Hepatitis C virus quantification in serum and saliva of HCV-infected patients

The hepatitis C virus (HCV) can be detected in blood and other bodily fluids, such as saliva, semen and gastric juices. The aim of this study was to compare the HCV viral loads in the serum and saliva of infected patients. Twenty-nine patients with detectable HCV RNA in their serum and saliva were included in this study. The HCV viral loads were determined through quantitative real-time polymerase chain reactions. The median viral RNA levels were 5.78 log10 copies in the serum and 3.32 log10 copies in the saliva. We observed that the salivary HCV viral load was significantly lower than the viral load in the serum. Further studies are required to understand the role of saliva in the diagnosis, management and potential transmission of HCV.     

Passive immunization reduces murine cytomegalovirus-induced brain pathology in newborn mice

Human cytomegalovirus (HCMV) is the most frequent cause of congenital viral infections in humans and frequently leads to long-term central nervous system (CNS) abnormalities that include learning disabilities, microcephaly, and hearing loss. The pathogenesis of the CNS infection has not been fully elucidated and may arise as a result of direct damage of CMV-infected neurons or indirectly secondary to inflammatory response to infection. We used a recently established model of mouse CMV (MCMV) infection in newborn mice to analyze the contribution of humoral immunity to virus clearance from the brain. In brains of MCMV-infected newborn mice treated with immune serum, the titer of infectious virus was reduced below detection limit, whereas in the brains of mice receiving control (nonimmune) serum significant amounts of virus were recovered. Moreover, histopathological and immunohistological analyses revealed significantly less CNS inflammation in mice treated with immune serum. Treatment with MCMV-specific monoclonal antibodies also resulted in the reduction of virus titer in the brain. Recipients of control serum or irrelevant antibodies had more viral foci, marked mononuclear cell infiltrates, and prominent glial nodules in their brains than mice treated with immune serum or MCMV-specific antibodies. In conclusion, our data indicate that virus-specific antibodies have a protective role in the development of CNS pathology in MCMV-infected newborn mice, suggesting that antiviral antibodies may be an important component of protective immunological responses during CMV infection of the developing CNS.     

无环鸟苷抗乙型肝炎病毒复制长短程疗法随访观察

慢性乙型肝炎病毒感染s5例,随机分无环鸟苷长程组22例,短程组11例和对照组22例。经一年随访观察,无环鸟苷治疗组见血清HBeAg、DNA-P和HBV-DNA有规律性阴转,短程组于12个月又有部分指标阳转,长程组阴转较多,其血清HBeAg、DNA-P和HBV-DNA阴转率与短程和对照组比较有显著性差异。一年结果四项病毒复制指标全部阴转例数与对照组有显著性差异,结果表明,无环鸟苷长疗程是有较好疗效。     

中枢神经系统感染患儿血清和脑脊液CRP、PCT、TNF-α及MMP-9水平及其临床意义

目的:探讨中枢神经系统感染患儿血清和脑脊液C反应蛋白(CRP)、降钙素原(PCT)、肿瘤坏死因子-α(TNF-α)及基质金属蛋白酶-9(MMP-9)水平及其临床意义。方法:选择2017年1月~2018年6月期间南京市第二医院收治的中枢神经系统感染患儿93例作为研究对象,其中化脓性脑膜炎62例记为化脓性脑膜炎组,病毒性脑炎31例记为病毒性脑炎组,另选取同期于我院治疗的非中枢神经系统感染患儿40例作为对照组,比较各组血清、脑脊液CRP、PCT、TNF-α、MMP-9水平及阳性率,并计算血清和脑脊液CRP、PCT、TNF-α、MMP-9诊断中枢神经系统感染的灵敏度、特异度及准确度。结果:化脓性脑膜炎组患儿血清、脑脊液CRP、PCT、TNF-α及MMP-9水平及阳性率高于病毒性脑炎组和对照组,病毒性脑炎组患儿血清、脑脊液CRP、TNF-α及MMP-9水平及阳性率高于对照组(P<0.05),病毒性脑炎组与对照组血清、脑脊液PCT水平及阳性率比较无统计学差异(P>0.05)。血清或脑脊液CRP+PCT+TNF-α+MMP-9联合检验对中枢神经系统感染具有一定的诊断价值。结论:中枢神经系统感染患儿血清、脑脊液CRP、TNF-α、PCT及MMP-9水平明显升高,其中化脓性脑膜炎患儿血清、脑脊液PCT水平高于病毒性脑炎患儿,血清或脑脊液CRP、PCT、TNF-α及MMP-9联合检验对儿童中枢神经系统感染的鉴别诊断具有较高的价值。     

Analysis of Hepatitis C Virus Decline during Treatment with the Protease Inhibitor Danoprevir Using a Multiscale Model

The current paradigm for studying hepatitis C virus (HCV) dynamics in patients utilizes a standard viral dynamic model that keeps track of uninfected (target) cells, infected cells, and virus. The model does not account for the dynamics of intracellular viral replication, which is the major target of direct-acting antiviral agents (DAAs). Here we describe and study a recently developed multiscale age-structured model that explicitly considers the potential effects of DAAs on intracellular viral RNA production, degradation, and secretion as virus into the circulation. We show that when therapy significantly blocks both intracellular viral RNA production and virus secretion, the serum viral load decline has three phases, with slopes reflecting the rate of serum viral clearance, the rate of loss of intracellular viral RNA, and the rate of loss of intracellular replication templates and infected cells, respectively. We also derive analytical approximations of the multiscale model and use one of them to analyze data from patients treated for 14 days with the HCV protease inhibitor danoprevir. Analysis suggests that danoprevir significantly blocks intracellular viral production (with mean effectiveness 99.2%), enhances intracellular viral RNA degradation about 5-fold, and moderately inhibits viral secretion (with mean effectiveness 56%). The multiscale model can be used to study viral dynamics in patients treated with other DAAs and explore their mechanisms of action in treatment of hepatitis C.     

Selenium, iron, copper, and zinc levels and copper-to-zinc ratios in serum of patients at different stages of viral hepatic diseases

Viral hepatic diseases, especially those induced by the hepatitis B virus, can progress into more serious pathological outcomes and eventually to hepatocellular carcinoma. A growing body of evidence indicates that many trace elements play important roles in a number of carcinogenic processes that proceed through various mechanisms. To examine the status of trace elements during the development of hepatic carcinoma, we determined the selenium, iron, copper, and zinc levels and copper-to-zinc ratios in the serum of patients at different stages of viral hepatic disease. We observed significant changes in the selenium, iron copper, and zinc levels in the serum of patients having hepatocellular carcinoma, relative to those of healthy controls (p<0.05). The mean serum copper level in patients with hepatocellular carcinoma was significantly higher than that of the control group. In contrast, the mean selenium, iron, and zinc levels in patients having hepatocellular carcinoma were significantly lower than those of the control group. In addition, the mean zinc level in the serum of patients with hepatic cirrhosis was significantly lower than that of the control group (p<0.05). Moreover, we found markedly elevated Cu: Zn ratios (p<0.05) in patients having hepatic cirrhosis or hepatocellular carcinoma. Our findings imply that the levels of some trace elements, such as selenium, iron, copper, and zinc, and Cu:Zn ratios, might serve as biomarkers for the increased severity of viral hepatic damage.     

病毒性心肌炎患者血清CK-MB、hs-CRP、IL-35 及cTnI水平的表达

目的:研究血清肌酸激酶同工酶(CK-MB)、超敏C-反应蛋白(hs-CRP)、白介素-35(IL-35)和肌钙蛋白(cTnI)对病毒性心肌炎的诊断意义。方法:研究对象为我院2012年1月至2015年1月间接受诊治的病毒性心肌炎病人63例,作为实验组,选取我院同时期健康体检人60例为对照组。对照两组血清CK-MB、hs-CRP、IL-35及cTnI的检测水平。结果:实验组CK-MB、hs-CRP及cTnI阳性率均明显高于对照组差异有统计学意义(P0.05);实验组的血清CK-MB、hs-CRP、IL-35及cTnI的检测结果:均明显高于对照组差异有统计学意义(P0.05);cTnI敏感度及特异度均是最高的,hs-CRP特异度高于CK-MB,但敏感度较低。结论:病毒性心肌炎患者血清CK-MB、hs-CRP、IL-35及cTnI可作为诊断的重要指标。     

Unbiased analysis by high throughput sequencing of the viral diversity in fetal bovine serum and trypsin used in cell culture

Fetal bovine serum (FBS) and trypsin are reagents used in cell culture and have been the source of viral contamination of pharmaceutical products. We performed high throughput sequencing (HTS) of two pools of commercial batches of FBS and three commercial batches of trypsin. Taxonomies were assigned by comparing sequences of contigs and singletons to the entire NCBI nucleic acid and protein databases. The same major viral species were evidenced between batches of a given reagent but the proportion of viral reads among total reads varied markedly between samples (from 0.002% to 22.7%). In FBS, the sequences found were mainly from bovine viral diarrhea virus (BVDV) 1 to 3 and bovine parvovirus 3 (BPV3). The BVDV sequences derived from FBS showed only minor discrepancies with primers generally used for the screening of BVDV. Viral sequences in trypsin were mainly from porcine circovirus type 2. Other known viral sequences at lower read counts and potential new viral species (bovine parvovirus and bovine pegivirus) were evidenced. The load of some known and new viruses detected by HTS could be quantified by qPCR. Results of HTS provide a framework for evaluating the pertinence of control measures including the design of PCRs, bioassays and inactivation procedures.     

Cellular functions are required for the synthesis and integration of avian sarcoma virus-specific DNA.

We have used two experimental strategies to test the role of cellular functions in the synthesis and integration of virus-specific DNA in cells infected by avian sarcoma virus.First, quail embryo fibroblasts, placed in stationary phase (G₀) by prolonged serum starvation, did not support the efficient synthesis of viral DNA during the first 24–48 hr after infection. Synthesis of viral DNA was impaired according to at least two parameters: the amount of DNA was diminished, particularly the amount of the plus-strand DNA (identical in polarity to the viral genome); and the length of both minus and plus strands was reduced in the stationary cells. In parallel cultures fed with fresh serum, over two thirds of the cells were able to reenter the cell cycle within 24 hr, and viral DNA of normal size was synthesized.Second, density labeling of viral and cellular DNA with BUdR was used to determine whether cellular DNA synthesis was required for integration of viral DNA. In both quail embryo fibroblasts released from G₀ by serum replacement and randomly growing duck embryo fibroblasts, viral DNA was integrated only into cellular DNA replicated during the infection.Our results indicate that serum-starved cells lack a factor (or factors) required for the efficient and complete synthesis of ASV-specific DNA. We have not been able to establish whether such factor(s) are present in growing cells only during S phase. Integration of viral DNA appears to require cellular DNA synthesis; this may be due to a requirement for a factor (or factors) present in adequate concentration only during S phase or to a requirement for the structural changes in cellular DNA that accompany replication.     

Regulation of DNA synthesis in division-arrested mouse C127 cells permissive for bovine papillomavirus DNA amplification.

Spontaneous amplification of bovine papillomavirus type 1 DNA occurs following a prolonged period of serum starvation of wild-type virus-transformed C127 cell lines and is associated with abundant viral E2 protein synthesis and a concomitant induction of viral oncogene (E5 and E6) expression. We show here that a subpopulation of the permissive cells incorporate bromo-deoxyuridine under conditions of cell growth arrest (serum starvation), whereas DNA synthesis is suppressed in the resting population of nonpermissive cells. Flow cytometric measurements of the cellular DNA content of the permissive cell population indicated that it contained predominantly a 4n DNA content, suggesting that these cells were blocked in the G2 phase of the cell cycle. In keeping with the hypothesis that viral DNA amplification is associated with the induction of a cellular S phase, we observed a specific induction of expression of two cell proliferation-related cellular antigens (PCNA and Ki67) in a subpopulation of permissive cells. C127 cell lines transformed by an E5-minus bovine papillomavirus type 1 mutant, which was competent for autonomous plasmid replication in mitotic cells, were completely defective for the induction of DNA synthesis and mutant viral DNA amplification under conditions of serum starvation. Moreover, the E5 protein is shown by immunofluorescence analysis to be expressed at a high level specifically in the permissive cell population. These results imply a dual role for the viral E5 protein in the C127 model system, both as a transforming protein and as a factor required for the induction of viral DNA amplification in postmitotic cells. We suggest that E5 acts at an early step in the induction of this process in C127 cells and may be required to turn on host cell DNA synthesis as a prerequisite for viral DNA amplification.     

Comparative approaches to the investigation of responses to stress and viral infection in cattle

Fatal bovine respiratory disease (BRD) is a major cause of financial losses in the cattle industry. A variety of stressors have been implicated as contributing to disease severity. However, it has proven difficult to determine the role these individual factors may play in the final outcome of this disease complex. The objective of the present investigation was to obtain proteomic, metabonomic, and elemental profiles of bovine serum samples from stressed and control animals before and after a primary viral infection to determine if these profiles could distinguish between responses to stressors and viral infection. Multivariate analysis revealed distinct differential trends in the distribution profile of proteins, metabolites, and elements following a stress response both before and after primary viral infection. A group of acute phase proteins, metabolites, and elements could be specifically linked to either a stress response (decreased serum amyloid A and Cu, increased apolipoprotein CIII, amino acids, LDL, P, and Mo) or a primary viral respiratory infection (increased apolipoprotein A1, haptoglobin, glucose, amino acids, LDL and Cu, decreased Lipid, and P). Thus, combined OMICS analysis of serum samples revealed that multimethod analysis could be used to discriminate between the complex biological responses to stress and viral infection.     

Serum procalcitonin and cerebrospinal fluid cytokines level in children with meningitis

AIMS: To determine the level of serum procalcitonin and cerebrospinal fluid cytokines in children with bacterial or viral meningitis and to document the use of these parameters in differential diagnosis. RESULTS: Before the start of antibiotic treatment, serum procalcitonin and tumor necrosis factor alpha levels were found to be higher in acute bacterial meningitis compared with viral meningitis and with the control group. Similarly, cerebrospinal fluid interleukin-6 levels were found to be significantly higher in children with acute bacterial meningitis compared with viral meningitis. However, no significant difference was determined between groups in respect to the cerebrospinal fluid interleukin-8 level. CONCLUSION: Serum procalcitonin and cerebrospinal fluid tumor necrosis factor alpha levels can be used in the early diagnosis of bacterial meningitis. Similarly, they may be useful adjuncts in differential diagnosis of bacterial and viral meningitis.     

Establishment, characterization, and viral susceptibility of two cell lines derived from goldfish Carassius auratus muscle and swim bladder

Goldfish Carassius auratus are common aquarium fish and have a significant economic and research value, having considerable worth to fisheries as a baitfish and the ability to adapt to a range of habitats. Two cell lines were established from goldfish muscle and swim bladder tissue, in order to create a biological monitoring tool for viral diseases. Cell lines were optimally maintained at 30 degrees C in Leibovitz-15 medium supplemented with 20% fetal bovine serum. Propagation of goldfish cells was serum dependent, with a low plating efficiency (>16%). Karyotyping analysis indicated that both cell lines remained diploid, with a mean chromosomal count of 104. Results of viral challenge assays revealed that both cell lines shared similar patterns of viral susceptibility and production to infectious hematopoietic necrosis virus, infectious pancreatic necrosis virus, snakehead rhabdovirus, and spring viremia carp virus. Both cell lines demonstrated a higher sensitivity and significantly larger viral production than control brown bullhead cells for channel catfish virus. These newly established cell lines will be used as a diagnostic tool for viral diseases in this fish species and also for the isolation and study of goldfish viruses in the future.     

CD40L-Adjuvanted DNA/Modified Vaccinia Virus Ankara Simian Immunodeficiency Virus (SIV) Vaccine Enhances Protection against Neutralization-Resistant Mucosal SIV Infection

Here, we show that a CD40L-adjuvanted DNA/modified vaccinia virus Ankara (MVA) simian immunodeficiency virus (SIV) vaccine enhances protection against a pathogenic neutralization-resistant mucosal SIV infection, improves long-term viral control, and prevents AIDS. Analyses of serum IgG antibodies to linear peptides of SIV Env revealed a strong response to V2, with targeting of fewer epitopes in the immunodominant region of gp41 (gp41-ID) and the V1 region as a correlate for enhanced protection. Greater expansion of antiviral CD8 T cells in the gut correlated with long-term viral control.     

阿魏酸钠联合双黄连注射液对病毒性心肌炎患者临床疗效及血清基质金属蛋白酶、锌铜水平的影响

目的:探究阿魏酸钠联合双黄连注射液对病毒性心肌炎患者临床疗效及血清基质金属蛋白酶、锌铜水平的影响。方法:选择在我院确诊为病毒性心肌炎的患者39例,随机分成实验组以及对照组。对照组18例给予阿魏酸钠治疗;实验组21例予阿魏酸钠联合双黄连注射液治疗。治疗14天后,评价和比较两组患者的临床疗效、心肌酶谱水平、血清基质金属蛋白酶水平及血清锌铜比值。结果:实验组总有效率为95.2%,显著高于对照组(72.2%),差异具统计学意义(P0.05)。与对照组比,实验组治疗后的血清、乳酸脱氢酶(LDH)水平、肌酸磷酸激酶同工酶(CK-MB)、天冬氨酸氨基转移酶(AST)、基质金属蛋白酶水平较低,血清铜锌比值较小,而左室射血分数(LVEF)水平较高,差异均具有统计学意义(P0.05)。结论:阿魏酸钠联合双黄连注射液治疗病毒性心肌炎患者能够有效提高其临床疗效,可能与其显著降低血清基质金属蛋白酶及铜锌水平有关。     

Latent membrane protein 1 of Epstein-Barr virus plays an important role in the serum starvation resistance of Epstein-Barr virus-immortalized B lymphocytes

We have previously shown that SNU-1103, which is a latency type III Epstein-Barr virus (EBV)-transformed lymphoblastoid cell line (LCL) that was developed from a Korean cancer patient, resists serum starvation-induced G(1) arrest. In this study, we examined the role of latent membrane protein-1 (LMP-1) in serum starvation resistance, since LMP-1 is known to be essential for EBV-mediated immortalization of human B lymphocytes. The LMP-1 gene from SNU-1103 was introduced into the EBV-negative BJAB cell line, and shown to be associated with resistance to G(1) arrest during serum starvation. Western blot analyses of the LMP-1-transfected cells revealed several protein alterations as compared to vector-transfected control cells. The expression of key cell-cycle regulatory proteins was affected in the G(1) phase: the expression of cyclin D3, CDK2, p27, and E2F-4 was up-regulated, and the expression of cyclin D2, CDK6, p21, and p103 was down-regulated during serum starvation. These results imply that of the several EBV viral genes expressed in EBV-negative B lymphoma cells, LMP-1 mediates resistance to serum starvation-induced G(1) arrest. However, we cannot rule out the possibility that other EBV genes are also involved in the cell-cycle progression of the EBV-transformed LCL during serum starvation, since the altered protein expression profile of the LMP-1 transfectants was distinct from that of the SNU-1103 cells that expressed all of the EBV viral proteins.     

Vaccination-induced noncytolytic effects in the acute phase of SHIV infection

Many studies have shown that vaccines inducing CD8+ T cell responses can reduce viral loads and preserve CD4+ T cell numbers in monkey models of HIV infection. The mechanism of viral control by the vaccine-induced CD8+ T cells is usually assumed to be cytolysis of infected cells. However, in addition to cytolysis of infected cells, CD8+ T cells secrete a range of soluble factors that suppress viral replication. We have studied the dynamics of virus and CD4+ T cells in a successful vaccination-challenge model of SHIV infection. We find that better viral control in the acute phase of infection is associated with slower decay of peak viral load. Comparing viral and CD4+ T cell dynamics in acute infection, we find that a cytolytic mode of viral control with direct killing of infected cells is inconsistent with the observed trends. On the other hand, comparison of the predicted effects of noncytolytic CD8+ effector function with the experimental data shows that non-cytolytic control provides a better explanation of the experimental results. Our analysis suggests that vaccine-induced CD8+ T cells control SHIV infection by non-cytolytic means.     

Surface-displayed viral antigens on Salmonella carrier vaccine

We have developed a recombinant live oral vaccine using the ice-nucleation protein (Inp) from Pseudomonas syringae to display viral antigens on the surface of Salmonella spp. Fusion proteins containing viral antigens were expressed in the oral vaccine strain, Salmonella typhi Ty21a. Surface localization was verified by immunoblotting and fluorescence-activated cell sorting. The immunogenicity of surface-displayed viral antigens on the recombinant live vaccine strain was assessed in mice inoculated intranasally and intraperitoneally. Inoculation resulted in significantly higher serum antibody level than those induced by viral antigens expressed intracellularly. Thus, this multivalent mucosal live vaccine may provide an effective means for inducing mucosal or systemic immune responses against multiple viral antigens.     

Antibody to a lytic cycle viral protein decreases gammaherpesvirus latency in B-cell-deficient mice

While antiviral antibody plays a key role in resistance to acute viral infection, the contribution of antibody to the control of latent virus infection is less well understood. Gammaherpesvirus 68 (gammaHV68) infection of mice provides a model well suited to defining contributions of specific immune system components to the control of viral latency. B cells play a critical role in regulating gammaHV68 latency, but the mechanism(s) by which B cells regulate latency is not known. In the experiments reported here, we determined the effect of passively transferred antibody on established gammaHV68 latency in B-cell-deficient (B-cell(-/-)) mice. Immune antibody decreased the frequency of cells reactivating ex vivo from latency in splenocytes (>10-fold) and peritoneal cells (>100-fold) and the frequency of cells carrying latent viral genome in splenocytes (>5-fold) and peritoneal cells (>50-fold). This effect required virus-specific antibody and was observed when total and virus-specific serum antibody concentrations in recipient B-cell(-/-) mice were <8% of those in normal mice during latent infection. Passive transfer of antibody specific for the lytic cycle gammaHV68 RCA protein, but not passive transfer of antibody specific for the v-cyclin protein or the latent protein M2, decreased both the frequency of cells reactivating ex vivo from latency and the frequency of cells carrying the latent viral genome. Therefore, antibody specific for lytic cycle viral antigens can play an important role in the control of gammaherpesvirus latency in immunocompromised hosts. Based on these findings, we propose a model in which ongoing productive replication is essential for maintaining high levels of latently infected cells in immunocompromised hosts. We confirmed this model by the treatment of latently infected B-cell(-/-) mice with the antiviral drug cidofovir.     

Truncated monocyte chemoattractant protein‐1 can alleviate cardiac injury in mice with viral myocarditis via infiltration of mononuclear cells

BALB/c mice inoculated intraperitoneally with coxsackievirus group B type 3 (CVB3) were allocated to five groups; namely, a viral myocarditis group infected with CVB3 alone (control group), an antibody intervention group that received intracardiac anti‐MCP‐1, an antibody intervention control group that received goat IgG, a tMCP‐1 intervention group that received plasmid pVMt expressing tMCP‐1, and a tMCP‐1 intervention control group that received plasmid pVAX1. There was also a normal control group. The ratio of murine heart weight to body weight, pathological score of myocardial tissue, serum creatine kinase‐MB titers and CVB3 loading of myocardial tissue were assessed. The cardiac lesions in mice that received 20, 40 or 60 µg pVMt (P < 0.05) were less severe than those in control mice with untreated viral myocarditis. In addition, fewer mononuclear cells had infiltrated the myocardium of mice who received 40 or 60 µg pVMt intramyocardially (P < 0.01), whereas there was no difference in mononuclear cell infiltration between mice with viral myocarditis and those that received 20 µg pVMt (P > 0.05). There was also no difference between mice that received anti‐MCP‐1 antibody and those that received 40 µg pVMt in ratio of HW/BW, serum CK‐MB titers and pathological score (P > 0.05). This study showed that tMCP‐1 can alleviate cardiac lesions and cardiac injury in mice with viral myocarditis via infiltration of mononuclear cells. Thus, tMCP‐1 may be an alternative to anti‐MCP‐1 antibody treatment of viral myocarditis. Further research is required.