人禽流感病毒H5N1多价重组痘苗病毒(天坛株)疫苗在小鼠中的免疫原性研究

Objective To develop an effective and broad immune protective H5N1 vaccine.Methods We first developed two recombinant vaccinia ( Tiantan strain) virus ( rTTV ) based H5N1 vaccines, which consisted of bicistron expressing the hemagglutinin(HA) and matrix protein 2(M2), or bicistron expressing the neuraminidase(NA) and matrix protein 1 (M1). The expression of H5N1 protein in rTTVs was confirmed. We immunized the BALB/c mice twice with two kind of dose ( 104 PFU, 107 PFU)using different combination. Subsequently, we assessed the humoral and cellular immune response in vaccinated mice. Results Our data showed that rTTV-based H5N1 vaccine induced rapidly robust HA- and NAspecific antibody level and IFN-γ secreting form cell(SFC) with either single dose of 107 PFU or twice dose of 104 PFU or 107 PFU. We also detected significant neutralizing antibody and matrix-specific immune response. In addition, we found that immunization with two kind of rTTV-based H5N1 vaccines induced much high level of M2-specific antibody than that with single of rTTV-based H5N1 vaccine. Conclusion rTTVbased H5N1 vaccines in this study elicited board array of immunity and our study offers a promising alternative H5N1 vaccine candidates with favorable potential to prevent various H5N1 pandemic.     

前S1蛋白（PreS1）在乙型肝炎诊断中的作用

目的探讨HBV感染者血清中PreS1与HBV-DNA、HBeAg三者间的关系,进而评估PreS1在HBV感染、复制、诊断中的作用。方法收集1750例乙型肝炎患者血清,按不同HBV-M进行分组：HBsAg（＋）、HBeAg（＋）、HBcAb（＋）为A组,HBsAg（＋）、HBeAb（＋）、HBcAb（＋）为B组,HBsAg（＋）、HBcAb（＋）为C组,HBsAg（＋）为D组,采用酶联免疫法（ELISA）检测乙肝病毒PreS1与HBV血清标志物,荧光定量PCR法检测HBV-DNA。结果以HBV-DNA＆gt;5×102拷贝/mL为阳性诊断标准,不同HBV-M模式中,PreS1与HBV-DNA的检出率无显著差异（P＆gt;0.01）,PreS1与HBV-DNA的总符合率高于HBeAg与HBV-DNA的符合率,在HBeAg（-）患者中仍可不同程度地检出PreS1与HBV-DNA。结论PreS1可作为HBV感染、复制的一项新指标,在乙型肝炎诊断中具有很大的应用价值。     

登革热病毒RNA实时荧光定量RT-PCR检测方法的建立

目的建立TaqMan探针实时荧光定量RT-PCR方法,测定登革热病毒（DV）及DV病毒的RNA拷贝数。方法利用TaqMan探针,建立实时荧光定量RT-PCR方法,通过对登革热病毒RNA定量外标准品的定量分析,优化反应体系,检测TaqMan探针实时荧光定量RT-PCR方法的灵敏度、特异性和重复性。结果该方法检测灵敏度可达1×103copies/mL,特异性及重复性良好,对同一样品进行5次重复检测,其循环阈值的平均标准偏差为0.792。结论TaqMan探针实时荧光定量RT-PCR法特异性、敏感性高,稳定性好,可用于定量测定登革热病毒及DVRNA载量。     

Prevalence of viral infection markers by polymerase chain reaction amplification and interferon-alpha measurements among patients undergoing lumbar puncture in an emergency department

Aseptic meningitis is a frequent diagnosis in emergency departments. Nevertheless, viral investigations are not carried out currently and the viral etiology in adult population has not been studied extensively. We conducted a prospective study including all consecutive patients undergoing lumbar puncture during a 15 months period in an adult emergency department. Bloody and purulent cerebrospinal fluid (CSF) were excluded. The main tests undertaken were: CSF genomic amplification by the polymerase chain reaction (PCR) for neurotropic viruses and serum and CSF interferon-alpha (IFN-alpha) measurements. Among 194 patients included, 45 had and 149 did not have aseptic meningitis. Of 45 patients with aseptic meningitis, 10 had alternative non-virological final diagnosis, and 35/45 were presumed to have neurological disorders of viral origin. Patients (27/35) completed virological analysis: 21/27 (78%) had either positive viral PCR (enterovirus: 8 patients, Varicella zoster virus (VZV): 5, Epstein-Barr virus (EBV): 2, herpes simplex virus (HSV): 1, human herpes virus 6: 1) or only raised serum or CSF IFN-alpha (4 patients). Overall, 59% of patients with a positive viral PCR had either CSF or serum raised IFN-alpha. Twentyone patients without meningitis had either positive viral PCR (enterovirus: 3 patients) or only high serum IFN-alpha level (18 patients). In the setting of aseptic meningitis diagnosed in an adult emergency department, viruses are the most common agents encountered, with enterovirus and VZV as the two main etiological agents. Current CSF viral genome amplification and IFN-alpha measurement are informative and could be useful to confirm the viral origin of various neurological disorders, although the sensitivity and specificity of IFN-alpha measurement for the diagnosis of viral infection need further confirmation.     

Kinetics of viremia and acute liver injury in relation to outcome of neonatal woodchuck hepatitis virus infection

The kinetics of serum viral responses and acute liver injury were studied during neonatal woodchuck hepatitis virus (WHV) infection in relation to the chronic or resolved outcome. The mean concentrations of serum WHV DNA and surface antigen were significantly higher by week 10 post infection in chronic infections compared to resolving infections, and diverged even further by the time of peak viral load development in serum (week 12). After week 12, these viral markers were detected less frequently with time and at lower concentrations in the resolved outcome. In both outcomes, mean serum activities of hepatic enzymes became increased significantly above baseline by weeks 10-12, peaked at week 14, and normalized by weeks 20-22, thus indicating transient acute liver injury. The increasing liver injury responses were comparable between outcomes at week 12, when serum viral load was markedly higher in the developing chronic infections. This suggested a deficiency in early non-cytolytic control of infection in the chronic outcome. At week 14, liver injury was significantly greater in the resolved outcome and associated with higher mean Fas ligand (FasL) and perforin messenger RNAs (mRNAs) in liver compared to the chronic outcome. This indicated greater immune-mediated killing of infected hepatocytes during resolution. Thus, chronicity as an outcome of neonatal WHV infection develops relatively early during the acute phase of infection due to reduced immune-mediated clearance of infected hepatocytes by both cytolytic and non-cytolytic processes.     

Variations in histological patterns of interstitial pneumonia between connective tissue disorders and their relationship to prognosis

Aims and methods: Pulmonary parenchymal disease is common in patients with connective tissue disorders (CTDs). However, most reports precede recognition of non‐specific interstitial pneumonia (NSIP). We have therefore reviewed 54 lung biopsies from 37 patients with polymyositis/dermatomyositis (PM/DM) (n = 13), Sjögren's syndrome (n = 5), rheumatoid arthritis (n = 17) and systemic lupus erythematosus (SLE) (n = 2) to assess the overall and relative frequencies of patterns of interstitial pneumonia and their impact on prognosis. Results and conclusions: NSIP was the most common pattern with an overall biopsy prevalence of 39% and patient prevalence of 41%. There was variation in prevalence between individual CTDs, with PM/DM commonly showing organizing pneumonia (n = 5), rheumatoid arthritis showing follicular bronchiolitis (n = 6) and Sjögren's syndrome showing chronic bronchiolitis (n = 4). These patterns presented either separately or in association with NSIP, occasionally with different patterns in biopsies from separate lobes. Only four patients showed a pattern of usual interstitial pneumonia (UIP): two with rheumatoid arthritis and one each with PM/DM and SLE. Overall mortality was 24%, the most frequently associated pattern being fibrotic NSIP (n = 5). In nine cases, pulmonary presentation preceded the systemic manifestation of the CTDs. When patients with CTDs present with chronic interstitial lung disease, the most common pattern is NSIP, although there is variation in pattern prevalence between individual disorders and patterns of interstitial pneumonia frequently overlap. These data suggest a different biology for intestitial pneumonias in CTDs when compared with the idiopathic interstitial pneumonias where UIP is the most common pattern. Mortality is similar to that seen in idiopathic NSIP and, coupled with pulmonary presentation occurring prior to the systemic manifestation of disease, this may have a bearing on the origin of some cases of putative idiopathic NSIP.     

A genomic map of infectious laryngotracheitis virus and the sequence and organization of genes present in the unique short and flanking regions

We present a genomic map of infectious laryngotracheitis virus (ILT) and an 18,912 bp sequence containing the entire unique short region and a portion of the flanking short repeats. In determining the genomic map, an 856 bp region repeated as many as 13 times was identified within the short repeats. The unique short sequence contains nine potential open reading frames (ORFs). Six of these ORFs show homology to other known herpesvirus unique short genes. Using the herpes simplex virus nomenclature, these genes are the US2, protein kinase, and glycoproteins G, D, I, and E (ORF 1, 2, 4, 6, 7, and 8, respectively). Interestingly, an open reading frame with homology to HSV-1 UL47 (ORF 3) is found in the unique short. One very large open reading frame (ORF 5) is present and contains a threonine-rich, degenerate repeat sequence. This gene appears to be unique to ILT among sequenced herpesviruses. Two ORFs were identified within the short repeat (SR) region. SRORF 1 is homologous to a gene (SORF3) found in the unique short region in both MDV and HVT, and appears to be specific to avian herpesviruses. SRORF 2 has homology to HSV US10.The nucleotide sequence data reported in this paper have been submitted to the GenBank nucleotide sequence data-base and have been assigned the accession number U28832.     

E. coli-expressed recombinant norovirus capsid proteins maintain authentic antigenicity and receptor binding capability

The baculovirus expression system has been widely used to produce the capsid proteins of Norovirus (NV) and the proteins form virus-like particles (VLPs) that are useful in many studies, such as immunology, diagnosis, and host-receptor interaction. We report here the application of the E. coli expression system in the production of recombinant NV capsid proteins. In a direct comparison of a previous well-characterized NV strain (VA387), we have demonstrated that the E. coli-expressed capsid proteins maintain the same antigenicity and receptor binding specificity as that of the baculovirus-expressed capsid, although the E. coli-expressed VA387 proteins did not form VLPs. Using the E. coli-expression system, we characterized the receptor-binding patterns of three additional NV strains (OIF1998, Parris Island and VA115), in which OIF1998 binds to HBGA of nonsecretors but did not bind or binds weakly to the HBGA of secretors, as seen in strain VA207. Parris Island binds to HBGA of types A and B but not type O secretors and nonsecretors. VA115 did not show specific binding to any A, B, O secretor nor nonsecretor, which is also observed when the capsid protein of this strain was expressed in baculovirus. Our data indicate that VLP formation is not required for receptor binding, and that the bacteria expression system offers a simple alternative for large production of NV capsid protein for various research purposes, particularly for strains generating low yields in the insect cells.     

Elevated interleukin-18 levels in bronchoalveolar lavage fluid of patients with eosinophilic pneumonia

BACKGROUND: Interleukin (IL)-18 can induce Th2 cytokine production particularly in collaboration with IL-2. Accumulation of Th2 cells and increased levels of Th2 cytokines are found in bronchoalveolar lavage fluid (BALF) from patients with eosinophilic pneumonia (EP). To evaluate the role of IL-18 in the pathogenesis of EP, we measured the concentration of IL-2, IL-12, IL-18, and Th2 cytokines in BALF from patients with EP. METHODS: The concentrations of interferon (IFN)-gamma, IL-2, IL-5, IL-10, IL-12, IL-13, and IL-18 in BALF were measured in patients with idiopathic acute eosinophilic pneumonia (AEP), with idiopathic chronic eosinophilic pneumonia (CEP), with sarcoidosis and healthy volunteers (HV). RESULTS: The BALF concentrations of Th2 cytokines, IL-5, IL-10, and IL-13, were higher in patients with EP than in sarcoidosis and control. The IL-2 level in BALF was higher in EP than in sarcoidosis and control. The IL-18 and IL-12 (p40 + p70) levels were higher in patients with EP than sarcoidosis, while the level of IL-12 (p70) was below the detection limit in patients with EP. There was a significant correlation between IL-2 level and both IL-5 and IL-13 in BALF of patients with EP. CONCLUSIONS: Our findings suggest that IL-18 may contribute to Th2 cytokine-dominant responses in patients with EP in collaboration with IL-2.     

Immunohistochemical and in situ detection of cytomegalovirus in lung autopsies of children immunocompromised by secondary interstitial pneumonia

Secondary interstitial pneumonia (SIP), a disease affecting patients immunocompromised by primary underlying diseases during their treatment in hospital, is frequently associated with cytomegalovirus (CMV) infection, a potentially treatable condition. However, in many cases, no infectious agent can be determined, and this clinical disease rapidly progresses to death. Theoretically, SIP could be caused by CMV, which may be present in such small amounts or such configuration that routine histopathological analysis or viral culture techniques cannot detect the virus. To test the hypothesis that immunohistochemistry (IH) and in situ detection by hybridization (ISH) provides more accurate results than the mere histological demonstration of CMV inclusions, these methods were applied to 37 autopsied lung sections obtained from children immunocompromised by primary underlying diseases and who died of SIP. As a result, the cases were subdivided into three groups: (1) children with SIP CMV inclusions (Diffuse alveolar damage-DAD-related) (n = 7); (2) children with SIP without classical viral inclusions (CMV-DAD-related) (n = 3); (3) children with SIP exhibiting nuclear cytopathic effect (not CMV-NSIP-related) (n = 27). In the first group, all three techniques yielded clearly positive results, whereas IH and ISH indicated that three of the children of the second group had CMV-related DAD without histological demonstration of CMV inclusions. In the third group, there were no positive CMV signals. These data indicate that DAD-related CMV infection is an important cause of SIP and of death in children immunosuppressed by primary underlying diseases, and that IH and in situ detection were more sensitive than the histological demonstration of CMV inclusions. A direct involvement of CMV in SIP exhibiting DAD is likely, but not in the non-specific interstitial pneumonia (NSIP) pattern. We conclude that all children with primary underlying diseases should be investigated for CMV SIP using sensitive IH and in situ tests in conjunction with histological routine procedures.