Detection and Localization of Viruses in Human Fetal Intestinal Organ Cultures by Immunofluorescence

Viral antigens from viruses belonging to four different viral groups were detected directly in human fetal intestinal organ cultures by the application of immunofluorescent techniques. The time of appearance and the cellular localization of fluorescent-stainable antigen varied with the type of virus under investigation. After infection with adenovirus or with adeno-associated virus, fluorescent-stainable antigen was seen in the epithelial cells of the explants, though no light microscopic changes could be observed. In infection with herpes simplex virus and echovirus, fluorescence was noted in both the epithelium and the lamina propria, along with histological changes throughout the organ culture. These techniques offer promise for the investigation of possible viral agents implicated in gastrointestinal disease.     

Rotavirus-Like, Calicivirus-Like, and 23-nm Virus-Like Particles Associated with Diarrhea in Young Pigs

Virus particles morphologically similar to caliciviruses and rotaviruses were detected by electron microscopy (EM) in the intestinal contents of a 27-day-old diarrheic nursing pig. A third small spherical 23-nm virus-like particle was also observed. Calicivirus-like particles averaged 33 nm in diameter. Similar to rotaviruses, rotavirus-like particles were present as single-capsid 55-nm forms or double-capsid 70-nm particles. Most gnotobiotic pigs orally exposed to samples containing these three viruses developed diarrhea and villous atrophy of the small intestine, and all shed the three viruses in their intestinal contents. Attempts to propagate these viruses in cell culture were unsuccessful. The antigenic relationship of the rotavirus-like particles to known rotaviruses was explored by immune EM and immunofluorescent staining. By these techniques, the rotavirus-like particles did not cross-react with antisera to porcine, bovine, or human rotaviruses or to reovirus type 3. Antisera from gnotobiotic pigs exposed to all three viruses had enzyme-linked immunosorbent assay and virus neutralization titers of <4 against porcine rotavirus. Previous infection of gnotobiotic pigs with the mixture containing rotavirus-like particles failed to protect them against a subsequent challenge with porcine rotavirus. The antigenic relationship of the calicivirus-like particles to known caliciviruses was investigated by immune EM and virus neutralization. By these tests, the calicivirus-like particles did not react with antisera against feline calicivirus strain 255 or M-8. In a study conducted at Plum Island Animal Disease Center, antiserum against the three combined agents did not specifically neutralize any serotype of swine vesicular exanthema virus.     

Replication of the pseudorabies virus in cultured pancreatic islet cells: further evidence of their paraneuronal nature.

Pancreatic B cells are known to be damaged by a wide range of viruses, causing diabetes. Though these viruses belong to different taxonomic groups, their single shared characteristic is neurotropism. In the present study, pseudorabies viral infection was modelled on fetal porcine islets cultivated in vitro. It was demonstrated that the endocrine cells of the pancreas, especially B cells, were infected in vitro and so served as a medium for the replication of the virus. All stages of the morphogenesis of the virus were observed ultrastructurally within the cells. The exocrine cells located close to the endocrine ones were free from attachment and invasion of the virus. The potential of the pseudorabies virus to develop within pancreatic endocrine cells is regarded as evidence of the paraneuronal nature of these cells.     

Tissue distribution of bovid herpesvirus-4 in inoculated rabbits and its detection by DNA hybridization and polymerase chain reaction

Summary A DNA hybridization technique, using the polyrepetitiveEcoRI L-fragment of bovid herpesvirus (BHV-4) as a probe, was developed to determine virus distribution in the tissues of BHV-4-infected pregnant rabbits. The cloned fragment did not react with the DNA of rabbits or of other herpesviruses, e.g., infectious bovine rhinotracheitis, bovine herpes virus mammillitis, and pseudorabies viruses. The detection limit was 10^–13g of DNA or approximately 600 genome equivalents of viral DNA, which indicates a level of sensitivity of one viral genome per 500 cells in our assay. Using conventional cell culture techniques, the virus was isolated from only one of fifteen infected rabbits and a few aborted fetuses. However, when organ culture or dot blot hybridization was used, BHV-4 was detected in all rabbits and their fetuses. Viral DNA was detected by DNA hybridization in spleen, ovary, uterus, lung, liver, salivary gland, lymph node, and placentome of adult rabbits and in a composite of fetal tissues. When polymerase chain reaction (PCR) was used, the virus was detected in several organs (including the nervous tissues) that were found negative by other techniques. These results indicate that blot hybridization and PCR are more sensitive than conventional techniques for studying the pathogenesis of BHV-4 in animals. The data obtained by these methods suggest that BHV-4 may be maintained in infected rabbits in a latent state in a variety of tissues including the nervous system.     

The propagation of “coronaviruses” in tissue-culture

Summary The conditions for the cultivation of the 229-E respiratory virus in tissue culture are described. The virus multiplies only in human tissue culture systems, but a variety of cell types are sensitive to it. The virus has been isolated in three continuous cell lines and a plaque assay has been developed in one of these. This cell line (L 132) derived from human embryo lung, was also used to isolate three other viruses of similar morphology which had previously been cultivated only in organ cultures of human respiratory epithelium.High rates of isolation of these viruses were made in L 132 cells using nasal washings from experimentally inoculated volunteers. It is concluded that this cell line is a valuable tool in the study of viruses of this group.     

Genetic reassortment of influenza A viruses in the intestinal tract of ducks

Genetic reassortment between influenza A viruses was shown to occur in the intestinal tract of ducks during mixed infection; this phenomenon was examined both in naturally and laboratory-infected ducks. Studies on cloacal samples from Canadian feral ducks demonstrated that 7% of these samples contained two or more antigenically distinguishable influenza viruses, indicating that mixed infections occur rather frequently in nature. The RNAs of multiple viruses isolated from one cloacal sample were separated by polyacrylamide gel electrophoresis; the RNA migration patterns showed that this sample contained a mixture of viruses, including antigenically identical viruses with heterogeneous RNAs. To determine if genetic reassortment may be responsible for the RNA heterogeneity, laboratory ducks were infected with two antigenically distinct avian influenza viruses (Hsw1N1 and Hav1Nav2). Antigenic recombinants (Hav1N1) were readily isolated, without selective antibody pressure, from the feces of these mixedly infected ducks. Comparisons between the RNA migration patterns of the parental viruses and multiple isolates from the feces showed that antigenically identical isolates (as the Hav1N1 recombinants) possessed different gene constellations. These findings show that genetic reassortment between viruses occurs readily in the duck's intestinal tract. Other parameters of virus infection in ducks were also examined. Juvenile ducks, inoculated with influenza virus, shed virus in their feces for 30 days, produced low levels of circulating antibodies (HI titer of 1:20), and were resistant to challenge with homologous virus. These studies illustrate the mechanism by which genetically diverse influenza A viruses may evolve in nature.     

Transport of viruses through fetal membranes: An in vitro model of perinatal transmission

A model system for perinatal transmission of viral infections was developed and transport of infectious virus particles through fetal membranes was investigated. Viruses of different families known to cause serious intrauterine infections were selected, including relevant and model viruses: the DNA-viruses HSV-1 and -2 as well as the animal herpes viruses BHV-1 and SHV-1, the RNA-virus BVDV as a model for hepatitis C virus, HIV-1 and -2, and PPV as a model for parvovirus B19. Migration of infectious virus from the maternal to the fetal side of the membrane could be detected as early as 20 min after the start of incubation. A peak of virus migration was observed after 1–2 hr. 0.02–1% of HSV-1 and 0.03–0.2% of HSV-2 were transported from the maternal side of the membrane to the fetal side. Only 0.01% of PPV migrated to the fetal side, whereas no transport of BVDV was observed. HIV-1 (1.4%) and HIV-2 (0.8%) seemed to be transported at higher rates. The concept of an active transport of infectious virus is compatible with the kinetics of penetration of the fetal membrane. The question of whether different receptors for the individual viruses on the cellular surface account for differences in virus transport will require further investigation. The fetal membrane acts as a protective barrier for the fetus, reducing greatly infectious titers or even preventing completely penetration of virus. J. Med. Virol. 54:313–319, 1998. © 1998 Wiley-Liss, Inc.     

Studies on the pathogenicity of enterovirus-like viruses in chickens.

FP3 and 612 viruses are enterovirus-like viruses. Antibody to these viruses is widespread in chicken flocks, but nothing is known about their pathogenicity. Seven experiments were carried out to investigate the tissue tropism and associated pathology of these novel fowl enterovirus-like viruses and to compare these with the effects of the previously studied enterovirus-like viruses, ELV-1 and avian nephritis (ANV). ANV is now classified as an astrovirus. Preliminary experiments were carried out with FP3 virus, 612 virus and ELV-1 to determine the distribution of viral antigen. Each preliminary experiment was followed by a larger experiment that included more birds and in which a greater range of tissues was studied. It was shown that all four viruses studied replicated in the intestine and had differing abilities to spread to other tissues. Histological changes were present in most antigen-positive tissues but they were usually relatively mild. ELV-1 was associated with the most severe intestinal lesions, followed by FP3 virus. FP3 virus produced lesions in the kidney that were marginally more severe than those caused by the G-4260 strain of ANV. FP3 virus also caused pancreatic lesions. The 612 virus was found to be only mildly pathogenic in specific pathogen free chickens.     

Development of a cell culture system susceptible to measles,canine distemper,and rinderpest viruses

Summary Three strains of chick embryo adapted canine distemper virus (Lederle, Wisconsin, and Onderstepoort strains) and chick embryo adapted LA strain of rinderpest virus were easily adapted to an established line of African green monkey kidney cells (Vero cells), which has been routinely employed for the titration of measles virus. By using these Vero cell adapted strains of canine distemper and rinderpest viruses, techniques of infectivity titration and virus neutralization in Vero cells were established. Comparative studies of cytopathology and growth characteristics of canine distemper, rinderpest, and measles viruses indicated that the behavior of the three viruses in Vero cells is almost the same. The Vero cell system was suggested as a suitable host for the comparative study of the serologic and biologic relationships among measles, canine distemper, and rinderpest viruses.     

Enhanced infectivity of H5N1 highly pathogenic avian influenza (HPAI) virus in pig ex vivo respiratory tract organ cultures following adaptation by in vitro passage

Pigs are thought to play a role in the adaptation of avian influenza (AI) viruses to mammalian hosts. To better understand this mechanism and to identify key mutations two highly pathogenic AI (HPAI) viruses (H5N1 and H7N7) were grown in pig cells, To mimic the pressure of an immune response, these viruses were grown in the presence of antiserum to the homologous virus or porcine IFN-γ. Mutations were identified in both viruses grown in vitro in the presence and absence of antisera or IFN-γ and included the PB2 mutations, E627K or 627E,D701N, described previously as requirements for the adaptation of AI viruses to mammalian species. Additional mutations were also identified in PB1, HA, NP and M genes for viruses passaged in the presence of immune pressure. The infectivity of these viruses was then assessed using ex vivo pig bronchi and lung organ cultures. For lung explants, higher levels of virus were detected in organ cultures infected with H5N1 HPAI viruses passaged in pig cell lines regardless of the presence or absence of homologous antisera or IFN-γ when compared with the wild-type parental viruses. No infection was observed for any of the H7N7 HPAI viruses. These results suggest that the mutations identified in H5N1 HPAI viruses may provide a replication or infection advantage in pigs in vivo and that pigs may continue to play an important role in the ecology of influenza A viruses including those of avian origin.     

Subgroup F adenovirus growth in foetal intestinal organ cultures

Summary An in vitro foetal intestinal organ culture system was employed to determine the permissiveness of human intestinal cells for subgroup F adenovirus infection. Ad 40 and Ad 41 growth, monitored through group-specific hexon antigen production, was poor in comparison to that of Ad 2 in these cultures, further demonstrating their fastidious nature in most human cells. The low growth capability of these viruses in culture, in relation to their association with gastrointestinal disease is discussed.     

Morphology of coronaviruses from different species of monkeys in the Sukhumi nursery

The ultrastructure of coronaviruses from 46 out of 111 monkeys examined (baboons, macaques, green monkeys, langurs) was studied by negative staining in homogenates of different parts of the intestinal tract, pancreatic gland, liver, kidneys, lungs, heart, and brain. Because of marked pleomorphism of coronaviruses it is suggested that morphological variants of the viruses may be distinguished. No relationship between pleomorphism of virus particles and species differences of monkeys and their organ pathology was established. Morphological signs distinguishing simian coronaviruses from those of man were noted.     

Heterogeneity of neuraminidase genetic information in an H1N2 reassortant influenza virus [X-7 (F1)]

Summary The sites of replication of influenza A viruses in ferrets and pigs were studied. The majority of the swine, equine, and avian influenza A viruses tested were recovered from the intestinal tract of ferrets as well as from the respiratory tract; most of the human influenza viruses studied were recovered only from the respiratory tract. In contrast with ferrets, only Hong Kong/1/68 (H 3 N 2) influenza virus was recovered from the intestinal tract of pigs. Despite the large biological variability found in ferrets and in pigs, the results do establish that the majority of influenza viruses have the potential to replicate in the intestinal tissues of some mammals. Additionally, the study suggests that there are differences among the influenza A viruses in tissue tropism in different mammals. Both viral and host genetic factors determine the tissue tropism of influenza viruses in mammals.     

Host Cell Range and Growth Characteristics of Bovine Parvoviruses

In assessing the host cell range of bovine parvoviruses, these viruses were found to replicate optimally in actively dividing bovine fetal lung and spleen cells. Other primary bovine fetal cells supported growth to a lesser extent, but bovine line cells and line cells of other animal species tested did not. Minimal infectivity remained after passage of bovine parvovirus in cells from chicken embryos and guinea pig fetuses. During bovine parvovirus replication in bovine fetal lung and spleen cells, production kinetics of infectious virus and hemagglutinins were determined. An eclipse period of 16 h occurred, and viral release from cells was not detected until 30 h after inoculation of bovine fetal lung cells and 36 h after inoculation of bovine fetal spleen cells. Cell-associated virus titers were always higher than extracellular virus titers. Hemagglutinins were detected in parallel to infectious virus.     

Rat cytomegalovirus replication in the salivary glands is exclusively confined to striated duct cells

The salivary gland is the preferred organ for cytomegalovirus (CMV) replication and viral persistence. In order to identify the nature of infected cells and to study viral replication in more detail, several experiments were conducted. Using the rat CMV (RCMV) model, acutely infected young adult rats (6 weeks of age) and new-born rats (3 days of age) were infected, and submandibular, parotid and sublingual glands were harvested at different time points after infection. For identification of the nature of infected cells, immunohistochemistry, in situ hybridisation and electron microscopic techniques were used. In young adult animals, the submandibular gland was the preferred organ for RCMV replication. The parotid and sublingual glands contained fewer viruses than the submandibular gland. In contrast, in new-born rats, the main site of RCMV replication was the sublingual gland, while the submandibular and parotid glands contained low amounts of virus. No virus could be detected in the parotid glands. In all glands of RCMV-infected animals, the infection was exclusively confined to striated duct cells. Infection resulted in a cellular inflammatory response which was mostly located in the interlobular duct region, whereas only few inflammatory cells were found in the neighbourhood of infected striated duct cells. This phenomenon may contribute to the long persistence of the virus in this organ. Received: 25 May 2000 / Accepted: 26 May 2000     

Transmissibility of influenza viruses in hamsters

Summary The growth characteristics of a series of influenza A viruses in the turbinates and lungs of hamsters was measured: in addition, the susceptibility of hamsters to infection by these viruses was also determined. These two criteria were used to give estimates of the growth potential of influenza viruses in hamsters, and the results were related to the incidence of transmission of virus from inoculated hamsters to cage-contacts. The results showed that strains of influenza virus reported as virulent for man tended to grow to higher titres in hamster nasal washings and lungs; were more infective for hamsters when inoculated by the intranasal route; and showed a high incidence of spread to cage-contacts. The methods could provide valuable measurements of virus attenuation and transmissibility for man, and the further exploitation of these techniques could facilitate the production and licencing of live, attenuated influenza virus vaccines.With 2 Figures     

High resolution three-dimensional imaging and measurement of lung,heart, liver,and diaphragmatic development in the fetal rat based on micro-computed tomography (micro-CT)

Understanding of normal fetal organ development is crucial for the evaluation of the pathogenesis of congenital anomalies. Various techniques have been used to generate imaging of fetal rat organogenesis, such as histological dissection with 3-dimensional reconstruction and scanning electron microscopy. However, these techniques did not imply quantitative measurements of developing organs (volumes, surface areas of organs). Furthermore, a partial or total destruction of the embryos prior to analysis was inevitable. Recently, micro-computed tomography (micro-CT) has been established as a novel tool to investigate embryonic development in non-dissected embryos of rodents. In this study, we used the micro-CT technique to generate 4D datasets of rat embryos aged between embryonic day 15–22 and newborns. Lungs, hearts, diaphragms, and livers were digitally segmented in order to measure organ volumes and analyze organ development as well as generate high-resolution 3D images. These data provide objective values compiling a 4D atlas of pulmonary, cardiac, diaphragmatic, and hepatic development in the fetal rat.     

One-step real-time quantitative PCR assays for the detection and field study of Sacbrood honeybee and Acute bee paralysis viruses

Two one-step real-time RT-PCR assays, based on SYBR Green (SG) chemistry, were developed or adapted respectively, for the detection, differentiation, and quantitation of two important honeybee viruses: Sacbrood virus (SBV) and Acute bee paralysis virus (ABPV). Both reactions were optimized to yield the highest sensitivity and specificity. The genome equivalent copies (GEC) detection limit per reaction was 389.3 for the ABPV RT-PCR. The GEC detection limit per reaction was 298.9 for the SBV RT-PCR. Viral detection and identification were confirmed by melting curve analysis and sequencing of the PCR products. Both techniques were used to evaluate Spanish field samples and establish the distribution of these viruses. Acute bee paralysis virus was not detected, and Sacbrood virus was present at low frequencies. The one-step real-time SG RT-PCR methods are fast, accurate, and useful for detecting and quantifying these honeybee viruses, which cause inapparent infections and contribute to the increasing depopulation of honeybee colonies.     

Organ culture in avian virology: a review

A review has been made of the applications of organ culture techniques in studies with viruses associated with avian disease. After a brief historical introduction, the discussion has been confined to viruses which relate directly to disease in domestic poultry, and has not included viruses which primarily affect man and other animals. The literature reviewed concerns a number of the recognised families of viruses, including poxviridae, herpetoviridae, adenoviridae, orthomyxoviridae, paramyxoviridae, coronaviridae and retroviridae. Up to the present time most studies of avian viruses in organ cultures have been concerned principally with those of Newcastle disease and avian infectious bronchitis, but the potential scope for the technique in avian virology is discussed. It is both likely and desirable that organ culture techniques will be used to a greater extent as the advantages of differentiated cell systems for the study of avian viruses are increasingly appreciated.