一起病毒性脑炎病因的分子生物学鉴定

目的探讨2003年山东济南地区病毒性脑炎（VE）流行病因。方法通过随机PCR及肠道病毒特异性的RT—PCR扩增获得病毒分离株特异性核酸片段，测序并进行同源性分析和多序列比对，确定病毒种类及分型。结果从患者标本中分离到5株病毒，以随机PCR获得其中1株病毒的核酸片段，经BLAST分析发现其与肠道病毒同源性最高，然后分别测得5株病毒5’端非编码区及部分VP1区核酸序列，经序列比对分析，确定5株病毒均为小RNA病毒科肠道病毒属的柯萨奇B5，并且均与2002--2004年间浙江无菌性脑膜炎流行期间分离的柯萨奇B5分离株同源性最高（95％以上）。结论柯萨奇B5是2003年济南地区VE流行的重要病因之一。     

Detection of viral RNA in experimental coxsackievirus B3 myocarditis of mice using the polymerase chain reaction.

The presence of the viral RNA in the myocardium in experimental coxsackievirus B3 myocarditis of mice was investigated using the polymerase chain reaction (PCR). Four-week-old C3H/He mice (n = 35) were inoculated with coxsackievirus B3 (Nancy strain, 10(5) plaque-forming units/mouse). We used a pair of primers, which encompass a part of the 5' end sequence of the coxsackievirus B3 genome and can also detect many enteroviral RNAs. We found that hearts were positive for the viral RNA from 2 to 21 days after virus inoculation by PCR, but negative after day 28 and in non-infected control mice (n = 5). The viral RNA were detected by PCR later than by culture. Thus, the detection of the viral RNA using enzymatic amplification is more rapid and easier and may be more useful for clinical diagnosis of viral myocarditis than conventional culture methods. However, virus persistence in the myocardium long after virus inoculation is unusual in this model.     

Specific interactions of mouse organ proteins with the 5'untranslated region of coxsackievirus B3: potential determinants of viral tissue tropism

Coxsackievirus B3 (CVB3) infects multiple organs of humans and causes different diseases such as myocarditis, pancreatitis, and meningitis. However, the mechanisms of organ-specific tropism are poorly understood. Coxsackievirus and adenovirus receptor (CAR) have been known to be important determinants for tissue tropism. However, current data on CAR mRNA expression in certain organs of mouse did not correlate well with the susceptibility of the respective tissues, suggesting that intracellular proteins may also play important roles in the regulation of viral infectivity through interaction with viral RNA. To search for such proteins and their interacting sites, we performed in situ hybridization to detect viral RNA in the organs of 4-week- and 10-week-old CVB3-infected mice and then correlated the data to patterns of host protein-viral RNA interactions. We found that heart and pancreas are the most heavily infected organs while the kidney remains highly resistant to the virus. The brain exhibited localized foci of viral replication, while the heart and liver showed random distribution of CVB3 RNA. The exocrine pancreas is highly susceptible to CVB3 infection but the endocrine cell type is resistant. In contrast to infections in other organs, mouse heart appears more resistant to CVB3 infection with increasing age. This resistance to infection in the kidney and older heart correlates well with the interaction of a 28 kDa mouse protein with the antisense sequence of nucleotides 210-529 of CVB3 5UTR. In addition, more intensified protein interactions were found within the nucleotides 530-630, a region that contains the internal ribosome entry site, which supports the previous findings that this segment plays critical roles in regulation of viral replication.     

Coxsackie B viruses use multiple receptors to infect human cardiac cells

Viral myocarditis is an inflammatory disease of the heart muscle that can be fatal. The primary viruses that have been linked to myocarditis and dilated cardiomyopathy are the human enteroviruses. The most common viruses associated with this disease are the Coxsackie B viruses and in particular Coxsackievirus B3 and Coxsackievirus B5. Early events in viral infection include attachment of the virus onto cell surface receptors. Even though, CD55 and Coxsackievirus adenovirus receptor protein (CAR) have been identified as receptors for Coxsackievirus B3, the exact mechanisms that Coxsackievirus B3 and B5 use to infect the cardiac muscle are not yet known. In this study, attempts were made to inhibit Coxsackievirus B3 and Coxsackievirus B5 infectivity of cardiac cells by using CAR and CD55 specific antibodies. The results show that these antibodies could not completely inhibit Coxsackievirus B3 and Coxsackievirus B5 binding or infectivity. Furthermore five new proteins have been identified that are used by Coxsackieviruses for binding to cardiac tissue and are distinct from CAR or CD55, leading us to believe that these viruses may use a different set of receptors for infection of cardiac muscle.     

Persistence of virus and viral genome in myocardium after coxsackievirus B3-induced murine myocarditis.

Following infection with Coxsackievirus B3 (CVB3), A-strain mice develop ongoing myocarditis that persists after the virus ceases to be cultivatable from heart tissue. We studied the natural history of this virus-induced but apparently autoimmune inflammation by means of in situ hybridization (ISH) and by polymerase chain reaction (PCR). Both ISH and culture allowed detection of virus up to 2 weeks post-infection in virtually all heart tissues. In contrast, PCR revealed the presence of viral genome for a substantially longer period of time, i.e. at least 34 days after CVB3 infection. Similarly, the majority of mice showed myocardial inflammation at this time point. However, the persistence of virus did not correlate with ongoing myocarditis, and vice versa. Most mice with ongoing myocarditis produced heart myosin autoantibodies, most probably as a result of tissue damage. The lack of correlation between presence of ongoing inflammation and persistence of virus supports our previous view that the late phase of CVB3-induced myocarditis is mediated by autoimmunological mechanisms.     

Change in the cells that express connective tissue growth factor in acute Coxsackievirus-induced myocardial fibrosis in mouse

Cardiac fibrosis and inflammation are major pathologic conditions that result from viral myocarditis. Connective tissue growth factor (CTGF) stimulates fibroblast proliferation and induces production of extracellular matrix molecules. We studied the correlation between CTGF and cardiac fibrosis in an acute Coxsackievirus B3 (CVB3) myocarditis animal model. Eight-week-old BALB/c mice were infected intraperitoneally with 10(4) plaque forming units (PFU) of CVB3. Myocardial inflammation peaked on day 7 and decreased markedly by day 14 post-infection (pi); cardiac fibrosis was noted from day 7 and peaked on day 14. By contrast, CTGF was weakly expressed by the interstitial cells in uninfected control hearts and also in the hearts of day 3 pi. CTGF expression measured by real-time PCR was elevated on day 3 and peaked on day 7 pi. TGF-beta expression peaked at day 7 pi. The cell type of CTGF expression changed from interstitial cells to myocytes after virus infection. On day 7, CTGF was strongly expressed by myocytes and inflammatory cells surrounding calcified necrotic areas. In addition, cardiac myocytes expressed CTGF on day 14. Our results, based on an acute CVB3 model of myocarditis, provide evidence that CTGF may mediate the development of fibrosis after viral myocarditis, and that the cells expressed CTGF changes during the course of viral myocarditis.     

Cellular immune responses in mice challenged with an amyocarditic variant of Coxsackievirus B3

An amyocarditic variant of a temperature-sensitive (ts) mutant derived from the parent myocarditic variant Coxsackievirus B3 (CVB3m) was studied in a murine model of CVB3m-induced myocarditis to assess virus-induced antigens and their possible role in the disease process. Amyocarditic variant ts5R induced a heart tissue antigen(s), extractable by hypertonic KC1, which inhibited migration of peritoneal exudate cells from CVB3-inoculated myocarditic mice in an agarose droplet cell-migration-inhibition assay. The ts5R variant was amyocarditic at inoculum doses of 10(3) to 10(8) plaque-forming units per mouse, but in cyclophosphamide-immunosuppressed mice, ts5R induced myocarditis. Viable ts5R served as a vaccine and protected mice against CVB3m-induced myocarditis. Murine neonatal skin fibroblasts (MNSF) infected with either virus served as in vitro targets and were lysed by splenic cytotoxic T lymphocytes from mice inoculated with either virus variant. ts5R and CVB3m replicated to similar titers in murine neonatal skin fibroblasts (MNSF) at 24 hr postinoculation (pi), but differences in titers were found by 72 hr pi. Levels of natural killer cell activities in spleens of ts5R-inoculated mice were slightly lower than in spleens of CVB3m-inoculated mice at 7 days pi. The data suggest that viral induction of new antigens on target cells and viral induction of specific cytotoxic T lymphocytes that recognize these antigenic changes do not always result in induction of myocarditis.     

Detection of enteroviral infection in myocardial tissues by polymerase chain reaction (PCR)

Objective: To determine if enteroviral infection is linked to myocarditis and dilated cardiomyopathy. Enteroviruses, especially coxsackieviruses, appear to be the most common agents of viral myocarditis.
Methods: We collected 53 endomyocardial biopsies and two autopsy specimens from 41 patients affected by myocarditis or dilated cardiomyopathy. The patients were diagnosed clinically, hemodynamically, virologically and histologically (Dallas classification). We tested for the presence of enteroviral sequences by PCR, using 5' non-coding (coxsackievirus B3, CB3, map position 450–474, 584–603) derived primers. Specificity was confirmed using the Southern blot. We used a fraction of CB3 acutely infected mouse myocardial tissue as a control.
Results: We detected enteroviral sequences in four patients with active myocarditis, borderline myocarditis or cardiomyopathy. The patient with active myocarditis had shown neutralizing antibodies in serologic analysis for coxsackievirus B3 and B5.
Conclusions: The data support a weak link of enteroviral infection to human myocarditis and dilated cardiomyopathy, at least when using a PCR assay on biopsies.     

Coxsackievirus B3-associated myocardial pathology and viral load reduced by recombinant soluble human decay-accelerating factor in mice

Coxsackievirus B3 (CVB3) infection can result in myocarditis, which in turn may lead to a protracted immune response and subsequent dilated cardiomyopathy. Human decay-accelerating factor (DAF), a binding receptor for CVB3, was synthesized as a soluble IgG1-Fc fusion protein (DAF-Fc). In vitro, DAF-Fc was able to inhibit complement activity and block infection by CVB3, although blockade of infection varied widely among strains of CVB3. To determine the effects of DAF-Fc in vivo, 40 adolescent A/J mice were infected with a myopathic strain of CVB3 and given DAF-Fc treatment 3 days before infection, during infection, or 3 days after infection; the mice were compared with virus alone and sham-infected animals. Sections of heart, spleen, kidney, pancreas, and liver were stained with hematoxylin and eosin and submitted to in situ hybridization for both positive-strand and negative-strand viral RNA to determine the extent of myocarditis and viral infection, respectively. Salient histopathologic features, including myocardial lesion area, cell death, calcification and inflammatory cell infiltration, pancreatitis, and hepatitis were scored without knowledge of the experimental groups. DAF-Fc treatment of mice either preceding or concurrent with CVB3 infection resulted in a significant decrease in myocardial lesion area and cell death and a reduction in the presence of viral RNA. All DAF-Fc treatment groups had reduced infectious CVB3 recoverable from the heart after infection. DAF-Fc may be a novel therapeutic agent for active myocarditis and acute dilated cardiomyopathy if given early in the infectious period, although more studies are needed to determine its mechanism and efficacy.     

Discovery of a novel human picornavirus in a stool sample from a pediatric patient presenting with fever of unknown origin

Fever of unknown origin (FUO) is a serious problem in the United States. An unidentified agent was cultured from the stool of an infant who presented with FUO. This virus showed growth in HFDK cells and suckling mice. Using DNase sequence-independent single-primer amplification, we identified several nucleotide sequences with a high homology to Theiler's murine encephalomyelitis virus. Nearly full-length viral genome sequencing and phylogenetic analysis demonstrate that this virus is a member of the Cardiovirus genus of the Picornaviridae family.     

Differential expression of coxsackievirus and adenovirus receptor on alveolar epithelial cells between fetal and adult mice determines their different susceptibility to coxsackievirus B infection

Coxsackievirus B (CVB) can cause aseptic meningitis, myocarditis and respiratory disease, especially in newborn infants. To compare the susceptibility to CVB infection of fetal and adult mice, we prepared primary alveolar epithelial cells (AECs) from lungs of BALB/c mice. In contrast to fetal mouse AECs, those of adults were less susceptible to CVB3 infection, as indicated by decreased cytopathic effects, and reduced levels of viral particles bound at the cell surface. In adult mouse AECs, amplification of the viral genome and virus capsid protein VP1 synthesis were concomitantly reduced. In addition, the cell-surface expression of coxsackievirus and adenovirus receptor (CAR), which plays a key role in the initiation of CVB and pulmonary infection, was downregulated in adult mouse AECs. These findings demonstrate that adult mouse AECs are less susceptible to CVB3 due to decreased CAR levels. Thus, these findings strongly indicate that the level of virus receptors on AECs is one of the crucial determinants for the age-dependence of CVB virulence in the mouse lung.     

Enteroviruses and idiopathic dilated cardiomyopathy.

Previous studies have suggested that some cases of idiopathic dilated cardiomyopathy (IDC) are due to persistent viral infection following an episode of viral myocarditis. Viral RNA sequences have recently been detected in material from patients with IDC using molecular biological techniques. We tested 40 samples from recipients' hearts explanted at cardiac transplantation for the presence of enteroviral RNA sequences, using a Northern blotting technique. Material from 19 cases of IDC and 21 cases of non-cardiomyopathic cardiac failure was examined together with Coxsackie-virus-infected neonatal mouse heart as a positive control and non-infected adult mouse heart as a negative control. A sharp band of viral RNA was detected in the positive control sample. No hybridization signal attributable to viral RNA was obtained for the negative control or for any of the test samples. We conclude that the role of enteroviruses in the pathogenesis of cardiomyopathy is not fully established and that further study is warranted.     

Detection of Coxsackievirus B3 RNA in myocardial tissues by the polymerase chain reaction.

Coxsackievirus B3 is a possible etiologic agent in some forms of myocarditis and idiopathic dilated cardiomyopathy. A method for the detection of coxsackievirus B3 RNA was developed using the polymerase chain reaction based on the amplification of a cDNA copy of the positive-strand viral RNA. The fidelity of the method was established in two murine models for coxsackie B3 myocarditis. All cardiac specimens with adequate RNA for study from coxsackie B3-infected mice contained detectable viral RNA, in contrast to none of control specimens from noninfected mice. The sensitivity of the technique was established at approximately 1 to 100 plaque-forming units of virus per gram of tissue, and the specificity was established as limited to the coxsackievirus B3 serotype among nine viruses tested. In patients with myocarditis, one of five specimens contained detectable viral RNA, whereas none of 11 specimens from patients with idiopathic dilated cardiomyopathy or 21 myocardial specimens from patients with a wide variety of other cardiac disorders contained detectable coxsackie B3 viral RNA. The results show that the polymerase chain reaction is a useful means for detecting coxsackie viral RNA and its application should help in the evaluation of hypotheses concerning the infectious etiology of human myocarditis and idiopathic dilated cardiomyopathy.     

A mechanism of immunoreceptor tyrosine-based activation motif (ITAM)-like sequences in the capsid protein VP2 in viral growth and pathogenesis of Coxsackievirus B3

Coxsackievirus B3 (CVB3) is an RNA virus that mainly causes myocarditis. We have reported previously that immunoreceptor tyrosine-based activation motif (ITAM)-like sequences are contained in the capsid protein VP2 of CVB3. The substitution of two tyrosines for phenylalanines in the ITAM-like region causes attenuation of CVB3, possibly via defective viral assembly. In this study, we found that Syk, a downstream molecule of ITAM, interacts with the wild-type (WT) CVB3 VP0 protein, but not with the mutant CVB3 VP0 (called YYFF), and that an inhibitor of Syk reduced the growth of CVB3. The WT CVB3 activated nuclear factor kappa B (NF-κB), a protein activated by ITAM, and eventually induced the production of interleukin-6 (IL-6)—one of the proinflammatory cytokines induced by NF-κB—in macrophages. However, the YYFF form did not. In addition, viral VP2 protein may be dependent on the phosphorylation of an ITAM-like region that affected the activation of NF-κB. Taken together, these results suggest that the ITAM-like sequences in CVB3 VP2 can not only affect viral structure but also act as signals in pathogenesis.     

EXPRESSION OF VASCULAR CELL ADHESION MOLECULE-1 IN MURINE HEARTS WITH ACUTE MYOCARDITIS CAUSED BY COXSACKIEVIRUS B3

Cell-mediated autoimmunity has been strongly implicated in the pathogenesis of the myocardial cell damage involved in viral myocarditis. Using a murine model of acute myocarditis caused by Coxsackievirus B3 (CVB3), perforin-expressing killer cells have been shown to infiltrate the heart, and intercellular adhesion molecular-1 (ICAM-1) together with major histocompatibility complex (MHC) antigen was induced on myocardial cells in acute viral myocarditis. To clarify the immunological mechanisms in more detail, the expression of vascular cell adhesion molecular-1 (VCAM-1) has been examined in the heart of acute myocarditis and on cultured cardiac myocytes treated with interferon-gamma (IFN-γ) and tumour necrosis factor-alpha (TNF-α). The effects of in vivo antibody treatment to VCAM-1 on myocardial damage involved in acute myocarditis were also analysed. CVB3-induced myocarditis resulted in enhanced expression of VCAM-1 on myocardial cells. VCAM-1 expression was also induced on cultured cardiac myocytes by treatment with IFN-γ and TNF-α. The in vivo antibody treatment to VCAM-1 decreased the myocardial damage to some extent, but the effects were not statistically significant. These data suggest that the expression of VCAM-1 on myocardial cells may play at least a partial role in the myocardial damage involved in acute viral myocarditis.     

Heart-specific autoantibodies induced by Coxsackievirus B3: identification of heart autoantigens

Postinfection sera from A.CA/SnJ A.SW/SnJ, B10.S/SgSf, and B10.PL/SgSf mouse strains which varied in their susceptibility to Coxsackievirus B3-induced immunopathology were suspected to contain autoantibodies against cardiac tissue. These sera were used to identify the target myocardial autoantigen(s). Sera pools were made during the peak of the early, virus-induced myocarditis at 5 and 7 days and during the peak of the late, immunopathic phase of myocarditis at Days 15 and 21 after infection. Only the A.CA/SnJ and A.SW/SnJ strains which develop the immunopathic heart disease had heart-specific autoantibodies as determined by indirect immunofluorescence. This panel of sera pools was then tested against solubilized extracts from whole heart and skeletal muscles. Results from Western immunoblotting analyses demonstrated that antibodies to myosin were a prominent feature in the sera of strains which developed immunopathic myocarditis. The immunopathic sera pools were subsequently assayed against low-salt, high-salt, and a number of detergent extracts of heart and skeletal muscle. Anti-myosin was still the most notable reactivity, even though other autoantigens were detected. Absorption with cardiac myosin removed the vast majority of heart reactivity from the pooled sera derived from the A.CA/SnJ and A.SW/SnJ strains as determined within the limitations of the immunofluorescent and immunochemical assays. Both sarcolemmal and A-band staining patterns were abolished by the cardiac myosin absorption. These studies suggest that myosin is one of the major autoantigens in Coxsackievirus B3-induced autoimmune myocarditis.     

Molecular screening by polymerase chain reaction detects panleukopenia virus DNA in formalin-fixed hearts from cats with idiopathic cardiomyopathy and myocarditis.

Viral myocarditis has been suggested as an etiology for cardiomyopathy in several mammalian species. Myocarditis and idiopathic cardiomyopathy have been reported in the domestic cat, although a viral etiology has not been demonstrated. Because of the continuing interest in the potential relationship between viral myocarditis and cardiomyopathy, we evaluated hearts from cats with spontaneous, idiopathic cardiomyopathy for viral genomic material within myocytes by polymerase chain reaction, and for the presence of myocarditis by light microscopy. Thirty-one (31) formalin-fixed hearts from domestic cats who died of idiopathic cardiomyopathy were randomly selected from pathology archives. Seventeen (17) formalin-fixed hearts from healthy cats were similarly selected as normal controls. The polymerase chain reaction (PCR) was used to evaluate myocardial tissue for the presence of viral genome from feline panleukopenia virus, herpes virus, calici virus, and corona virus. Hearts were examined using light microscopy for histologic evidence of myocarditis according to the Dallas criteria. Panleukopenia virus was identified by PCR in 10 of 31 cats with cardiomyopathy but in none of the controls. Neither cardiomyopathic or control cats tested positive by PCR for herpes virus, calici virus, and corona virus. Myocarditis was detected by histologic examination in 18 of 31 cardiomyopathic cats and in none of 17 control cats. Myocarditis and or feline panleukopenia virus genome was detected in felines with idiopathic hypertrophic, dilated, and restrictive cardiomyopathy, suggesting a possible role of viral infection and inflammation in the pathogenesis of cardiomyopathy in this species.