrated that the viral envelops had been broken, but the cylindrical nucleocapsids could be observed clearly. The nucleocapsid of average 62 nm × 314 nm was composed of a helix system of capsomers, giving rise to an open stacked ring structure and repeating approximately every third ring. The number of repeating unit was 13 to 15. We propose that the virus described here could be designated as Non-Occluded Shrimp Virus (NOSV) or Penaeus chinensis Baculo-Like Virus (PcBLV). Received January 15, 1997 Accepted June 5, 1997     

Coordination of the contractile activity of bile canaliculi. Evidence from calcium microinjection of triplet hepatocytes

The discovery that bile canaliculi are capable of spontaneous contractile activity has led to their use in the investigation of the physiology of the liver cell. The contraction of a bile canaliculus is dependent on the network of actin, which is found in the pericanalicular region of the hepatocyte, and agents that inhibit actin filaments interfere with canalicular contraction. Injection of calcium directly into the cytoplasm of one hepatocyte of a cell pair results in contraction. Injection of calcium directly into the cytoplasm of one hepatocyte of a cell pair results in contraction of the canaliculus. To determine whether the contractile activity of adjacent bile canaliculi was coordinated, calcium was injected into one cell of a group of three hepatocytes that formed two neighboring bile canaliculi. Calcium microinjection resulted in contraction of the bile canaliculus contiguous with the microinjected cell and was followed by a contraction of the second, adjacent canaliculus. These secondary contractions occurred after an interval of approximately 45 seconds. The finding that bile canalicular contractions are coordinated with contractions occurring in a sequential fashion supports the hypothesis that, within the liver lobule, transport of bile within the canalicular network results from the coordinated contractions of the canaliculi.     

Intracellular lumina and bile canaliculi in rat hepatocytes in vitro--a cytochemical study

Within a few hours after being placed in culture, isolated rat hepatocytes reassociate into clusters and differentiate intercellular cavities bordered by junctional complexes. These structures bear a strong resemblance to the bile canaliculi found in the liver. If the plating is impaired by permanent agitation, intracellular lumina surrounded by microvilli appear in the cytoplasm of some isolated hepatocytes. The formation of these structures which mostly contain an osmiophilic substance is enhanced by cAMP and they probably result from modifications in the functioning of the Golgi complexes as suggested by thiamine pyrophosphatase cytochemistry. It may be speculated that these intracellular lumina may be functionally related to new bile canaliculi differentiated between reassociated cells.     

Electron microscopic observation of potato virus A using murine monoclonal antibodies.

Six monoclonal antibodies (MoAbs) against potato virus A (PVA) were prepared and used in enzyme-linked immunosorbent assay (ELISA), immunoblot analysis and electron microscopic study of the virus. Four MoAbs, 151, 290, 328 and 634, reacted with purified virus preparation in dot blot test and showed strong reaction also with virus coat protein (CP) denatured by sodium dodecyl sulphate (SDS), while two MoAbs, 534 and 187, gave significantly weaker reaction with denatured CP than with purified virus. On electron micrographs, MoAb 534 effected binding only on few separate locations of the virus surface after prolonged storage. We presume that this MoAb recognized a conformation-dependent epitope.     

Peripheral immunofluorescence of hepatocytes. Relation to smooth muscle antibodies and bile canaliculi.

By means of indirect immunofluorescence microscopy, a peripheral fluorescence reaction with hepatocytes was found, surrounding the entire cell, with sera from patients with cancer and acute hepatitis and from normal blood donors. It was proved that this reaction was not related to bile canaliculi, contrary to bile canaliculi antibody demonstrated previously from patients with chronic active liver disease. By using fluorescein-conjugated anti-human IgG, the reaction with the periphery of the hepatocytes was produced to a more or less pronounced degree with almost all sera studied. The reaction seems not to be directed against contractile proteins on the liver cell membrane as only a minor part of these sera had also IgG smooth-muscle antibody, and anti actomyosin antibody obtained by affinity chromatografy did not react with the hepatocyte. When applying fluorescein-conjugated anti-human IgM the reaction with the periphery of the hepatocytes was observed in one fourth of the patients with acute hepatitis and only sera which contained also IgM smooth-muscle antibody.     

Electron microscopic observation of African swine fever virus development in Vero cells.

African swine fever virus emerges from infected Vero cells either from areas of smooth cell surface or from microvilli. The two patterns may occur at different sites on the same cell and are unique for this virus. The scanning electron micrographs supplement regular thin section views.     

Phalloidin-induced alterations of bile canaliculi

Phalloidin, a toxin from the plant Amanita phalloides, irreversibly polymerizes actin filaments and causes cholestasis. Three-dimensional structural changes induced by phalloidin in the bile canaliculi and the intra-acinar localization of these changes were studied in the rat liver by scanning and transmission electron microscopy. After 3 days of treatment, canalicular changes appeared mainly in zones 2 and 3 of Rappaport's acinus, but after 7 days of treatment changes occurred in bile canaliculi of the whole acinus. The changes in the bile canaliculi included tortuosity, saccular dilatation, loss of microvilli, bleb formation and elongation of canalicular side branches. Some side branches extended near to Disse's space, leaving only a thin cytoplasmic rim between the canalicular lumen and Disse's space. Kupffer cells were occasionally situated near such extended bile canaliculi and protruded their processes into the hepatic cord. These results suggest that bile canaliculi in zone 3 are more susceptible to phalloidin toxicity than those in zone 1 and that biliary constituents may leak from such altered bile canaliculi.     

Electron microscopic examination of congenital cytomegalovirus hepatitis

Summary The electron microscopic features of cytomegalovirus hepatitis in the liver biopsy of a three-week-old infant were studied.The liver cells did not contain virus, but severe alterations similar to virus hepatitis were observed. In the bile duct cells, nuclear and cytoplasmic virus inclusions were demonstrated. In the nuclear inclusions virus particles of various degrees of maturity were embedded in dense granular material. The cytoplasm of the infected cells contained vacuoles with mature viruses. The Golgi zone seemed to play an important role in vacuole formation. In another type of infected cell, viruses were lying free in the cytoplasm and passed into the lumen of the bile ducts. It is concluded that viruses are eliminimated by the bile. Based on this electron microscopical observations, the examination of duodenal fluid is recommended as a new diagnostic procedure for demonstrating viruses.     

Replication of hepatitis B virus in culture systems with adult human hepatocytes

We developed a technique for isolation and primary culture of adult human hepatocytes from surgical liver biopsy specimens by in situ perfusion and a shaking method. Cultured hepatocytes were maintained in monolayers for more than three weeks and showed morphological and functional characteristics in vivo. The cultured human hepatocytes were inoculated with hepatitis B virus (HBV). Hepatitis B surface antigen (HBsAg) in the medium was detected for about three weeks after inoculation, which was longer than that reported in previous studies. In one case of high attachment efficiency, hepatitis B e antigen (HBeAg) was detected in the medium five to eight days after inoculation. HBsAg and HBeAg were also detected in the extracts of inoculated human hepatocytes. Immunofluorescence study revealed HBsAg in 20-30% of hepatocytes and hepatitis B core antigen (HBcAg) in 2-3% of the cultured human hepatocytes four days after inoculation. Free HBV DNA was identified in the human hepatocytes for at least two weeks after inoculation, although single-stranded HBV DNA was not detected. These studies suggest that HBsAg was actively produced and that HBV replicated in a small number of inoculated adult human hepatocytes in primary culture. However, further improvement of culture systems is needed for active replication of HBV in vitro.     

Electron microscopic study of sindbis virus morphogenesis

Summary An electron microscopic study of the growth and development of Sindbis virus in chick embryo fibroblast cultures demonstrated that the morphogenesis of this virus is essentially like that of other group A arboviruses hitherto studied.     

Electron microscopic characterization of duck plague virus

A virus [duck plague Long Island virulent virus (DPLIVV)] was isolated from ducks in a recent disease outbreak on farms on Long Island, New York. This disease exhibited characteristics similar to duck plague, a disease in ducks endemic to the Netherlands and India and not previously reported in the United States. An attenuated strain of the duck plague virus from the Netherlands was studied morphologically by electron microscopy. Its infectivity for duck embryo cell cultures was also studied. The DPLIVV and the Holland strain were morphologically identical. Furthermore, enzymatic digestion of methacrylate-embedded thin sections of mature virus particles showed that both Holland duck plague-attenuated virus and DPLIVV contained deoxyribonucleic acid. The dimensions of the mature cytoplasmic particle; nucleocapsid diameter ca. 75 micron, envelope diameter ca. 181 micron, and other characteristics would presumably place duck plague virus in the herpesvirus group.