Analysis of Jembrana disease virus replication dynamics in vivo reveals strain variation and atypical responses to infection

Jembrana disease virus (JDV) is an acute lentiviral infection of Bali cattle in Indonesia. Data generated during a series of cattle infection experiments was examined and significant differences were identified in the mean plasma viral load on the first and second days of the febrile response in cattle infected with JDV_TAB/87 compared to those infected with JDV_PUL/01. The peak and total viral loads ≥ 10⁶ genome copies/ml during the acute stage of the disease were significantly higher in JDV_TAB/87 infected cattle. JDV_PUL/01 infected cattle developed peak rectal temperatures earlier than the JDV_TAB/87 cattle but there were no differences in the duration of the febrile responses observed for the 2 groups of animals. The plasma viremia was above 10⁶ genome copies/ml for almost 3 days longer in JDV_TAB/87 compared to JDV_PUL/01 infected cattle. Atypical responses to infection occurred in approximately 15% of experimentally infected animals, characterized by reduced viral loads, lower or absent febrile responses and absence of p26-specific antibody responses. Most of these cattle developed normal Tm-specific antibody responses between 4-12 weeks post-infection.     

Assessment of viral loads in patients with chronic hepatitis C with AMPLICOR HCV MONITOR version 1.0, COBAS HCV MONITOR version 2.0, and QUANTIPLEX HCV RNA version 2.0 assays

The correlation between response to antiviral therapy and pretreatment viral load in patients with chronic hepatitis C has prompted the development of quantitative assays to measure viral load. The aim of our study was to assess the clinical relevance of the newly developed semiautomated PCR system COBAS HCV MONITOR version 2.0 in comparison with (i) the AMPLICOR HCV MONITOR version 1.0 assay, which underestimates RNA concentration of hepatitis C virus (HCV) genotypes 2 to 6, and (ii) the QUANTIPLEX HCV RNA version 2.0 assay, which achieves equivalent quantification for each HCV genotype, with samples from 174 patients diagnosed with chronic hepatitis C before therapy. The level and range of quantification measured with AMPLICOR HCV MONITOR version 1.0 were 1 log lower than when measured with the COBAS HCV MONITOR version 2.0, at 0.261 x 10(6) RNA copies/ml (range, 0.001 x 10(6) to 2.50 x 10(6) RNA copies/ml) and 4.032 x 10(6) RNA copies/ml (range, 0.026 x 10(6) to 72.6 x 10(6) RNA copies/ml), respectively. The two assays showed a poor correlation (r(2) = 0.175). The level and range of quantification were similar when measured with the COBAS HCV MONITOR version 2.0 and QUANTIPLEX HCV RNA version 2.0 assays, at 3.03 x 10(6) RNA copies/ml (range, 0.023 x 10(6) to 72.6 x 10(6) RNA copies/ml) and 4.91 Meq/ml (range, 0.200 to 49.5 Meq/ml), respectively. The two assays showed a strong correlation (r(2) = 0. 686) for each HCV genotype. The duration of treatment (6 or 12 months) is modulated according to HCV genotype and viral load. Our results indicate that COBAS HCV MONITOR version 2.0 and QUANTIPLEX HCV RNA version 2.0 assays showing an equal dynamic range for each HCV genotype are suitable tools to assess patients before therapy.     

Prior bovine immunodeficiency virus infection does not inhibit subsequent superinfection by the acutely pathogenic Jembrana disease virus

In cattle the interaction between the two genetically and antigenically related bovine lentiviruses, the acutely pathogenic Jembrana disease virus (JDV) and the non-pathogenic Bovine immunodeficiency virus (BIV) has not been reported although both JDV and a BIV-like virus have been reported in the Bali cattle (Bos javanicus) population in Indonesia. The outcome of infection of Bali cattle with the R29 strain of BIV prior to superinfection 42 days later with JDV_TAB/87 was determined. All BIV-inoculated cattle were successfully infected and developed an antibody response to the TM and CA proteins. BIV infection did not prevent subsequent infection with JDV or ameliorate the clinical signs of Jembrana disease in the infected cattle. It did, however, modify the dynamics of the JDV infection with an earlier onset and end of the acute disease process, and a reduction in the duration of viremia that exceeded 10⁶ genome copies/ml of plasma.     

实时定量PCR检测马传染性贫血病毒载量方法的建立和应用

目的建立实时定量PCR检测血浆病毒载量的方法，对马传染性贫血病毒（equine infectious anemia virus，EIAV）强毒株攻毒马和疫苗免疫攻毒马血浆中病毒载量进行了跟踪检测，探讨病毒载量和临床疾病状态的相关性。方法以EIAV强毒株LN40序列为标准，在gag保守区设计1对引物和Taqman探针，用于实时定量PCR扩增，用含扩增目的基因的体外转录RNA作标准品，获得标准曲线，对扩增样品进行准确定量。强毒株LN40直接攻毒，或者疫苗株DLV免疫马6个月后用强毒株LN40进行攻毒，跟踪检测攻毒马及免疫攻毒马血浆中EIAV载量情况。结果反应在10^1～10^9copies／ml之间具有良好的线性关系，反应的检出下限为10copies／ml。强毒攻毒马出现发热并最终死亡，发热期间马血浆中EIAV载量与体温呈正相关，载量最高达10^7copies／ml。免疫攻毒马未出现发热，其血浆EIAV载量的总体水平低于强毒攻毒马，攻毒后3个月低至10copies／ml以下。结论成功建立了实时定量PCR检测血浆EIAV载量的方法，并证实了用实时荧光定量PER检测EIAV病毒载量的方法来监测动物感染状态具有可行性，为EIAV致病机制研究和弱毒疫苗免疫保护机制的研究提供良好的技术平台。     

实时荧光定量RT-PCR监测恒河猴体内人/猴免疫缺陷病毒载量在传代过程中的变化

目的为分析中国SHIV/猕猴AIDS模型的病毒载量变化趋势,建立一种实时、灵敏、特异的针对人/猴免疫缺陷病毒的定量检测方法。方法体外转录制备RNA标准品,利用TaqManEZRT-PCR试剂盒的反应体系和针对SHIVgag保守区91个碱基的TaqMan探针和引物,建立一步法实时荧光定量RT-PCR。提取126份来自SHIV-CN97001感染恒河猴血浆病毒RNA并定量检测。结果利用梯度稀释的RNA标准品对反应体系进行优化,标准曲线下限达到2×102拷贝/ml,相关性(r>0.99)及重复性(CV=4.14%)均能达到测定要求。病毒载量的检测结果表明SHIV-CN97001在猴体内传代过程中病毒载量有先升后降的趋势,病毒载量通常在接种病毒或感染猴的全血后第14天达到高峰。血浆载量可达到105~106拷贝/ml。结论成功地建立了一步法定量SHIVRNA的实时荧光定量RT-PCR,为SHIV/恒河猴AIDS模型的建立与应用提供了灵敏的病毒载量检测方法。SHIV-CN97001的体内繁殖能力在猴体内传代过程中有所增强。     

Prolonged fecal shedding of hepatitis E virus (HEV) during sporadic acute hepatitis E: evaluation of infectivity of HEV in fecal specimens in a cell culture system

To investigate the duration of fecal shedding and changing loads of hepatitis E virus (HEV) in feces and serum from patients with acute HEV infection, HEV RNA was quantitated in periodic serum and fecal specimens obtained from 11 patients with sporadic acute hepatitis E. All 11 patients had detectable HEV RNA in serum at admission, with the highest viral load being 1.9 x 10(3) to 1.7 x 10(7) copies/ml, and HEV viremia lasted until days 17 to 48 (mean, 28.3) after the onset of hepatitis. Even at the initial examination on days 10 to 29 (mean, 17.6), the HEV load in fecal supernatant was less than 5.7 x 10(4) copies/ml for 10 of the 11 patients, while for the remaining patient (patient 1) it was markedly high, 2.0 x 10(7) copies/ml on day 22. In addition, although HEV RNA in fecal supernatant continued to be positive until days 14 to 33 (mean, 22.4) for patients 2 to 11, that for patient 1 was detectable even on day 121. HEVs in fecal specimens obtained on days 22, 24, 26, 28, and 30, but not day 121, from patient 1 grew efficiently in PLC/PRF/5 cells, reaching the highest titer of up to 10(7) copies/ml in culture medium on day 50 postinoculation. The HEV genome recovered from patient 1 had 29 unique nucleotides that were not seen in any of the 25 reported HEV isolates of the same genotype over the entire genome, with six amino acid substitutions in the ORF1 protein.     

Evaluation of a real-time two-step RT-PCR assay for quantitation of Chronic bee paralysis virus (CBPV) genome in experimentally-infected bee tissues and in life stages of a symptomatic colony

A two-step real-time RT-PCR assay, based on TaqMan technology using a fluorescent probe (FAM-TAMRA) was developed to quantify Chronic bee paralysis virus (CBPV) genome in bee samples. Standard curves obtained from a CBPV control RNA and from a plasmid containing a partial sequence of CBPV showed that this assay provided linear detection over a 7-log range (R(2)>0.99) with a limit of detection of 100 copies, and reliable inter-assay and intra-assay reproducibility. Standardisation including RNA purification and cDNAs synthesis was also validated. The CBPV TaqMan methodology was first evaluated by quantifying the CBPV genomic load in bee samples from an experimental infection obtained by topical application. Up to 1.9 x 10(10) CBPV copies per segment of insect body (head, thorax and abdomen) were revealed whereas a lower CBPV genomic load was detected in dissected organs such as mandibular and hypopharyngeal glands, brain and alimentary canal (up to 7.2 x 10(6) CBPV copies). The CBPV genomic loads in different categories of bees from a hive presenting the trembling symptoms typical of Chronic paralysis were then quantified. Significantly higher CBPV loads were found in guard, symptomatic and dead bees (up to 1.9 x 10(13) CBPV copies) than in forager, drones and house bees (up to 3.4 x 10(6) CBPV copies). The results obtained for symptomatic or dead bees support the correlation between high CBPV genomic load and pathology expression. Moreover, the high CBPV genomic load revealed in guard bees highlights the possible pivotal role played by this category of bees in CBPV infection.     

Monitoring herpesvirus DNA in three cases of acute retinal necrosis by real-time PCR.

BACKGROUND: It is not clear whether quantitative analysis of viral DNA in ocular specimens is correlated with disease activities of acute retinal necrosis (ARN). OBJECTIVES: To monitor viral load in ocular specimens collected from patients with ARN by real-time polymerase chain reaction (PCR). STUDY DESIGN: Ocular samples (aqueous humor and vitreous) were serially collected from three patients with ARN. Viral load in those samples was evaluated by real-time PCR. RESULTS AND CONCLUSION: In case 1, large amounts of varicella zoster virus (VZV) DNA (4.8 x 10(6) to 5.5 x 10(6) copies/ml) were detected in aqueous humor during the first 2 weeks after admission. The viral load in vitreous was higher than that in aqueous humor at the time of vitrectomy. As ophthamoscopic findings and visual acuity improved through acyclovir (ACV) treatment, the viral load in aqueous humor decreased dramatically. In case 2, the patient was treated with intravenous ACV at first, but clinical features did not improve. The herpes simplex virus (HSV)-2 viral load in aqueous humor remained stable (2.3 x 10(3) to 2.8 x 10(3) copies/ml) during the first 3 weeks after admission. The amount of HSV-2 DNA in vitreous was again higher than that in aqueous humor. Although neither clinical features nor viral load had changed by ACV, intra-ocular ganciclovir (GCV) injection improved clinical features, and decreased viral load to undetectable levels. In case 3, the patient developed ARN within 1 month after the onset of varicella and demonstrated only mild clinical symptoms. She was treated with ACV administration alone and recovered quickly. In contrast to case 1, the copy number of VZV DNA at the time of admission was low (9 x 10(2) copies/ml), and decreased quickly in response to the treatment. Correlation between viral load in ocular specimens and clinical course of the disease was demonstrated in these patients.     

Use of ELISA procedures for the detection of Derzsy's disease virus of geese and of antibodies produced against it

We have developed a double antibody sandwich (antigen capture) ELISA for the detection of Derzsy's disease virus (GPV-1) antigen produced in cell cultures. This provided unambiguous quantification of virus whereas evaluation of cytopathic effect was sometimes difficult. A blocking ELISA was developed to quantify anti-GPV-1 antibodies in goose sera. This method was rapid, simple, easily standardized and correlated well with a virus neutralization test.     

High virus loads in naturally and experimentally SIVagm-infected African green monkeys

A quantitative RT-PCR assay was developed for SIVagm and was used to measure the levels of viral RNA in the plasma of experimentally and naturally infected African green monkeys. The number of productively infected PBMCs and the number of cells carrying integrated provirus were also measured. Plasma virus loads in experimentally infected animals peaked at 2 weeks postinfection, ranging from 2.9 x 10(5) to 4.2 x 10(7) RNA copies/ml plasma. Set points of 2.1 x 10(3) to 2.8 x 10(6) RNA copies/ml plasma were maintained for one year. Similarly, the number of cells carrying integrated SIVagm provirus remained relatively stable in individual animals for one year with set points ranging from 73 to 810 proviral copies per 10(6) PBMC. However, the number of productively infected cells fluctuated considerably during this period. Virus loads in the 26 naturally infected AGMs ranged from 8.3 x 10(3) to 1.1 x 10(7) (mean 1.7 x 10(6)) RNA copies/ml plasma. These levels of viremia are similar to those seen in pathogenic systems (HIV-1, SIVmac), indicating that control of SIVagm replication is not the reason for the natural host's resistance to disease progression.     

High TT virus load as an independent factor associated with the occurrence of hepatocellular carcinoma among patients with hepatitis C virus-related chronic liver disease

The TT virus (TTV) load was estimated in sera obtained from 237 patients with hepatitis C virus (HCV)-related chronic liver disease including 42 patients with hepatocellular carcinoma (HCC), by real-time detection PCR using primers and a probe derived from the well-conserved untranslated region of the TTV genome, which can detect all known TTV genotypes. Of the 237 patients studied, 18 (8%) were negative for TTV DNA, 87 (37%) had low TTV viremia (1.3 x 10(2)-9.9 x 10(3) copies/ml), and 132 (56%) had high TTV viremia (1.0 x 10(4)-2.1 x 10(6) copies/ml). Various features were compared between the patients with high TTV load (n = 132) and those with no TTV viremia or low viral load (n = 105). High TTV viremia (> or =10(4) copies/ml) was significantly associated with higher age (P < 0.05), past history of blood transfusion (P < 0.001), complication of cirrhosis (P < 0.05) or HCC (P < 0.0005), lower HCV RNA titer (P < 0.05), and lower platelet count (P < 0.01). On multivariate logistic regression analysis, high TTV viral load was a significant risk factor for HCC (P < 0.05), independent from known risk factors such as complication of liver cirrhosis (P < 0.0001) and high age (> or =65 years, P < 0.05), among all 237 patients. Furthermore, high TTV viral load was an independent risk factor for HCC among the 90 cirrhotic patients (P < 0.05). These results suggest that a high TTV viral load is associated independently with the complication of HCC and may have prognostic significance in patients with HCV-related chronic liver disease, although whether high TTV viremia mediates the progression of HCV-related chronic liver disease remains to be defined.     

Vaccination reduces the viral load and the risk of transmission of Jembrana disease virus in Bali cattle

The efficacy of a tissue-derived vaccine, which is currently used in Indonesia to control the spread of Jembrana disease in Bali cattle, was determined by quantifying the viral load in plasma following experimental infection with Jembrana disease virus. Virus transmission is most likely to occur during the acute phase of infection when viral titers are greater than 10⁶ genomes/ml. Vaccinated cattle were found to have a 96% reduction in viral load above this threshold compared to control cattle. This would reduce the chance of virus transmission as the number of days above the threshold in the vaccinated cattle was reduced by 33%. Viral loads at the onset and resolution of fever were significantly lower in the vaccinated cattle and immune function was maintained with the development of antibody responses to Env proteins within 10-24 days post challenge. There was, however, no significant reduction in the duration of the febrile period in vaccinated animals. The duration and severity of clinical parameters were found to be variable within each group of cattle but the quantification of viral load revealed the benefits of vaccinating to reduce the risk of virus transmission as well as to ameliorate disease.     

Comparison between serum and saliva for the detection of hepatitis A virus RNA

Due to the ease of collection, oral fluid is being investigated as an alternative to serum for diagnostic and epidemiological purposes. However, for prospective studies involving hepatitis A virus (HAV) RNA detection, a standard methodology must be developed. In the present study, nested RT-PCR and real-time PCR were optimized and evaluated for HAV detection and quantification, using oral fluid from healthy volunteers (n=20) and paired serum/oral fluid samples from individuals involved in a hepatitis A outbreak (n=78). Using nested RT-PCR, HAV RNA was detected in 50% of oral fluid and in 42% of serum samples from acute cases, as well as in 12% of all samples from cases without IgM and total anti-HAV. Using real-time PCR, HAV RNA was detected in 61% of oral fluid and in 71% of serum samples from acute cases, as well as in 17 and 12%, respectively, from patients without HAV markers. Mean viral loads were 1.7+/-3.24 x 10(3)copies/ml in oral fluid and 2.8+/-6.46 x 10(3)copies/ml in serum. Although nested RT-PCR and real-time PCR both detected HAV RNA in oral fluid, real-time PCR was more sensitive. Oral fluid sample testing could be used as a noninvasive method of detecting HAV RNA during HAV outbreaks.