Enhancement of antiviral capacity of transgenic silkworms against cytoplasmic polyhedrosis virus via knockdown of multiple viral genes.

Bombyx mori cytoplasmic polyhedrosis virus (BmCPV), a major pathogen of silkworms, causes serious economic losses in sericulture. The BmCPV genome contains 10 discrete dsRNA segments; among these, S1, S2, S3, S4, S6, and S7 encode virus structural proteins, whereas S5, S8, S9, and S10 encode nonstructural proteins. In an attempt to create an anti-BmCPV silkworm strain, we constructed transgenic RNAi vector pb-CNS for knockdown of S5, S8, S9, and S10, and pb-SNS targeting S1, S2, S4, S5, and S8. Transgenic silkworm line CNS and SNS were generated via microinjection of the practical diapause silkworm strain Furong. Following infection via the oral administration of a high dose of BmCPV, the mortality rates of the nontransgenic control, CNS, and SNS were 91%, 37%, and 41%, respectively. qPCR showed that the viral mRNA content in CNS and SNS was significantly lower than that in the nontransgenic line. The economic traits of CNS and SNS were not affected. These results suggest that the knockdown of multiple BmCPV genes significantly enhances the antiviral capacity of the silkworm.

Z-FA-FMK as a novel potent inhibitor of reovirus pathogenesis and oncolysis in vivo.

BACKGROUND: Respiratory enteric orphan (reo)virus is a promising oncolytic viral candidate. Reoviral anticancer therapy is currently undergoing multiple clinical trials targeting various human cancers; however, there is no effective reoviral inhibitor that can be used to block unwanted reovirus replication during reoviral anticancer therapy. METHODS: Studies were conducted with transformed or normal cells in vitro and in vivo to characterize viral replication in the presence or absence of chemical inhibitors. RESULTS: We have identified a protease inhibitor that is very effective in the inhibition of viral replication. The dipeptide benzyloxycarbonyl-Phe-Ala-fluoromethyl ketone (Z-FA-FMK) effectively inhibited reovirus replication in a susceptible host and cured cells of a persistent infection with reovirus in vitro. Electron microscopic analysis of Z-FA-FMK-treated cells revealed that internalized reovirus virions, retained in a perinuclear localization, no longer undergo further processing into viral factories following Z-FA-FMK treatment, suggesting that Z-FA-FMK specifically affects a reovirus virion maturation step. Animal studies showed that reovirus infection of Ras oncogenic tumours and host heart tissues is completely blocked by Z-FA-FMK treatment in severe combined immunodeficiency mice. CONCLUSIONS: Z-FA-FMK is a very effective viral inhibitor that can prevent reovirus replication in vitro and reovirus-mediated myocarditis, as well as reovirus-mediated oncolysis, in vivo. A potential application of this drug for inhibition of reovirus infection is suggested.

Morbidity in immunosuppressed (SCID/NOD) mice treated with reovirus (dearing 3) as an anti-cancer biotherapeutic.

Specific viral oncolysis of cancer cells has aroused great interest as a potential anti-cancer therapy. Reovirus was proposed as an anti-cancer biotherapeutic several years ago, as it elicits virus-mediated death of human cancer cells both in vitro and in mouse model systems. A common model system for reovirus oncolysis is the NOD/ LtSz-scid/scid (SCID/NOD) immunocomprimised mouse. While human tumour xenografts are effectively killed by intra-tumour injections of reovirus, the mice often exhibit discoloration and necrosis of extremities including feet, distal leg, tail and ears several weeks after injection. This phenomenon never occurs in sham-injected mice, nor is it observed in wild type or nude mice. The pathogenesis of this "Black Foot" lesion has not yet been described, but may be of relevance for future human studies of biotherapeutics. Examination of SCID/NOD mice was performed at various time points following intratumoral injection of reovirus. Immunohistological evaluation of tissues reveals infection of cardiac myocytes and venous endothelial cells at approximately 2 days post infection. Over time, venules and veins showed a mixed inflammatory vasculitis and thrombus formation. Synchronously, the heart showed diffuse myocyte death, with dystrophic calcification. The results indicate that the "Black Foot" syndrome is likely due to venous vasculitis secondary to reovirus infection, on a background of reovirus myocarditis and heart failure. The rationale for the selective susceptibility of venous over arterial endothelium to reovirus infection is currently unknown. The results of this study may be relevant to the use of oncolytic viruses, particularly reovirus, in the anti-cancer therapy of immunosuppressed patients.

Reovirus type 2 isolated from cerebrospinal fluid.

An 8-week-old female infant presented with a history of active varicella complicated by Escherichia coli sepsis, oral thrush, hypoalbuminemia, intermittent fevers, diarrhea and feeding intolerance. Rhesus monkey kidney cells inoculated with cerebrospinal fluid revealed reovirus-like particles by electron microscopy. Virus neutralization and RNA-gel electrophoresis studies identified the isolated pathogen as reovirus serotype 2. This report represents one of only a few to isolate reovirus from the central nervous system in humans.

Studies of reovirus pathogenesis reveal potential sites for antiviral intervention.

Pathogenesis studies in animals can uncover details concerning viral replication, growth, and access to target organs, in vivo. This, in turn, reveals opportunities for antiviral intervention that may be otherwise missed by limiting analysis to growth of virus in tissue culture. In this report, reovirus infection of mice is used as a model. Three general aspects of reovirus behavior in mice are presented and each demonstrates a property of the virus that could easily have been missed by studies in tissue culture.

Thymic atrophy in type 2 reovirus infected mice: immunosuppression and effects of thymic hormone. Thymic atrophy caused by reo-2.

Suckling mice infected with reovirus type 2 showed a thymic atrophy followed by a marked suppression of the antibody, production to SRBC (a T cell dependent antigen) and bacterial LPS, when measured by the splenic PFC assay. The PFCs produced were sometimes less than 1% of uninfected control animals. Histologically the thymus was usually smaller than normal, and atrophy of the cortex and increased number of Hassal's bodies were observed. Number of nucleated cells in the thymus of infected mice showed 90% decrease as compared to uninfected mice. The spleen, although larger in size, showed depletion of lymphocytes from the thymus-dependent and follicular areas. No viral replication was detected in lymphatic organs using virological methods. Virus-infected mice transferred with the splenocytes or thymocytes from age-matched uninfected mice restored the antibody production against SRBC to normal levels. Thymocytes were more efficient than splenocytes in enhancing the antibody production in virus-infected mice. Injection of several different kinds of immunopotentiating agents enhanced the antibody production to SRBC, although LPS exacerbated the unresponsiveness. Thymic hormones such as FTS and TP5 enhanced antibody production to SRBC and LPS more efficiently than MDP. Flow cytometric analysis showed that percentage of CD4+ single positive cells was slightly increased in virus-infected mice treated with FTS, while there was no difference in the phenotypic distributions of thymocyte subpopulations among virus-infected mice, FTS-untreated and FTS-treated normal mice.

Genetic and molecular mechanisms of viral pathogenesis: implications for prevention and treatment.

The pathogenesis of infection of mice by the mammalian reoviruses involves several discrete steps. Each of the three viral outer capsid proteins has a highly distinct and specialized role: one protein (sigma 1) binds to cell surface receptors; a second protein (mu 1C) determines the capacity for viral growth at mucosal surfaces; and the third protein (sigma 3) is responsible for inhibiting cell macromolecular synthesis. A detailed picture of the molecular basis of reovirus virulence and attention is now emerging.

Glucose vs sucrose in oral rehydration solutions for infants and young children with rotavirus-associated diarrhea.

The use of oral rehydration solutions containing essential electrolytes and either glucose or sucrose of equal osmolality was compared in a double-blind sequential trial of 784 children with rotavirus-associated diarrhea treated at a center in rural Bangladesh. The oral fluid failure rate was 11.5% for the sucrose-containing solution group and 7.3% for the glucose-containing group (P = NS). Vomiting was a significantly more common cause of failure for the group treated with sucrose-containing oral rehydration solution and was associated with an increased rate of intake of the sweeter sucrose-containing solution. The purging rate was not different for the two groups. The oral fluid failure rates for children in the most underweight category (less than 60% of expected weight for age) were not different from those for other groups, although, as assessed by purging rate and initial dehydration, the stool losses of members of this group constituted a greater proportion of their body weight. Glucose is the preferred carbohydrate for oral electrolyte solutions, although sucrose can be substituted with only minimum loss of efficacy.

[Rotavirus enteritis in children]. / Entérites à rotavirus chez l'enfant.

Recent clinical experience with twelve cases of rotavirus gastroenteritis has enabled us to review this subject. After a historical introduction and a review of the literature, two patients are described in detail. The clinical and laboratory data of all the twelve children are reviewed and compared with cases reported in the literature. These observations suggest several conclusions: The disease is very contagious. The patients present with diarrhoea lasting a few days, vomiting and fever. They recover without complications usually within 6 days. All our cases occurred in winter. The blood count revealed a neutrophilia at the onset of the illness followed after 4-5 days by a lymphocytosis with 1-3% plasmocytes. The finding of increased serum transaminase levels indicates a hepatic involvement. These levels returned to normal values only several weeks after the clinical improvement. Treatment consisting only of rehydration and a usual diet for acute gastroenteritis was successful in all the patients.

Oral rehydration therapy for treatment of rotavirus diarrhoea in a rural treatment centre in Bangladesh.

In November 1977, an enzyme-linked immunosorbent assay for detecting rotavirus antigen was introduced in the laboratory of a rural treatment centre in Bangladesh. During the next 40 days rotavirus without other pathogens was found in the stools of 216 (45%) of 480 children under age 5 years who visited the centre with a gastrointestinal illness. 188 (87%) of these children were treated with oral rehydration alone, using the solution currently recommended by the World Health Organisation, while 28 (13%) also required some intravenous rehydration; there were no deaths. Oral rehydration treatment was judged successful in 205 (95%) of the rotavirus patients and was not associated with any serious side effects. Oral rehydration treatment, with this solution, has been used extensively and successfully in the treatment of enterotoxin-mediated diarrhoea and can also safely be used for treating rotavirus diarrhoea in infants and young children.

PIP: The outcome of a rehydration treatment used during a 40-day period at a WHO Center in Bangladesh on 216 children under age 5 is reported. In addition, an enzyme-linked immunosorbentassay (ELISA) designed to detect rotavirus in stool specimens is described and its application explained. The ELISA assay was adaptable to use in a rural treatment center. In a 40-day period, using the new virus-detecting assay, rotavirus without other pathogens was found in stools of 216 (45%) of 480 children who attended the center with gastrointestinal illness. Of these 216 children with only rotavirus pathogen, 188 were treated with oral rehydration alone (oral glucose solution prepared according to WHO procedures); 28 required additional intravenous rehydration therapy. No deaths occurred. 95% of the cases were judged successful on oral rehydration alone for gastrointestinal effects of rotavirus infection. No serious side effects were reported. This oral glucose solution is now indcated in E. coli (enter otoxin)-mediated diarrhea as well as in rotavirus-induced diarrhea.

An epidemic of rotavirus-associated gastroenteritis in a nursing home for the elderly.

92 cases of acute gastroenteritis were registered among 256 individuals during an outbreak in a nursing home for the elderly, the majority of patients being between 70 and 90 years of age. Most cases appeared in 4 of the 8 wards; 66% of the inmates of these wards became ill. Characteristic symptoms were initial nausea and vomiting followed by diarrhoea and low fever. A number of patients were severely ill. One patient died. Rotavirus infection was diagnosed by virus detection and/or antibody titre rise during the acute phase of the illness in 13 of 16 patients examined. At the end of the outbreak, high titres of complement-fixing antibodies against rotavirus (greater than or equal to 64) were detected in serum from 21/22 patients convalescing from the disease, as compared to only 5/45 individuals with no signs of disease. It is tentatively suggested that the outbreak became extensive and rather severe because of lowered immunity against rotavirus infection among the elderly.