Shan Shan Zhou

Experimental Coinfection of Rhesus Macaques with Rhesus Cytomegalovirus and Simian Immunodeficiency Virus: Pathogenesis

ABSTRACT Human cytomegalovirus (HCMV) possesses low pathogenic potential in an immunocompetent host. In the immunosuppressed host, however, a wide spectrum of infection outcomes, ranging from asymptomatic to life threatening, can follow either primary or nonprimary infection. The variability in the manifestations of HCMV infection in immunosuppressed individuals implies that there is a threshold of host antiviral immunity that can effectively limit disease potential. We used a nonhuman primate model of CMV infection to assess the relationship between CMV disease and the levels of developing anti-CMV immunity. Naive rhesus macaques were inoculated with rhesus cytomegalovirus (RhCMV) followed 2 or 11 weeks later by inoculation with pathogenic simian immunodeficiency virus SIVmac239. Two of four monkeys inoculated with SIV at 2 weeks after inoculation with RhCMV died within 11 weeks with simian AIDS (SAIDS), including activated RhCMV infection. Neither animal had detectable anti-SIV antibodies. The other two animals died 17 and 27 weeks after SIV inoculation with either SAIDS or early lymphoid depletion, although no histological evidence of activated RhCMV was observed. Both had weak anti-SIV antibody titers. RhCMV antibody responses for this group of monkeys were significantly below those of control animals inoculated with only RhCMV. In addition, all animals of this group had persistent RhCMV DNA in plasma and high copy numbers of RhCMV in tissues. In contrast, animals that were inoculated with SIV at 11 weeks after RhCMV infection rarely exhibited RhCMV DNA in plasma, had low copy numbers of RhCMV DNA in most tissues, and did not develop early onset of SAIDS or activated RhCMV. SIV antibody titers were mostly robust and sustained in these monkeys. SIV inoculation blunted further development of RhCMV humoral responses, unlike the normal pattern of development in control monkeys following RhCMV inoculation. Anti-RhCMV immunoglobulin G levels and avidity were slightly below control values, but levels maintained were higher than those observed following SIV infection at 2 weeks after RhCMV inoculation. These findings demonstrate that SIV produces long-lasting insults to the humoral immune system beginning very early after SIV infection. The results also indicate that anti-RhCMV immune development at 11 weeks after infection was sufficient to protect the host from acute RhCMV sequelae following SIV infection, in contrast to the lack of protection afforded by only 2 weeks of immune response to RhCMV. As previously observed, monkeys that were not able to mount a significant immune response to SIV were the most susceptible to SAIDS, including activated RhCMV infection. Rapid development of SAIDS in animals inoculated with SIV 2 weeks after RhCMV inoculation suggests that RhCMV can augment SIV pathogenesis, particularly during primary infection by both viruses.

A Recombinant Rhesus Cytomegalovirus Expressing Enhanced Green Fluorescent Protein Retains the Wild-Type Phenotype and Pathogenicity in Fetal Macaques

ABSTRACT To facilitate identification of rhesus cytomegalovirus (RhCMV)-infected cells, a recombinant virus expressing enhanced green fluorescent protein (EGFP), designated RhCMV-EGFP, was constructed. An expression cassette for EGFP under the control of the simian virus 40 (SV40) early promoter was inserted into the intergenic region between unique short 1 (US1) and US2 of the RhCMV genome by homologous recombination. RhCMV-EGFP exhibited comparable growth kinetics to that of wild-type virus in rhesus fibroblast cultures and retained its pathogenicity in monkey fetuses. Typical neurologic syndromes caused by CMV infection were observed in all fetuses experimentally inoculated with RhCMV-EGFP, as evidenced by sonographic and gross examinations. Systemic RhCMV infections were established in all fetuses, as viral antigen was detected in multiple organs and virus was isolated from fetal blood samples. The engineered viral genome was stable following rapid serial passages in vitro and multiple rounds of replication in vivo. Infected cells could be readily distinguished by green fluorescence both in tissue cultures and in the fetuses. In addition, EGFP expression was detected in various cell types that were permissive to RhCMV infection, consistent with a broad tissue tropism of the SV40 promoter. These results demonstrate that RhCMV can be successfully engineered without loss of wild-type replication and pathogenic potential. Further, the spectrum of cortical anomalies and the distribution of infected cells in the brain tissues indicated that RhCMV may have preferentially targeted immature neuronal cells. The pattern of RhCMV infection in the central nervous system may offer an explanation for the severe developmental outcomes associated with congenital human CMV infection early in gestation.

Immunogenicity and Protective Efficacy of DNA Vaccines Expressing Rhesus Cytomegalovirus Glycoprotein B, Phosphoprotein 65-2, and Viral Interleukin-10 in Rhesus Macaques

ABSTRACT Rhesus cytomegalovirus (RhCMV) infection of macaques exhibits strong similarities to human CMV (HCMV) persistence and pathogenesis. The immunogenicity of DNA vaccines encoding three RhCMV proteins (a truncated version of glycoprotein B lacking the transmembrane region and endodomain [gBΔTM], phosphoprotein 65-2 [pp65-2], and viral interleukin-10 [vIL-10]) was evaluated in rhesus macaques. Two groups of monkeys (four per group) were genetically immunized four times with a mixture of either pp65-2 and gBΔTM or pp65-2, vIL-10, and gBΔTM. The vaccinees developed anti-gB and anti-pp65-2 antibodies in addition to pp65-2 cellular responses after the second booster immunization, with rapid responses observed with subsequent DNA injections. Weak vIL-10 immune responses were detected in two of the four immunized animals. Neutralizing antibodies were detected in seven monkeys, although titers were weak compared to those observed in naturally infected animals. The immunized monkeys and naïve controls were challenged intravenously with 105 PFU of RhCMV. Anamnestic binding and neutralizing antibody responses were observed 1 week postchallenge in the vaccinees. DNA vaccination-induced immune responses significantly decreased peak viral loads in the immunized animals compared to those in the controls. No difference in peak viral loads was observed between the pp65-2/gBΔTM DNA- and pp65-2/vIL-10/gBΔTM-vaccinated groups. Antibody responses to nonvaccine antigens were lower postchallenge in both vaccine groups than in the controls, suggesting long-term control of RhCMV protein expression. These data demonstrated that DNA vaccines targeting the RhCMV homologues of HCMV gB and pp65 altered the course of acute and persistent RhCMV infection in a primate host.

Prenatal Exposure to Toluene Results in Abnormal Neurogenesis and Migration in Rat Somatosensory Cortex

Toluene inhalant abuse during pregnancy may result in growth-retarded microcephalic newborns who subsequently demonstrate developmental impairment. By using a rat model of toluene-abuse embryopathy, we studied the effects of prenatal toluene exposure on the generation and migration of cortical neurons. Dams were exposed by gavage to either corn oil or toluene diluted in corn oil on d 6–21 of gestation. The time of origin of cortical neurons was determined in the mature pups of dams injected with the thymidine analogue 5′-bromodeoxyuridine on 1 d during the period from d 13–21 of gestation. 5′-Bromodeoxyuridine–labeled neurons were identified by immunohistochemistry in a 400-μm-wide column of somatosensory cortex. The brains of the toluene-exposed pups had a significant reduction in the number of neurons within each cortical layer (p < 0.001). Depending on the cortical layer, the generation of neurons in the toluene-exposed pups was delayed by 1 or 2 d. In addition, the brains of the toluene-exposed pups also showed evidence of abnormal neuronal migration. However, there were no differences in either brain weight or body weight between the control and toluene-exposed pups. These observations suggest that although prenatal toluene exposure results in abnormal neuronal proliferation and migration, brain weight in the toluene-exposed pups may be preserved by enhanced development of glia or the neuropil.

Development of a Rat Model of Toluene-Abuse Embryopathy

A rat model was developed to study toluene-abuse embryopathy, a clinical syndrome which occurs in offspring of women who abuse toluene during pregnancy. On d 6-19 of gestation, eight dams received a daily gavage dose of toluene, 650 mg/kg body weight, diluted in corn oil, whereas eight control dams and eight pair-fed dams received corn oil. The fetuses were delivered on d 19 of gestation. In the toluene-exposed group, the weights of the fetuses were reduced by 21.6% (p < 0.001), and a delay in skeletal ossification was demonstrated. Toluene exposure significantly reduced the weight of the fetal brain by 11.9% (p < 0.001), as well as the weights of the heart, liver, and kidney. Organ weight/body weight ratios did not differ significantly. Morphometric analysis of brain sections demonstrated that toluene exposure resulted in smaller brains together with an increase in the size of the ventricular system and a reduction in the size of the caudate nucleus. Although toluene exposure resulted in a 13.7% reduction in maternal food consumption, the observations made in the pair-fed group did not differ from those made in the control group. These findings suggest that prenatal exposure to toluene results in generalized fetal growth retardation, and that these effects are not due to the reduction in maternal food consumption.