Pathogenesis of H-1 virus infection of embryonic hamster bone in organ culture.

H-1 virus infection of hamsters has been shown to produce runting, microcephaly, cranial lacunae, and deformed teeth in animals inoculated during the suckling period and to cause various abnormalities, including skeletal defects, in embryos infected transplacentally. To explore the pathogenesis of these effects of viral infection on bone, the response of embryonic hamster tibiae in organ culture to inoculation with the H-1 strain of picodna virus was studied. This system made possible the direct observation of the reaction of bone to virus in a regulated environment. During a period of 7-17 days after inoculation the following observations were made: (a) H-1 virus was found to infect and replicate in bone. (b) Infected bones became more translucent, slender, and elongated than control bones. (c) Bone growth as measured by increase in wet weight was reduced in infected tibiae. (d) Infected bones showed periosteal and perichondral degeneration and diminished deposits of subperiosteal bone. It was concluded that the skeletal abnormalities which develop in embryonic and suckling hamsters after H-1 virus inoculation are the direct result of viral replication in bone, and that indirect phenomena such as those associated with chronic infection need not be postulated to explain the deformities seen in these animals.

Effect of prior immunization on induction of cervial cancer in mice by herpes simplex virus type 2.

Previous studies at this laboratory showed that repeated application of inactivated herpes simplex virus type 2 to the mouse cevix produced premalignant and malignant lesions. In the present study mice were inoculated with inactivated herpes simplex virus type 2 or control solution and Freund's adjuvant by intraperitoneal and subcuaneous routes before exposure of the cervix to inactivated virus. It appears that immunization with inactivated virus conferred a protection against the induction of cervical carcinoma.

Roles of cytomegalovirus and Chlamydia trachomatis in the induction of cervical neoplasia in the mouse.

Cytomegalovirus and Chlamydia trachomatis are prevalent sexually transmissible pathogens. They produce persistent infections of the cervix and have been associated with cervical neoplasia. Cytomegalovirus has also been shown to induce transformation of cells in culture. Because of the high prevalence of genital infections with these pathogens and evidence that they may have oncogenic effects on the cervix, cytomegalovirus (strain AD-169) and C. trachomatis (serovar LGV-2) were tested for oncogenicity in a mouse model in which induction of cervical neoplasia by repeated exposure to inactivated herpes simplex viruses has been demonstrated previously. Cotton tampons, saturated with UV-inactivated cytomegalovirus, C. trachomatis, or corresponding control fluids, were inserted into the vaginas of virgin C57 mice 3 times a week. Smears of vaginal aspirates for cytological examination were obtained every 5 weeks. After 75-90 weeks of exposure, the mice were sacrificed and serial sections of their reproductive tracts were examined. Cervical dysplasia was detected by histological examination in 51% and cervical carcinoma in 10% of mice exposed to cytomegalovirus. In control mice, in contrast, dysplasia developed in 3% and carcinoma in none. The progression from normal cervical epithelium to dysplasia to carcinoma observed with cytomegalovirus exposure was similar to that observed previously in this model after exposure of mice to herpes simplex virus types 1 and 2. The frequencies of cervical abnormalities in mice exposed to C. trachomatis or corresponding control fluid were low, and differences between the two groups were not statistically significant. These data indicate that strain AD-169 of cytomegalovirus is oncogenic for the mouse cervix and suggest that the LGV-2 serovar of C. trachomatis is not.

Identification and quantification of ureaplasmas colonizing the respiratory tract and assessment of their role in the development of chronic lung disease in preterm infants.

BACKGROUND: The role of Ureaplasma urealyticum in the development of chronic lung disease (CLD) in preterm infants continues to be disputed. Recently U. urealyticum has been found to consist of two species, U. urealyticum and Ureaplasma parvum, a finding that has not been considered in previous studies of CLD. This study examined the possible relationships between development of CLD and respiratory colonization by these newly redefined species, their concentrations in lower respiratory secretions and the effect of pulmonary surfactant treatment on these relationships in preterm infants with birth weights < 1500 g. METHODS: Endotracheal aspirates (ETA) were collected from intubated infants when airway suctioning was medically required. ETA were stored at -80 degrees C until quantitative cultures for ureaplasmas and Mycoplasma hominis were performed. Culture results were correlated with development of CLD. RESULTS: Of 475 infants (birth weights < 1500 g) admitted during the 2-year study period, 272 were excluded because they were not intubated or were extubated before ETA could be obtained. An additional 28 infants died, were discharged or were transferred before they could be assessed for CLD. From the remaining 175 infants ureaplasmas were isolated from 66 (38%). No statistically significant associations were identified between development of CLD and the Ureaplasma species isolated, or concentration of ureaplasmas in lower respiratory secretions. These findings were not altered by treatment with pulmonary surfactant (Survanta). CONCLUSION: Lower respiratory colonization by ureaplasmas does not appear to be a contributory cause of CLD in preterm infants.

Cervical carcinogenesis with herpes simplex virus, type 2.

In the past few years there have been a number of reports correlating a high frequency of herpes simplex virus type 2(HSV-2) infection with lesions of the uterine cervix. These studies have used a clinical history of herpetic infection or the demonstration of herpetic antibodies in the cancer patients. The present study was performed to evaluate any possible carcinogenic activity of the formalin-inoculated herpes simplex virus type 2 in the reproductive tract of the female mouse. This approach to the study was selected because of previous experience with a model system of carcinogenesis of the cervix uteri using coal tar hydrocarbons. Cytologic and histologic preparations from experimental animals and controls are presented to demonstrate the mucosal alterations and tumors observed in the animals. Noninvasive lesions of the cervix were identified in 76.8% and invasive adenocarcinoma detected in 30.2% of the mice.

Rapid detection of herpes simplex virus in culture by in situ hybridization.

Detection of herpes simplex virus (HSV) by a newly developed HSV-DNA in situ hybridization (ISH) technique (Diagnostic Hybrids, Inc., Athens, OH, USA) was compared with a reference standard that combines observation for cytopathic effect in shell vial cultures with subsequent identification of virus by staining with fluorescein-labeled HSV-specific monoclonal antibody. The new technique utilizes a probe consisting of an alkaline phosphatase direct labeled, cloned, single stranded DNA fragment that is common to HSV-1 and HSV-2. Both methods include enhancement of infection by centrifugation. In concurrent testing of 98 freshly collected specimens, HSV was detected in 17 by culture and 16 by ISH. In testing of 57 frozen positive specimens, HSV was detected in 54 by culture and 53 by ISH. Of the total isolates, 22 were HSV-1 and 49 were HSV-2. HSV was detected after 24 hrs of incubation by the DNA probe technique. Using the shell vial method as a reference standard, the new HSV-DNA ISH method had a sensitivity of 97.2%, specificity of 100%, positive predictive value of 100%, and a negative predictive value of 97.7%. HSV-DNA ISH appears to be a practical, sensitive, and specific technique for detection of HSV.

Resistance of mouse nasopharynx to induction of neoplasia by herpes simplex virus.

In previous studies, carcinoma of the uterine cervix of the mouse was induced by repeated vaginal exposure to inactivated herpes simplex viruses, type 1 or 2. To determine if the mouse nasopharynx is also susceptible to the carcinogenic effects of these viruses, animals were inoculated intranasally with suspensions of formaldehyde-inactivated herpes simplex viruses, type 1 or 2, or control material, four to five times a week. After exposure periods of 12, 20, and 32 weeks, groups of animals were killed randomly and the nasopharynx, along with the larynx, trachea, bronchi, and lungs, were examined histologically. No preinvasive or invasive lesions were detected. These data suggest that the nasopharynx and cervix of the mouse differ in susceptibility to induction of carcinogenesis by inactivated herpes simplex viruses.

Incidence and etiology of conjunctivitis in Navy recruits.

Recruit sick call at the Naval Training Center, Great Lakes, Illinois was monitored for cases of conjunctivitis during two 2-week periods in March in 1981 and 1982. Twenty-three cases were detected. The incidence of conjunctivitis was 1.1 cases per 1000 recruits per week. Peak incidence occurred during the third and fourth weeks of training and two recruit companies had multiple cases. Conjunctival cultures for viruses and Chlamydia trachomatis were negative in all cases. Concurrent cultures of conjunctival exudate were obtained from 12 cases. Haemophilus influenzae was isolated from three of these cases and Streptococcus pneumoniae from one. Despite the high percentage of negative cultures, the clinical characteristics and pattern of occurrence of conjunctivitis in Navy recruits suggest that it is caused by an infectious agent or agents.

Pathogenesis of the rubella exanthem: distribution of rubella virus in the skin during rubella with and without rash.

In a previous assessment of the role of rubella virus in the pathogenesis of the rubella exanthem, virus was consistently isolated from cell cultures of skin biopsy specimens of the rash, and it was concluded that presence of virus in the skin was essential to evolution of the rash. For determination of whether virus is present in the skin only in association with rash, punch biopsies were performed concurrently on areas of skin with and without rash. Among paired skin specimens of 16 patients, virus was isolated from sites of rash in 12 and from the uninvolved skin in 10. In another patient, shown by serologic response and recovery of virus from the pharynx to have rubella without a rash, virus was also isolated from the skin. It is concluded that rubella virus is widely disseminated in the skin of patients with rubella irrespective of the presence or distribution of the rash, and that the presence of virus in the skin, although a constant feature of the disease, is only one of the factors involved in the pathogenesis of the exanthem.

Growth inhibition of human embryonic and fetal rat bones in organ culture by rubella virus.

Paired organ cultures of metacarpal, metatarsal, and long bones of previable human embryos of 7 to 12 weeks' gestation and tibias of 17-day rat fetuses with inoculated with live or ultraviolet-inactivated rubella virus or control fluids and the growth of the bones was measured by increase in wet weight. In several cultures the ability of the human bones to incorporate 35S, a measure of rate of mucopolysaccharide synthesis, was tested. Growth of human and rat bones was retarded in cultures inoculated with live virus but not in cultures inoculated with inactivated virus or control fluids. Mean 35S uptake was increased by approximately 25% in virus-inoculated cultures of bones of 9- to 12-week human embryos. No histological abnormalities were seen. These findings suggest that (1) defective bone growth in congenital rubella is a direct effect of viral infection of bone, (2) a disorder of mucopolysaccharide syntheses may contribute to the osseous lesions that occur in this disease, and (3) organ cultures of human embryonic and fetal rat bones may serve as convenient models for studying the pathogenesis of this virus-induced congenital osteopathy.

Effects of viral exposure of the two-cell mouse embryo on cleavage and blastocyst formation in vitro.

The effect of viral exposure of two-cell mouse embryos on their capacity to undergo subsequent cleavage and blastocyst formation in vitro was determined. Exposure to Coxsackie viruses B-4 and B-6, reovirus type 2, influenza virus type A, mouse cytomegalovirus, adenovirus type 5, and mouse adenovirus resulted in statistically significant inhibition of blastocyst formation. Development in vitro was unaffected by exposure to ECHO virus type 11, attenuated poliomyelitis virus type 2, parainfluenza virus type 1, mumps, rubella, and herpes simplex viruses types 1 and 2. Blastocyst formation was also unaffected by exposure of embryos to mouse interferon in a concentration 24 units/ml of culture fluid. Coxsackie virus B-4 was recovered from exposed embryos.

Frequency and significance of isolation of Ureaplasma urealyticum and Mycoplasma hominis from cerebrospinal fluid and tracheal aspirate specimens from low birth weight infants.

To investigate the pathogenicity of Ureaplasma urealyticum and Mycoplasma hominis in preterm infants, we conducted a study to determine (1) frequency of isolation from cerebrospinal fluid and tracheal aspirate specimens and (2) clinical outcomes and effect of erythromycin treatment in ureaplasma-colonized infants. From the cerebrospinal fluid of 920 infants, U. urealyticum was isolated from 2 (0.2%) and M. hominis from none. From tracheal aspirate specimens from 224 infants, U. urealyticum was recovered from 37 (17%) and M. hominis from 4 (2%). Demographic characteristics and clinical outcomes were compared in very low birth weight infants (< 1500 gm) who were culture-positive or -negative for U. urealyticum. Although infants with positive results were less mature than their cohorts with negative results, there were no substantive differences in clinical outcomes between the two groups. Initiation of erythromycin treatment of infants with positive ureaplasma culture results at a mean age of 16.4 days did not appear to alter the clinical outcome. We conclude that in preterm infants (1) infection of the cerebrospinal fluid by U. urealyticum is infrequent, (2) ureaplasma organisms are frequently present in tracheal aspirate specimens but do not appear to be related to the presence or the subsequent development of respiratory disease, and (3) initiation of erythromycin treatment at 1 to 3 weeks of age does not alter the clinical course.

Cell-mediated immune responses to Chlamydia trachomatis in mothers and infants.

Cell-mediated immunity to Chlamydia trachomatis was studied in pregnant women with chlamydial infection of the cervix, in infants born vaginally to these women, and in infants presenting with chlamydial conjunctivitis. Uninfected pregnant women and their infants were studied as controls. McCoy cell cultures were used to isolate C. trachomatis from clinical specimens. Cell-mediated immunity was measured by lymphocyte proliferative responses in vitro to stimulation by chlamydial antigens. Chlamydial IgG antibody in serum specimens was detected by a microenzyme-linked immunosorbent assay technique. The mean lymphocyte proliferative responses to chlamydial antigens were greater in infected women than in uninfected women both during pregnancy and in the postpartum period. Lymphocyte responsiveness in infected pregnant women, however, was less than in postpartum women. Despite failure to detect chlamydial infection in exposed infants, lymphocyte proliferative responses were greater in umbilical cord blood and later in peripheral blood samples from neonates born to infected mothers than in infants born to uninfected mothers. These responses were also greater in infants with chlamydial conjunctivitis than in infants of uninfected mothers. These data suggest that cellular immune responses to chlamydial antigens are increased in infected mothers and infants and that infants may acquire chlamydial cell-mediated immunity transplacentally.

Chlamydia trachomatis infection in mothers and infants. A prospective study.

The incidence of chlamydia trachomatis infection of the cervix during pregnancy was found to be 18% in a group of 1,327 women attending the prenatal clinic of a large urban hospital. There were no statistically significant differences between infected and uninfected women in the type or frequency of complications of pregnancy. Chlamydial infection was demonstrated in 27 (28%) of 95 infants born vaginally to infected mothers. Conjunctival infection in these infants was detected earlier than nasopharyngeal infection and the conjunctivae appeared to be the usual portal of entry for the organism. Infants were observed through the age of 12 weeks. Conjunctivae, but the chlamydial pneumonia syndrome occurred in only three (17%) of 18 infants with nasopharyngeal infection.

Induction of cervical neoplasia in the mouse by herpes simplex virus type 2 DNA.

Induction of cervical neoplasia in the mouse cervix by herpes simplex virus types 1 (HSV-1) and 2 (HSV-2) has been reported. The present study was done to determine if transfection with DNA of HSV-2 can induce carcinogenesis in this animal model. Genomic HSV-2 DNA was isolated from infected HEp-2 cells and separated from host cell DNA by cesium chloride density gradient centrifugation. The DNA was applied to mouse cervix for periods of 80-100 weeks. Experimental controls were treated with uninfected genomic HEp-2 cell DNA or with calf thymus DNA. Vaginal cytological preparations from all animals were examined monthly to detect epithelial abnormalities. Animals were sacrificed and histopathology studies were done when cellular changes indicative of premalignant or malignant lesions were seen on vaginal smears. Cytologic and histologic materials were coded and evaluated without knowledge of whether they were from animals treated with virus or control DNA. Premalignant and malignant cervical lesions similar to those that occur in women were detected in 61% of the histologic specimens obtained from animals exposed to HSV-2 DNA. The yield of invasive cancers was 21% in animals treated with HSV-2 DNA. No cancers were detected in mice treated with either HEp-2 or calf thymus DNA. Dysplasia was detected in only one of these control animals.