A model of lytic, latent, and reactivating varicella-zoster virus infections in isolated enteric neurons

Because human primary afferent neurons are not readily obtained, we sought to develop a model in which the lytic, latent, and reactivating phases of varicella-zoster virus (VZV) infection were recapitulated in neurons from an animal source. Enteric neurons were obtained from the small intestine of adult guinea pigs and from the bowel of fetal mice. Latency was established when these neurons were infected by cell-free VZV in the absence of fibroblasts or other cells of mesodermal origin. In contrast, lytic infection ensued when fibroblasts were present or when the enteric neurons were infected by cell-associated VZV. Latency was associated with the expression of a limited subset of viral genes, the products of which were restricted to the cytoplasm. Lysis was associated with the expression of viral glycoproteins, nuclear translocation of latency-associated gene products, and rapid cell death. Reactivation was accomplished by expressing VZV open reading frame (ORF) 61p or herpes simplex virus ICP0 in latently infected neurons. Isolated enteric neurons from guinea pigs and mice recapitulate latent gene expression in human cranial nerve and dorsal root ganglia. Expression of latency-associated VZV gene products was detected in 88% of samples of adult human intestine, suggesting that VZV not only infects enteric neurons but also is latent in the human enteric nervous system. This in vitro model should facilitate further understanding of latency and reactivation of VZV.     

Testing antiretroviral drug efficacy in conventional mice infected with chimeric HIV-1

OBJECTIVE: We previously described chimeric HIV-1, EcoHIV, which can infect mouse cells in culture and cause spreading infection in conventional immunocompetant mice. We have now applied this system as a model for preclinical evaluation of anti-retroviral drugs. DESIGN AND METHODS: We used chimeric virus EcoHIV/NDK constructed on the backbone of subtype D NDK. EcoHIV/NDK expression in mice was characterized 5-10 days after infection by testing viral DNA, RNA, and protein burdens in spleen and macrophages by real-time PCR (QPCR), RT-PCR, and p24 ELISA. For antiviral evaluation, groups of 5-7 mice were pretreated with 2',3'-dideoxycytidine (ddC), abacavir, or vehicle; mice were then infected with EcoHIV/NDK, treatment maintained for additional 48 h, and tested for viral DNA and RNA burdens in spleens and macrophages by QPCR. RESULTS: EcoHIV/NDK infected mice reproducibly showed viral burdens of up to 1.4 x 10 viral DNA copies and 200 pg p24 per 10 spleen cells and expressed spliced Vif RNA and mature p24 in macrophages 5-10 days after infection. Treatment of mice with 60 or 300 mg ddC/kg/day blocked EcoHIV/NDK infection in a dose-dependent manner with significantly lower viral DNA and RNA burdens at both drug doses (P < 0.001) in the spleens of infected mice. Abacavir tested at 100 mg/kg/day caused 96% inhibition of viral DNA synthesis in spleen and it almost completely abolished viral spliced RNA synthesis in spleens and macrophages. CONCLUSIONS: The system of chimeric HIV-1 infection of mice permits rapid, statistically powerful, and inexpensive evaluation of antiretroviral drugs in vivo.     

Sequence of events in the transformation process in cells infected with a temperature-sensitive transformation mutant of Moloney murine sarcoma virus.

Normal rat kidney cells infected with the temperature-sensitive transformation mutant of Moloney murine sarcoma virus were used to study the biochemical and morphological changes that occur during transformation. The infected cells exhibited a normal morphology at the nonpermissive temperature (39 degrees C) and a transformed morphology at the permissive temperature (33 degrees C). A new viral protein was detected 2 hr after shift to the permissive temperature as a polyprotein with an estimated Mr of 85,000 (p85). Scanning electron microscopy of the cells within 5 hr after shifting them to the permissive temperature showed that they became smaller and rounded with numerous elongated microvilli. In an earlier study, changes in hexose uptake were found to occur 8-12 hr after the shift [Horn, J. P., Wood, T. G., Blair, D. G. & Arlinghaus, R. B. (1980) Virology 105, 516-525]. By 48 hr, the cells had the morphology of a fully transformed cell. Concomitant with the changes in the morphology were alterations in the cytoplasmic microtubule complex. At the nonpermissive temperature, the complex consisted of a lacy network of microtubules. Within 5 hr at the permissive temperature, the lacy network was still present but the microtubules were more diffusely stained and less discernible. By 48 hr, the microtubules were so diffuse that the lacy network could not be recognized. Alterations in the F-actin cables did not occur until 24 hr after shifting the cells to the permissive temperature. Enucleation of the cells at the nonpermissive temperature and shifting the cytoplasts to the permissive temperature did not result in the synthesis of detectable p85 or in any alteration of the cytoplast morphology or microtubule complex, suggesting that the temperature-sensitive lesion affects some event occurring in the nucleus.     

Direct spread of reovirus from the intestinal lumen to the central nervous system through vagal autonomic nerve fibers.

A crucial event in the pathogenesis of systemic enteric virus infections is entry of virus into the nervous system. Whether enteric virus spreads from the intestinal tract to the central nervous system through nerves or through the bloodstream was examined using a serotype 3 reovirus strain. After peroral inoculation of newborn mice with reovirus, serial histologic sections of small intestine, brain and spinal cord were prepared and stained by immunoperoxidase to detect viral antigen. Three days after inoculation, viral antigen was observed in mononuclear cells of ileal Peyer's patches and in neurons of the adjacent myenteric plexus. Infection first appeared in the central nervous system 1-2 days later in neurons of the dorsal motor nucleus of the vagus nerve. Endothelial cells, meninges, choroid plexus, hypothalamus, and area postrema were not infected, indicating neural rather than bloodborne spread from the intestine. Staining of neurons in the dorsal motor nucleus of the vagus nerve depended on the route of virus inoculation and was independent of the amount of virus in the bloodstream. These results demonstrate that an enteric virus entering a host from the intestinal lumen can spread to the central nervous system through nerve fiber innervating the intestine.     

Impairment of immunocompetent mouse spleen cell functions by infection with coxsackievirus B3

Early after infection, mice inoculated with coxsackievirus B3 showed consistent reduction of antibody responsiveness. Beginning one week after infection they also evidenced progressive spleen atrophy. The cellular basis of the reduced antibody response exhibited in vitro by spleen cells of infected mice at the onset of atrophy was investigated by the use of different antigens, by supplementation with different subpopulations of immunocompetent cells from normal donors, and by cross-recombination with normal lymphoid cells. Whereas B cell functions appeared to be preserved, a deficit of macrophage accessory functions was clearly evident, possibly at the level of antigen presentation. In infected spleens, nonspecific suppressor T cells were also observed, but their activity did not correlate strictly with the degree of immunodepression. Because no evidence for a direct effect of virus on, or for viral replication in, immunocompetent cells was found, these alterations were tentatively ascribed to activation of the host's suppressor system. Such changes may have implications in the pathogenesis of coxsackievirus infections.     

Effect of Preinjury Large Bowel Emptying on the Inhibition of Upper Gastrointestinal Motility After Spinal Cord Injury in Rats

Spinal cord transection (SCT) inhibits gastrointestinal motility in rats. We evaluated the effect of preinjury large bowel emptying on this phenomenon. Male Wistar rats (N = 52) were fasted for 24 or 48 hr with water ad libitum and pretreated with lactose (0.8 g) or saline. Next, laminectomy followed or not by complete SCT between T4 and T5 vertebrae was performed. Phenol red recovery in the stomach and proximal, medial, and distal small intestine was determined 1 day later. In animals submitted to 24 hr fasting + saline, SCT increased gastric recovery by 42.8% decreased medial small intestine recovery by 56.2%, while 48 hr fasting + saline or 24 hr fasting + lactose prevented the inhibition of gastric emptying (GE) in SCT animals. The 48 hr fasting + lactose prevented the inhibition of both GE and gastrointestinal transit. SCT-induced inhibition of upper gastrointestinal motility may involve enhancement of inhibitory reflexes, which can be prevented by large bowel emptying.     

Ethanol decreases natural killer cell activation but only minimally affects anatomical distribution after administration of polyinosinic:polycytidylic acid: role in resistance to B16F10 melanoma

BACKGROUND: Natural killer (NK) cells are critical in resistance to B16F10 lung metastases in B6C3F1 mice. Activation of NK cells by polyinosinic:polycytidylic acid (poly I:C; 0.1 mg, intraperitoneally) increases resistance to B16F10 cells. This effect is reduced after administration of ethanol (EtOH; 6 g/kg by oral gavage). The present study was conducted to determine whether decreased resistance is due to alteration of the distribution and/or the activation of NK cells. METHODS: These parameters were measured in the spleen, lungs, and peripheral blood 4 and 12 hr after EtOH and poly I:C administration. For assessing the time after poly I:C administration during which NK cells are important in resistance to B16F10 cells, anti-NK1.1 antibody was used to deplete NK cells in vivo 48 hr before and 0, 6, 12, and 24 hr after intravenous injection of B16F10 tumor cells. RESULTS: Depletion of NK cells at any time up to 12 hr after B16F10 administration significantly increased the number of tumor nodules in the lungs, but depletion at 24 hr had a smaller effect. Flow cytometry revealed that there was a small but significant increase in the percentage of NK cells in the lungs at 12 hr, which was not changed by EtOH. Corresponding NK cell lytic function in the lungs was increased significantly at both 4 and 12 hr by poly I:C. However, the increase was not significantly different from the naive control value at 4 hr in mice treated with poly I:C plus EtOH, indicating that EtOH decreased activation of NK cells in the lungs at 4 hr. In the spleen, no treatment significantly altered the percentage of NK cells at 4 or 12 hr. However, poly I:C significantly enhanced lytic function, and this enhancement was suppressed by EtOH (by approximately 50%). In the blood, the only significant change in NK cell percentage or lytic activity was an increase in the percentage of NK cells at 12 hr, which was equivalent in the poly I:C and the poly I:C plus EtOH groups. CONCLUSIONS: These results demonstrate that EtOH partially abrogates the poly I:C-induced enhancement of resistance to B16F10 cells and that decreased activation of NK cells in the lungs at a critical time early in the response to poly I:C may contribute to this effect. Other parameters could also contribute, but there was little support for an important role for changes in NK cell distribution.     

柯萨奇病毒性心肌炎小鼠细胞外基质基因表达变化

目的　探讨细胞外基质 (ECM)基因在实验性病毒性心肌炎小鼠心肌中的表达变化。方法　以柯萨奇病毒B3感染BALB C小鼠建立心肌炎动物模型 ,同时设正常对照 ,以包容 80 0 0余条基因的cDNA微矩阵芯片分析其心脏组织特异的mRNA表达 ,筛选出高表达的ECM基因后以RT PCR方法验证筛选结果。结果　芯片筛选发现心肌炎小鼠心脏中 9条ECM基因表达增高 ,经RT PCR验证其中 5条ECM基因为高表达基因 ,这 5条基因是诱导型生长因子 15基因、Ilk、β1 整合素结合蛋白基因、Lamr1和ADAMTS 1。结论　急性柯萨奇病毒性心肌炎的发病与某些ECM成分基因表达异常有关。     

Burn and Starvation Increase Programmed Cell Death in Small Bowel Epithelial Cells

Maintenance of gut mucosal homeostasis depends on a balance between cell proliferation and cell death. Gut mucosal integrity is impaired after severe burn and during starvation. We determined the effect of burn, starvation, and the combination of both on small bowel epithelial apoptosis and proliferation. Fifty adult male Fischer 344 rats (260–300 g) received a 60% full-thickness scald burn and were randomly divided into fed and starved groups. Small intestine was taken at 12, 24, and 48 hr after injury. All animals in the 12-hr group were starved while recovering from anesthesia. Apoptosis was quantified by immunohistochemical staining (TUNEL) and mucosal proliferation was determined by bromodeoxyuridine (BrdU) incorporation. The apoptotic index was higher in burned rats compared to controls at 12 hr after burn; both these groups were starved (P < 0.05). At 24 and 48 hr after burn, apoptosis was highest in the starved groups, with no additional effects of burn (P < 0.05). Mucosal epithelial cell proliferation was not different between groups at any time point. In conclusion, burn and starvation both increase apoptosis in the small bowel mucosa; however, these effects are not additive. Apoptosis could be attenuated by enteral feeding, which delineates the importance of early enteral feeding initiation after injury to maintain mucosal integrity.     

弓形虫感染小鼠血清对小鼠B16黑色素瘤细胞增殖和凋亡的影响

目的观察RH株弓形虫感染小鼠血清在体外对小鼠B16黑色素瘤细胞增殖以及凋亡的影响。方法B16细胞在含5%、10%和20%RH株弓形虫感染小鼠血清RPM1640中培养24和48 h,四甲基氮噻唑蓝（MTT）法检测B16细胞增殖抑制率;在10%、20%感染小鼠血清中培养24 h,Annexin V/PI染色,流式细胞检测细胞凋亡,HE染色观察细胞形态改变。结果正常血清能促进B16细胞增殖。5%、10%和20%弓形虫感染小鼠血清与B16细胞共孵育24和48 h,细胞生长抑制率分别为（9.06±0.36）%（、14.77±0.94）%（、23.74±0.5）%和（14.82±0.77）%（、24.56±1.04）%、（39.77±2.82）%,与对照组比较差异有统计学意义（P〈0.05）;10%、20%弓形虫感染鼠血清组B16细胞24 h凋亡率分别为（26.01±3.27）%和（44.55±2.87）%,显著高于对照组凋亡率（5.01±2.62）（P〈0.05）。弓形虫感染鼠血清作用的B16细胞生长密度明显降低,细胞核固缩、浓染。结论RH株弓形虫感染血清能够抑制B16细胞增殖并促进其凋亡。     

Coxsackievirus B cardiopathy and angiopathy in the hypercholesterolemic host.

Studies on the pathogenic potential of the human cardiotropic enterovirus, coxsackievirus B5, show that this agent localizes and replicates in the aorta of mice. Nutritionally-induced hypercholesterolemia leads to an increased replication and persistence of virus in tissues, specifically the aorta. Coxsackievirus B cardiopathy is markedly augmented in the hypercholesterolemic host, resulting in a persistent cardiomyolysis which is not evident in virus-infected animals with normal cholesterol levels. Pathological changes in the aorta become evident only months after the acute infection, and only in hypercholesterolemic animals previously infected with coxsackievirus B5. Our findings of coxsackievirus B-induced angiopathy and cardiopathy in the hypercholesterolemic host extend the known pathogenic range of these human viruses, and further emphasizes their potential as etiological agents of cardiovascular disease.     

T-cell receptor Vβ8. 1 peptide reduces coxsackievirus-induced cardiopathology in aged mice

Viral myocarditis is an important cause of heart failure and cardiomyopathy. Immunosenescence, characterized by a dramatic reduction in immune responsiveness, can increase susceptibility to cardiopathology from viral infections. The T-cell receptor (TCR) Vβ 8.1 peptide, a 16-merpeptide, has shown immuno-regulating and immunostimulating effects in viral-induced immunodeficiency. In our study, 18-mo-old C57BI/6 female mice were treated twice with TCR Vβ8.1 peptide and 10 d before sacrifice were injected ip with coxsackievirus B3. Cardiac histopathology was assessed for lesion severity. Splenocyte cytokine production (interleukin-2,-4,-6, interferon-γ) and heart viral titers were determined. Our data suggest that immunosenescence suppressed both T helper (Th1) and Th2 cytokine production and that treatment with TCR Vβ8.1 peptide induced cytokine stimulation close to levels seen in young mice. Nontreated aged mice developed some degree of myocarditis (75% mild and 25% severe), whereas only 35% of the peptide-treated aged group developed cardiopathology, with 25% being mild and 10% severe. Heart tissue from nontreated aged mice infected with coxsackievirus had a higher viral titer than hearts of aged mice equally infected but treated with the peptide. In conclusion, TCR Vβ8.1 peptide induced immunoregulation, and inhibited or reduced coxsackievirus B3-induced, cardiopathology in aged mice.     

Soluble recombinant coxsackievirus and adenovirus receptor abrogates coxsackievirus b3-mediated pancreatitis and myocarditis in mice

BACKGROUND: Group B coxsackievirus infection can result in organ injury and inflammation. The coxsackievirus and adenovirus receptor (CAR) and decay-accelerating factor (DAF; CD55) have both been identified as receptors for coxsackievirus B3 (CVB3). We have shown elsewhere that early DAF-Fc treatment attenuates CVB3-induced myocarditis and virus replication. METHODS: CAR was synthesized as a soluble IgG1-Fc fusion protein (CAR-Fc). In vitro, CAR-Fc blocked infection by 2 different strains of CVB3. A/J mice were infected in vivo with CVB3 and were administered CAR-Fc either 3 days before infection, during infection, or 3 days after infection and were compared with mice infected with virus alone and control animals. RESULTS: All CAR-Fc treatment groups had reduced recoverable infectious virus in the heart. CAR-Fc treatment of mice, either preceding or concurrent with CVB3 infection, resulted in complete inhibition of myocardial lesion area, cell death and inflammation, and viral RNA. Early treatment also completely blocked inflammation and cell death in the pancreas, an organ that is normally very sensitive to infection. CONCLUSION: To our knowledge, CAR-Fc is the only protein that has been shown to block coxsackievirus infection of the pancreas. However, regardless of the efficacy of the test protein, target tissue cannot be rescued after day 3 of infection in the A/J mouse model.     

In situ detection of enteroviral genomes in myocardial cells by nucleic acid hybridization: an approach to the diagnosis of viral heart disease.

We have developed an in situ hybridization assay capable of detecting enteroviral RNA in myocardial cells, using molecularly cloned coxsackievirus B3 cDNA as a diagnostic probe. Because of the high degree of nucleic acid sequence homology among the numerous enteroviral serotypes, including the group A and B coxsackieviruses and the echoviruses, detection of these various agents commonly implicated in human viral heart disease is possible in a single hybridization assay. We demonstrate the considerable potential of this method for an unequivocal diagnosis of enteroviral heart disease as well as for pathogenicity studies. Using athymic mice persistently infected with coxsackievirus B3 as a model system, we show that the myocardium is affected in a disseminated, multifocal manner.     

Immunological mechanisms in experimental coxsackievirus B3 myocarditis in mice.

To address unresolved questions, experimental models of viral myocarditis may be of great value. In this study, immunological mechanisms of myocardial damage in coxsackievirus B3 myocarditis in mice were investigated. The results showed that susceptibility to viral infection is primarily determined by the genetic background of the host, that the severity of myocarditis depends not upon B cells but upon T cells, and that antigen-specific T cells play a pivotal role in the pathogenesis of acute coxsackievirus B3 myocarditis.     

Small intestinal epithelial cell kinetics and protozoal infection in mice

The effects of chronic protozoal infection on small intestinal architecture have been examined in mice, infected with Giardia muris and Hexamita muris. Techniques used were conventional histology, quantitation of intraepithelial lymphocytes, microdissection and measurement of individual villi and crypts, and epithelial cell kinetic studies. The histology of small intestine from infected mice appeared normal apart from the intraepithelial lymphocyte numbers. Mean intraepithelial lymphocyte counts in two groups of uninfected mice were 11.6 and 13.6 per 100 epithelial cells, and in two groups of infected mice were 17.6 and 21.8. Dynamic studies showed that protozoal infection doubled the cell production per crypt per hour from mean values of 6.2, 7.3, and 8.2 in three groups of uninfected animals, to 11.8, 13.4, and 17.1 in groups of chronically infected mice. Cell production per villus was also influenced by protozoal infection, with values of 93, 99, and 101 cells per hr in groups of uninfected animals whereas in infected mice the values were 155, 162, and 180 cells per hr. Although there was no reduction in villus height in the infected animals, radioautography using [3H]thymidine confirmed that the enterocytes moved rapidly up the sides of the villi than was the case for uninfected mice.     

禽流感病毒感染小鼠模型病理损伤及抗原定位研究

目的通过建立高致病性禽流感H5N1病毒感染小鼠模型,研究病毒感染致小鼠组织病理损伤、病毒抗原在体内分布及复制特点,为禽流感病毒传播途径及致病机理研究提供实验依据。方法采用A/vietnam/1194/2004(H5N1)病毒株,滴鼻感染BALB/c小鼠,5 d后取小鼠肺、脑、脾、肠、心、肝、肾等组织,制备石蜡切片并进行组织病理损伤和抗原定位研究。结果小鼠肺组织表现为弥漫性肺损伤,脑组织出现轻度炎症反应,脾组织结构完整但脾小体可见凋亡细胞,其它脏器组织结构未查见组织病理损伤;利用M2、NS1单抗,采用免疫组织化学方法检测发现小鼠肺、脑、肠等均有M2、NS1抗原分布。结论高致病性H5N1病毒感染可在小鼠肺、脑、肠等多种组织中复制,引起严重的肺组织病理损伤及轻度的脑组织及脾组织病理损伤。     

Age-dependent pathogenicity of group B coxsackieviruses in Swiss-Webster mice: infectivity for myocardium and pancreas

Coxsackieviruses B1-B4 were inoculated intraperitoneally into 48-hr-old, 14-day-old, and three- to five-month-old Swiss-Webster mice. Immediate death occurred only among mice less than 48 hr old, which died from fulminant encephalitis. Older mice usually survived. Myocarditis ensued in mice less than 48 hr old that were infected with coxsackieviruses B1 and B4. Several of the surviving mice developed left ventricular aneurysms, which resulted from transmural necrotizing myocarditis. In this group (coxsackieviruses B1 and B4), pathologic changes in the heart were synchronous with maximal cardiac titers of virus. Fourteen-day-old mice infected with coxsackieviruses B2 and B3 developed nontransmural necrotizing myocarditis in which maximal pathologic changes followed peak cardiac titers of virus by several days, whereas three- to five-month-old mice infected with coxsackieviruses B1, B2, B3, or B4 showed maximal susceptibility to destructive lesions in the exocrine glandular pancreas. Therefore, specific susceptibilities to infection with coxsackieviruses group B vary with age of the mouse, virus type (and strain), and organ.