Enhanced green fluorescent protein as a marker for localizing murine cytomegalovirus in acute and latent infection.

A recombinant murine cytomegalovirus (mCMV) that expresses enhanced green fluorescent protein (EGFP) under control of the native immediate-early 1/3 promoter was constructed to detect directly sites of viral activity in latent and reactivated infections. The recombinant virus had acute and latent infection characteristics similar to those of wild-type mCMV. Rare green-fluorescing foci were observed in paraffin sections from lungs and spleens infected latently. Positive immunoperoxidase staining for EGFP in sections of the same lung tissues suggests that these cells may be sites of restricted viral gene expression. EGFP was detected easily in tissue explants reactivating from latent infection in vitro. Morphology and adhesion characteristics of fluorescing cells suggest that viral reactivation occurs in tissue macrophages in explant cultures. The observations presented in this study demonstrate the usefulness of EGFP-expressing recombinants as tools for direct tracking of mCMV activity in vivo and in vitro.

Mouse cytomegalovirus reactivation in severe combined immune deficient mice after implantation of latently infected salivary gland.

The hypothesis that replication-competent mouse cytomegalovirus (MCMV) is detectable in severe combined immunodeficient (scid) mice after implantation of latently infected tissue was examined. Sections of latently infected salivary gland from 5 BALB/c mice were implanted into 20 C.B-17 scid mice. Recipient scid mice were sacrificed weekly for 5 weeks, and MCMV infection was detected in target organs using culture and DNA polymerase chain reaction (PCR). All donors were negative by standard culture but positive by DNA PCR. Replicating MCMV was recovered from 9 of 15 recipient scid mouse salivary gland, lung, liver, or spleen samples at postoperative weeks 2-5. No virus was recovered from recipient scid mice at weeks 1 or 12. Transplantation of latently infected tissues into scid mice resulted in rapid reactivation and dissemination of the virus. Further study of this model promises insight into the mechanisms of CMV latency and reactivation.

Electron microscopic study of the African strain of malignant catarrhal fever virus in bovine cell cultures.

Malignant catarrhal fever (African strain) is a viral disease of ruminants which is considered an exotic disease in the United States. Viral isolates obtained from one clinically ill gaur (Bos gaurus) and a greater kudu (Tragelaphus strepsiceros) located in a zoologic park in Oklahoma, and from one heifer (Bos taurus) and a domestic white-tailed deer (Odocoileus virginianus) experimentally inoculated with the isolated and identified African strain of malignant catarrhal fever virus (MCFV), were each studied in bovine cell cultures by electron microscopy. Certain of the viral isolated were previously characterized as MCFV by serologic and morphologic examinations, their cytopathic effect in cell cultures, and their ability to reproduce disease in a ruminant host. The virions of MCFV (African) examined by electron microscopy were icosahedral similar to herpes-virus, were between 98 and 194 nm, developed in the nucleus and matured in the cytoplasm of the cell, and exhibited budding. The virus in infected cells passed through the nuclear and plasma membranes and also into cytoplasmic vesicles from which it acquired one or more envelopes. Virions of malignant catarrhal fever were closely associated with the cellular endoplasmic reticulum, and aberrant morphologic forms of MCFV were observed in virus-infected cells.

Malignant catarrhal fever in an Indian gaur and greater kudu: experimental transmission, isolation, and identification of a herpesvirus.

Herpesviruses were isolated in bovine cell cultures from buffy coat cells obtained from an Indian gaur (Bos gaurus) and a greater kudu (Tragelaphus strepsiceros) with clinical signs of the head and eye form of malignant catarrhal fever (MCF). Both animals were from herds housed in a zoologic park in Oklahoma. Serial transmission of the head and eye form of MCF was accomplished by using whole blood from the gaur into a Hereford-Angus heifer, then whole blood from the heifer into a Holstein calf, and finally, whole blood from the calf into a white-tailed deer (Odocoileus virginianus). A herpesvirus was isolated in bovine cell cultures inoculated with buffy coat cells from the heifer, and 2 deer inoculated with this herpesvirus developed the head and eye form of MCF. A deer inoculated with whole blood from the greater kudu also developed clinical signs of MCF, and a herpesvirus was subsequently recovered from the deer. Clinical signs of MCF included a mucopurulent catarrh, pyrexia (38.8 to 42.1 C), anorexia, and corneal opacity, and death occurred between postinoculation days 15 and 21.

Inhibition of antigodies binding polyinosinic-polycytidylic acid in human and mouse lupus sera by viral and synthetic ribonucleic acids.

The specificities of anti-RNA antibodies of diverse origin were studied by inhibition of the binding of radioative polyinosinic polycytidylic acid. The antibodies were from human patients with systemic lupus erythematosus (SLE), older NZB/NZW F(1) mice who have SLE, and young NZB/NZW F(1) mice immunized with either synthetic or viral double-stranded (ds) RNA. The inhibitors were two viral ds and two synthetic ds RNAs, ribosomal RNA and transfer RNA. The human sera were more heterogeneous than the mouse lupus sera, and had greatest specificity for reovirus RNA. The mouse lupus sera were more homogeneous and, in general, were inhibited efficiently by all four ds RNAs. Sera from mice immunized with synthetic RNA reacted poorly with viral RNA, whereas sera from mice immunized with viral RNA reacted with all four ds RNAs and resembled the lupus sera. These results suggest a role for viruses in the induction of anti-RNA antibodies, and are compatible with the concept that virus infection as well as excessive antibody responses are involved in the pathogenesis of SLE.

Tolerance to polyinosinic-polycytidylic acid in NZB-NZW mice.

Immunological tolerance to polyinosinic * polycytidylic acid was induced in NZB/ NZW mice. This is the first experimental induction of tolerance to a nucleic antigen.