Model of coxsackievirus B3 persistent infection in orally-inoculated BALB/c mouse

Many mouse models of human enterovirus disease have been pro- posed, concerning both acute and persistent infection. However, rather paradoxically since the usual way of contamination is fecal-oral, most of them used a systemic route of infection. The aim of the present work was to follow the development of an experimental enterovirus infection and to study the viral persistence at the organ level. Twenty-eight female 3-week old BALB/c mice were infected with 5 x 10(4) TCID(50) of coxsackievirus B3 (CV-B3), Nancy strain, by oral route using a rigid cannula introduced into the stomach. The kinetics of infection was studied by sacrificing 2 animals at different times post infection (from 1 hour to 90 days). The presence of the virus in various organs (small intestine, heart, pancreas, lung, spleen, kidney, liver) was studied by cell culture and RT-PCR. As soon as one hour post infection, the virus was detected in the small intestine. In the heart, the virus was present at 24 and 48 hours post infection by RT-PCR and culture, respectively. At 5 days post infection, all the organs but the liver were found infected. The virus was detected up to 15 days in kidney, 21 days in pancreas, 30 days in lung and spleen, and 45 days in intestine, by both culture and PCR. The heart was still found infected 90 days post infection by both techniques. These results show the dramatic cardiotropism of CV-B3 inoculated by oral route, with a detection of the virus very soon in the course of infection (24 hours) and a persistence of the virus for more than 3 months. The intestine, the initial target of enterovirus infection, can also be considered as a site of viral persistence.     

In situ study on the pathogenesis and immune reaction of equine herpesvirus type 1 (EHV-1) infections in mice.

The mouse model was used to study the pathogenesis of equine herpesvirus type 1 (EHV-1) after primary and secondary intranasal infections. Within a few hours after infection, EHV-1 was found in nasal and olfactorial epithelium and sub-epithelial cells of the respiratory mucosa, but antigen-specific immune cells were never detected. Next to the lung, EHV-1 was transmitted early and directly to the brain, both via the olfactory route and the trigeminal nerve, but traces of degenerative or inflammatory processes were not detected there. In the lung, the immune cells residing or invading the parenchyma did not contain viral DNA or proteins. The primary immune response in the lungs was an alveolar and interstitial inflammation, dominated by the sequential appearance of neutrophils and macrophages, while the number of T and B lymphocytes remained unaltered. Within 24 hr after re-infection, lymphocytes accumulated around the blood vessels, outnumbering monocytes more than twofold, without neutrophils appearing. The lymphocytes comprised of little more B than T cells and the T cells were predominantly CD8+ cells. Those and B cells infiltrated the parenchyma. These results show the route of virus distribution and demonstrate the lack of antigen-specific immune cells in the lungs of mice after primary intranasal infection with EHV-1.     

Infectious bursal disease virus polyprotein expression arrests growth and mitogenic stimulation of B lymphocytes

Summary. Infectious bursal disease virus (IBDV) causes lymphocytolysis and immunosuppression in infected poultry. The IBDV genome encodes a polyprotein VP243 that is post-translationally cleaved by the VP4 protease into the two structural proteins pVP2 and VP3. The objective of the present study was to determine if IBDV polyprotein induced suppression of bursal B lymphocyte growth and their capacity for proliferation. Bursal B cells were examined both for chickens infected with IBDV and for chickens orally inoculated with a DNA construct expressing IBDV VP243 polyprotein. Bursae were collected at 0, 12, 24 and 48 hours after inoculation. Proliferation of bursal B cells (purified AvBu1⁺ cells) in response to concanavalin A mitogenic stimulation was significantly suppressed by infection at 1 day old with either the classical STC or variant E strains of IBDV. Oral administration of DNA constructs expressing the IBDV VP243 polyprotein from either the classical STC or variant E strains in the pCR3.1 vector resulted in persistent, moderate levels of construct in the bursa until at least 48 hours after inoculation. The VP243 DNA construct similarly induced suppression of proliferation for bursal lymphocytes independently of the virus infection. Expression of VP243 polyprotein in transiently transfected DT40 B lymphocyte culture also suppressed cell growth and proliferative responses to mitogen stimulation. Polyprotein expression did not affect cell viability and suppression of proliferation probably occurred by means of cell cycle arrest. The expression of the mature viral proteins VP2, VP4 or VP3 did not change the rate of cell proliferation or response of B cell cultures to mitogen. The results suggested that IBDV polyprotein is a mediator of immunosuppression.     

Persistent detection of varicella-zoster virus DNA in a previously healthy child after severe chickenpox

In immunocompetent children with primary varicella-zoster virus (VZV) infection, peak viral loads are detected in peripheral blood near the onset of the vesicular rash. VZV DNA concentrations normally diminish and become undetectable within 3 weeks after the appearance of the exanthem. Here, we present a previously healthy, human immunodeficiency virus-negative, 4-year-old boy admitted with severe varicella. High viral loads (>340,000 copies/ml) were found in his blood, and the viral loads remained high for at least 1.5 years. Clinical recovery preceded complete clearance of the virus. General and VZV-specific immune reactivity were intact. NK cells and CD8(+) T cells were activated during acute infection, and VZV-specific CD4(+) T cells were detected at high frequencies. VZV DNA was initially detected in B cells, NK cells, and both CD4(+) and CD8(+) T cells. In contrast, during the persistent phase of VZV DNA detection, the viral DNA was primarily located in CD8(+) T cells. For the first time, we describe the persistent detection of VZV DNA in a previously healthy child.     

Coxsackievirus B3 replication is related to activation of the late extracellular signal-regulated kinase (ERK) signal

MAP kinase signaling has been implicated in coxsackievirus B3 (CVB3) pathogenesis and as necessary in the virus lifecycle. We studied the correlation with extracellular signal-regulated kinase 1/2 (ERK1/2) signaling and virus replication in the presence of coxsackievirus and adenovirus receptor (CAR). In CHO cells that do not expressed CAR, specific ERK1/2 phosphorylation (pERK1/2) was not detected, and progeny virus was not produced after infection. By contrast, in HeLa and CHO-CAR cells, which expressed CAR, the specific early and late pERK1/2 at 0.5 and 8 h were induced, and progeny viruses were produced progressively through 24 h after infection. However, when CHO-CAR cells were infected with replication-defective CVB3, specific pERK1/2 was not detected. In addition, when late pERK1/2 is inhibited by the MEK1 inhibitor PD98059, at 4 h after infection, virus replication significantly decreased. Therefore, our findings suggest that early pERK1/2 is a response to virus binding to CAR, whereas late pERK1/2 is related to the viral replication.     

Persistent parvovirus B19 infections in immunocompromised children

Immunocompromised patients have been shown to suffer from prolonged viral infections often without detectable immune response. Here chronic infections with low virus levels can be frequently observed. In these patients viral DNA can be detected over long periods by polymerase chain reaction (PCR). In this study parvovirus B19 presence was assessed by PCR, immunoblot and enzyme-linked immunosorbent assay in sera from children with mainly oncological and hematological diseases. In 45% of sera B19 DNA was observed. Of the children 25% had IgG antibodies to viral protein 1 and 2 (VP1/2) and 15% to nonstructural protein 1 (NS1). In 6% of children IgM antibodies to VP1/2 were detected. These results indicate that the number of children with immune response to B19 proteins is distinctly lower than the number of children with B19 DNA. Transfusions of blood products might have been a possible route for B19 infection. Establishment and maintenance of a persistent parvovirus B19 infection with or without immune response are enhanced in the analyzed immunocompromised children in comparison with immunocompetent children. A persistence of B19 DNA was demonstrated up to 10 months in patients sera. Received: 22 September 1997     

Rotavirus and coxsackievirus infection activated different profiles of toll-like receptors and chemokines in intestinal epithelial cells

Objective  To understand the inflammatory-immune response in intestinal epithelial cells after infection of rotavirus and coxsackievirus B3. Methods  We examined by quantitative PCR the expression profiles of genes encoding five toll-like receptors (TLR) and levels of three chemokines in response to rotavirus and coxsackievirus B3 infection in a human intestinal epithelial cell line (HT-29 cells). Results  We demonstrated that rotavirus induced significantly increased levels of mRNA expression for TLR2, TLR3, TLR7 and TLR8 in HT-29 cells in a time-dependent manner. In contrast, coxsackievirus B3 did not stimulate mRNA expression for TLR3. Rotavirus and coxsackievirus B3 also induced higher levels of mRNA expression for RANTES, IP-10 and IL-8 during the period of infection in a different manner. Finally, significantly elevated levels of RANTES, IP-10 and IL-8 were detected by ELISA in rotavirus-infected cells from 24 to 48 h. Conclusion  Our findings suggest that different patterns of TLRs and chemokines were induced in the initiation and modulation of immune response to rotavirus and coxsackievirus B3 infection.     

Myocarditis susceptibility in female mice depends upon ovarian cycle phase at infection

Female BALB/c mice were infected with coxsackievirus B3 in the diestrus, proestrus, estrus, or metestrus phases of the ovarian cycle. Cycle stage was determined by vaginal smear. All mice were killed 7 days after infection. Females infected in the diestrus and especially the proestrus phases developed myocarditis. CD4+ T cells expressing interferon-gamma (IFNgamma) infiltrate the myocardium in these two phases, whereas CD4+ T cells expressing IL-4 are more frequent during estrus. Cardiac virus titers were determined 15 h and 7 days after infection. No differences in virus titer were seen at 7 days. These studies show that natural hormone variations can have substantial effects on viral pathogenicity presumably due to hormone effects on the immune system.     

婴儿肠道不同部位T细胞亚群及其表面分子检测——婴儿肠道HIV-感染和复制的可能性分析

通过对婴儿外周血(PB)、肠系膜淋巴结(MLN)和肠上皮内(IE)来源淋巴细胞的比较性研究,以分析婴儿肠道HIV-1感染容许性和HIV-1复制支持性的细胞与分子基础。分离婴儿外周血单个核细胞(PBMC),肠系膜淋巴结淋巴细胞(MLNL)和肠上皮间淋巴细胞(iIEL),制备单细胞悬液,运用流式细胞术结合荧光抗体染色技术:(1)检测不同部位的CD3+T细胞中CD4+辅助性T细胞和CD8+杀伤性T细胞比例;(2)检测不同部位的CD4+T细胞CCR5和CXCR4以及CD69和HLA-DR的表达水平。结果发现:(1)iIEL中的CD4+辅助性T细胞占CD3+T细胞的比例明显低于PBMC和MLNL,而CD8+杀伤性T细胞占CD3+T细胞的比例明显高于PBMC和MLNL;(2)iIEL中CD4+T细胞CCR5和CXCR4以及CD69和HLA-DR的表达水平均明显高于PBMC和MLNL。结果表明:婴儿iIEL中CD4+T淋巴细胞与PBMC和MLNL的CD4+T淋巴细胞相比较,高表达HIV-1入胞共受体CCR5和CXCR4,提示iIEL更易被HIV-1感染;iIEL中CD4+T淋巴细胞高表达细胞活化标记分子CD69和HLA-DR,说明其更有利于HIV-1复制,这种高基础活化率可能与其不断接触小肠来源的食物和共生菌抗原有关。由此可见,婴儿肠道具有了HIV-1感染容许性和HIV-1复制支持性的细胞和分子基础,因此成了HIV-1最易感的解剖部位,这可能是围产期HIV感染的婴儿病毒载量明显高于成年人,且病情进展也比成年人快的原因。     

Chicken infectious anaemia vaccinal strain persists in the spleen and thymus of young chicks and induces thymic lymphoid cell disorders

The chicken infectious anaemia virus (CIAV) infection may induce immunosuppression and persistent infection. The use of vaccination in young chicks is still controversial due to its low immune efficiency. In order to verify the viral persistency of a vaccinal strain of CIAV and its associated-lymphoid cell disorders, 54 1-day-old specific pathogen free chicks were vaccinated (CIAV-VAC^®; Intervet, Millsboro, Delaware, USA) and haematologic examination, expression of viral VP3 gene, humoral response and phenotyping of lymphoid cells were studied in lymphoid organs at various times post vaccination (p.v.). No clinical signs were observed but light heteropaenia was detected in CIAV-vaccinated chicks. The VP3 gene of CIAV was detected by polymerase chain reaction in the thymus and spleen from day 7 until 28 days p.v. Thymic larger CD4⁺CD8⁺ cells increased only at 7 days p.v. while smaller CD4⁺CD8⁺ cells decreased after 14 and 28 days in CIAV-vaccinated birds. The CD4 expression, in contrast to that seen for CD8, decreased in thymocytes from the CIAV-vaccinated group. In the spleen and bursa, the percentage of CD8⁺ cells increased at 7 and 28 days p.v. only, while CD4⁺ cells decreased simultaneously. The vaccinated chicks also exhibited a higher number of splenic CD3^–CD8⁺ cells (natural killer cells). The anti-CIAV antibody responses, however, remained low in most vaccinated chicks and did not persist up to 18 days p.v. These results suggest that the vaccinal virus strain is clinically attenuated but persists in the thymus and spleen in some birds, inducing a low humoral immune response and altering thymopoiesis.     

Enhanced cellular immune response and reduced CD8(+) lymphocyte apoptosis in acutely SIV-infected Rhesus macaques after short-term antiretroviral treatment

Losing the decisive virus-specific functions of both CD4(+) and CD8(+) T lymphocytes in the first weeks after immunodeficiency virus infection ultimately leads to AIDS. The SIV/rhesus monkey model for AIDS was used to demonstrate that a 4-week chemotherapeutic reduction of viral load during acute SIV infection of macaques allowed the development of a competent immune response able to control virus replication after discontinuation of treatment in two of five monkeys. Increasing SIV-specific CD4(+) T-helper-cell proliferation was found in all macaques several weeks after treatment, independent of their viral load. However, only macaques with low viral loads showed persistent T-cell reactivity of lymph node cells. In contrast to animals with higher viral loads, T-helper-cell counts and memory T-helper cells did not decline in the two macaques controlling viral replication. Lymphocyte apoptosis was consistently low in all treated macaques. In contrast, high CD8(+) lymphocyte death but only slightly increased CD4(+) lymphocyte apoptosis were observed during the first weeks after infection in untreated control animals, indicating that early apoptotic death of virus-specific CTL could be an important factor for disease development. Antiretroviral treatment early after infection obviously retained virus-specific and competent T lymphocytes, whereby a virus-specific immune response could develop in two animals able to control the viral replication after cessation of treatment.     

Early events in Epstein-Barr virus infection of human B lymphocytes.

The sequence of Epstein-Barr virus (EBV) and B lymphocyte changes in the 3 days following acute infection was analyzed. By 16 hr the average infected lymphocyte had 1 EBV episome. Nuclear protein-2 (EBNA-2) and EBNA-leader protein (-LP) were detected by 12 hr, and by 32 hr were at the levels of stable EBV infection in lymphoblastoid cell lines (LCLs). At 12 hr, all EBNA-LP and EBNA-2 RNAs were initiated from the Pw promoter. By 36 hr a significant EBNA-LP and EBNA-2 RNA fraction initiated from the upstream Pc promoter. Throughout acute infection, a similar fraction of potentially bicistronic EBNA-LP mRNAs had first exon splices which would result in EBNA-LP translation. By 36 hr c-myc RNA was transiently induced, and CD21 and CD23 RNAs were beginning to increase. This coincided with low-level EBNA-1, EBNA-3A, B, and C, and latent membrane protein-1 (LMP-1) expression. By 46 hr, EBNA-1, the EBNA-3s, and LMP-1 were near the levels ordinarily found in LCLs and a substantial fraction of lymphocytes were in S phase. These results are compatible with a key role for EBNA-2 (or EBNA-LP) in regulating virus and cell gene expression. High-level expression of the EBV-encoded small RNAs, EBERs, was delayed beyond 36 hr and may, therefore, be activated by other virus or cell genes. A 65-kDa virion protein persisted in acutely infected cells. This protein could be a mediator of virus or cell gene expression.     

CD4 T lymphocyte activation in BLV-induced persistent B lymphocytosis in cattle

Bovine leukemia virus (BLV) is an oncogenic retrovirus in the human T cell leukemia virus family. BLV infects B lymphocytes and induces a nonmalignant persistent lymphocytosis (PL) and leukemia/lymphoma in cattle. There is evidence that CD4 T lymphocytes are activated during BLV infection and promote the development of PL. How CD4 T lymphocytes are activated by BLV infection is not known. We observed that CD4 T lymphocytes from PL cattle proliferated in the presence of autologous, irradiated peripheral blood mononuclear cells (PBMC), whereas no proliferation occurred in cell cultures from BLV-infected non-PL cattle. Proliferation required direct contact with metabolically active irradiated PBMC but was not associated with viral protein expression or inhibited by antibodies to BLV. Unexpectedly, B lymphocytes alone failed to account for the irradiated PBMC stimulation of CD4 T lymphocytes. These observations and the magnitude of the proliferative response suggest that activation is polyclonal and involves mechanisms other than BLV antigen-specific stimulation.     

Analysis of the B and T cell response in guinea pigs induced with recombinant vaccinia expressing foot-and-mouth disease virus structural proteins

Summary.  Recombinant vaccinia viruses expressing foot-and-mouth disease virus (FMDV) P1 and VP1 genes have been used to study the immune response induced by these viral polypeptides in guinea pigs. Anti-FMDV antibodies, but not neutralizing activity, were detected in the sera from immunized animals. The results indicate that both CD4+ and CD8+ FMDV-specific T cells were induced by the vaccinia recombinants. Consistently with the activation of CD4+ T cells, lymphocytes from immunized animals specifically proliferated in vitro in response to whole virus. The induction of virus-specific CD8+ T cells was determined by CTL assay of immune splenocytes restimulated in vitro with FMDV infected cells. Altogether, the results obtained indicate that both B and T cell immune responses to FMDV are elicited upon immunization of guinea pigs with vaccinia recombinants expressing FMDV structural polypeptides. Accepted September 18, 1997 Received June 19, 1997     

Coxsackievirus B4 infection alters thymic, splenic, and peripheral lymphocyte repertoire preceding onset of hyperglycemia in mice.

Diabetogenic Coxsackievirus B4 infection may trigger autoimmune islet loss in diabetes-susceptible mice, resulting in hyperglycemia in nearly 90% of the animals at 6-8 weeks postinfection (p.i.). To ascertain whether changes in lymphocyte repertoire following infection could predispose these animals to diabetes, alterations in their thymic, splenic, and peripheral lymphocytes were analyzed. Additionally, lymphocyte changes were correlated with the virus load in these tissues and with lymphocyte migration to the inflammatory pancreas. Splenic B lymphocytes more than doubled at 72 hr p.i. and then continuously decreased by 16% of the noninfected controls at 8 weeks p.i. T lymphocytes (CD4+ + CD8+) decreased by about 50% at 72 hr and then increased to the control level by 8 weeks p.i.; CD8+ subset continuously decreased by 40% of the control at 8 weeks, resulting in a 67% increase in CD4+/CD8+ ratio. Macrophages and CD5+ B subset increased at 72 hr and then dipped by 93% and 84%, respectively, at 8 weeks. In contrast, peripheral B lymphocytes increased by 74% and T lymphocytes decreased by 11% at 8 weeks p.i. Macrophages increased by twofold at 72 hr and then dipped slightly (6%) at 8 weeks, whereas CD5+ B subset increased by 245%. Most prominent thymic T lymphocyte alteration was reflected by about 150% increase in CD4- CD8- cells at 8 weeks p.i. The peak viremia occurred at 72 hr p.i., with highest and lowest virus in the spleen and thymus, respectively. The thymus cleared virus by 3 days, the other tissues by 7 days. Insulitis and acinar necrosis followed infection; infiltrating lymphocytes were mostly CD4+. Virus-induced abnormal lymphocyte maturation may contribute to the development of insulitis and hyperglycemia.     

A subset of anti-rotavirus antibodies directed against the viral protein VP7 predicts the onset of celiac disease and induces typical features of the disease in the intestinal epithelial cell line T84

Celiac disease (CD) is an autoimmune disorder of the small intestine triggered by environmental factors in genetically predisposed individuals. A strong association between type 1 diabetes (T1DM) and CD has been reported. We have previously shown that rotavirus infection may be involved in the pathogenesis of CD through a mechanism of molecular mimicry. Indeed, we identified a subset of anti-transglutaminase IgA antibodies that recognize the rotavirus viral protein VP7. In this study, we aimed at evaluating whether such antibodies may predict the onset of CD in children affected by T1DM. Moreover, to further analyze the link between rotavirus infection and pathogenesis of CD, we analyzed the effect of anti-rotavirus VP7 antibodies on T84 intestinal epithelial cells using the gene-array technique, complemented by the analysis of molecules secreted in the supernatant of stimulated cells. We found that anti-rotavirus VP7 antibodies are present in the vast majority (81 %) of T1DM-CD tested sera, but are detectable also in a fraction (27 %) of T1DM children without CD. Moreover, we found that anti-rotavirus VP7 antibodies are present before the CD onset, preceding the detection of anti-tTG and anti-endomysium antibodies. The gene-array analysis showed that purified anti-rotavirus VP7 antibodies modulate genes that are involved in apoptosis, inflammation, and alteration of the epithelial barrier integrity in intestinal epithelial cells, all typical features of CD. Taken together, these new data further support the involvement of rotavirus infection in the pathogenesis of CD and suggest a predictive role of anti-rotavirus VP7 antibodies.