Recovery of Swedish Equine arteritis viruses from semen by cell culture isolation and RNA transfection

Recovery of infectious Equine arteritis virus (EAV) from the semen of persistently infected Swedish stallions was attempted by classical cell culture isolation and by transfection of extracted total RNA. Whereas virus from semen samples stored for several months at -20 degrees C or from extended semen could only be recovered by transfection of extracted RNA, isolation in cell culture was achieved readily with fresh, unextended semen stored at -70 degrees C or directly used after sampling. In parallel, the viruses were examined in the variable region of the large glycoprotein GP5 by nested RT-PCR and direct nucleotide sequencing. The resulting sequences were placed into a large phylogenetic tree from this region, demonstrating that Swedish strains belonged to very diverse phylogenetic groups. This represents the first report of recovery of infectious EAV from archived semen samples by RNA transfection.     

三种不同转染方法拯救病毒的比较研究

目的比较3种不同转染方法,探索最佳的拯救病毒策略。方法3种转染方法包括：①含T7RNA聚合酶启动子的全长肠道病毒71（EV71）感染性质粒（P—EV71）和T7RNA聚合酶真核表达质粒（VR-1a）共转染Vero细胞;②含T7RNA聚合酶启动子的EV71全长基因片段（PCR产物）与VR-1a共转染Vero细胞;③利用体外转录技术得到EV71病毒全长RNA,直接转染Vero细胞。分别观察3种方法转染后产生细胞病变效应（CPE）时间及病变情况,用RT—PCR扩增拯救病毒核酸,测序验证其正确性,用蛋白印迹方法检测拯救病毒抗原。结果3种方法均可成功转染Vero细胞得到拯救病毒,三种转染方法转染效果比较：方法③优于②、②优于①。结论三种转染方法均可成功拯救EV71病毒,以病毒RNA直接转染效果最好。     

Direct detection of highly pathogenic avian influenza A/H5N1 virus from mud specimens

Contaminated mud and soil may play roles as reservoirs and sources of transmission for avian influenza A virus. However, the persistence of highly pathogenic avian influenza (HPAI) H5N1 virus in soil or mud has not been well documented, and specific methods of H5N1 virus detection in mud and soil specimens have not been described. The aim of this work was to evaluate the capacities of five different commercial kits and one elution-concentration technique to extract nucleic acids from H5N1 virus and to detect infectious viral particles in experimentally infected mud specimens. The viral RNA detection thresholds for the QIAamp kit, Trizol LS and the MagNA Pure LC kit were 5 × 10(2)RNA copies per gram of mud. Trizol reagent and the RNA PowerSoil? kit were unsuccessful in recovering any viral RNA from mud. When the elution-concentration technique was performed prior to nucleic acid extraction, the performance of the MagNA Pure kit increased to a level that allowed the detection of H5N1 nucleic acids in naturally contaminated environmental samples that had previously tested negative after direct extraction using commercial kits. The levels of detection of infectious virus after inoculation into embryonated eggs were higher in concentrates than in eluates.     

Mechanisms of herpes simplex virus infectivity enhanced by ultracentrifugal inoculation

Ultracentrifugation of very dilute suspensions of herpes simplex virus directly onto monolayer cells grown in centrifuge tubes was studied. Enhanced infectivity by ultracentrifugation was similar at 4 degrees C and at 35 to 37 degrees C. The high infectivity levels of cultures centrifuged at 4 degrees C were further examined by infectious center assays. At 4 degrees C, the numbers of infectious centers in control (noncentrifuged) cultures were almost 100-fold fewer than in control cultures at 37 degrees C. However, the numbers of infectious centers in cultures ultracentrifuged at 4 degrees C were similar to those ultracentrifuged at 37 degrees C. The great difference in the numbers of infectious centers between 4 and 37 degrees C control cultures, in contrast to the similarity between 4 and 37 degrees C ultracentrifuged cultures, indicated that ultracentrifugation at 4 degrees C enhanced infectivity possibly by facilitation of herpes simplex virus penetration into monolayer cells.     

Quantification of human immunodeficiency virus type 1 RNA from dried plasma spots collected on filter paper.

To assess dried plasma spots (DPSs) as a source of material for virus quantification, human immunodeficiency virus type 1 (HIV-1) RNA levels were quantified in matched DPS and liquid plasma samples from 73 infected patients, including 5 neonates and 4 adult patients with acute HIV-1 infection. Quantifications were performed by commercially available assays (NASBA [nucleic acid sequence-based amplification] or Amplicor, or both). There was a strong correlation between HIV-1 RNA levels in plasma and DPSs. More importantly, there was no decline in HIV-1 RNA levels in DPSs stored for as long as 2 weeks at 20 degrees C. Similarly, storage of DPSs for 3 days at 37 degrees C resulted in no decrease in viral RNA levels. For patients with primary infection, the DPS method allowed for the measurement of RNA levels in plasma during the initial spike in the level of viremia and in the subsequent period of suppressed viral replication. DPS quantification was equally informative in the neonatal setting, with all five newborns showing HIV-1 RNA loads of greater than 4.991 log10 copies/ml. We conclude that the viral RNA levels in DPSs are equivalent to those measured in fresh-frozen plasma. The ease and economy of DPS sampling, the minute volumes required, and the unexpected stability of dried RNA suggest that the use of DPSs will be particularly valuable for small-volume neonatal samples and large, population-based studies in which cold storage and transportation present special problems, as is often the case in developing countries. The ability to measure viral changes during primary infection suggests that the method will be useful for assessing vaccine efficacy in large field trials.     

Assessment of hepatitis C virus RNA stability in serum by the Quantiplex branched DNA assay.

OBJECTIVES: Quantification of hepatitis C virus (HCV) RNA in serum is used to assess the probability of treatment response and to monitor antiviral therapy. Since serum specimens often are shipped to central sites for HCV RNA testing, it is important to define conditions that preserve HCV RNA integrity. METHODS: We evaluated the stability of HCV RNA in 25 previously frozen (PF) and 11 fresh, never previously frozen (NPF) specimens subjected to handling and short-term storage conditions that mimic those encountered during interlaboratory shipping. All sera were separated within 4 h of collection. PF samples covering a approximately 3 log10 HCV RNA dynamic range were thawed, divided into aliquots, incubated at 4, 23, and 37 degrees C (+/- 1.5 degrees C) for 24, 48, 72 and 96 h (+/- 2 h), and then refrozen at -70 degrees C prior to testing with the Quantiplex HCV RNA 2.0 assay. Eleven NPF samples were stored at -70, -20, and 4 degrees C (+/- 1.5 degrees C) for up to 1 month prior to testing. RESULTS: Linear regression analysis showed no HCV RNA degradation in PF specimens kept at 4 degrees C over 4 days. However, HCV RNA levels in PF specimens decreased over 4 days by 20 and 105% at 23 and 37 degrees C, respectively. Three independent statistical methods showed that the probability of specimen failure in PF specimens, defined as a loss of 20% or more of HCV RNA, was lowest at 4 degrees C and increased with increasing temperature. The HCV RNA quantification of the 11 NPF specimens stored at 4 degrees C was similar to their frozen controls. CONCLUSION: HCV RNA in separated serum specimens is stable for at least 4 days at 4 degrees C.     

贵州白纹伊蚊对登革病毒易感性的研究

目的 用细胞、分子生物学技术进行贵州省白纹伊蚊不同地理株对登革病毒(DEN)易感性的研究。方法 采集贵州省9个地(州)市共计15个县(区)白纹伊蚊幼虫标本，饲养为成蚊；取羽化后3～5日龄期的贵州不同地理株白纹伊蚊，用不同型别的DEN分别经口连续感染3d，于首次感染后的4、7、10、14d收集感染成蚊标本；制备蚊悬液，碘化钠法提取RNA，用DENNS1基因区通用引物经逆转录．聚合酶链反应(RT-PCR)检测DEN核酸；蚊悬液接种C6／36细胞进行病毒分离，制作细胞抗原片，经间接免疫荧光法检测DEN抗原；同时感染白纹伊蚊海南株作为对照。结果 DEN1-4型国际参考株感染白纹伊蚊贵州省不同地方株，其感染比率分别为12／15、12／15、8／15和13／15。结论 白纹伊蚊贵州省不同地方株对DEN1-4型国际参考株普遍易感，表明贵州省具备引起登革热流行的条件。     

Detection and quantitation of infectious pancreatic necrosis virus by real-time reverse transcriptase-polymerase chain reaction using lethal and non-lethal tissue sampling

Infectious pancreatic necrosis virus (IPNV) is a bisegmented double-stranded RNA virus belonging to the family Birnaviridae, genus Aquabirnavirus, which is a major viral pathogen of salmonid fish. The virus infects wild and cultured salmonids, causing high mortality in juvenile trout and salmon. A highly sensitive and specific real-time RT-PCR assay using the fluorogenic dye SYBR((R)) Green I was developed for the detection and quantitation of IPNV in rainbow trout (Oncorhynchus mykiss). Rainbow trout were infected experimentally with IPNV in the laboratory by injection or immersion and then pectoral fin, spleen, and head kidney samples were collected for analysis. The corresponding cDNA was synthesized using DNase I-treated total RNA and then real-time RT-PCR was performed using primers based on the IPNV non-structural protein gene, designated as either NS or VP4. Rainbow trout beta-actin and elongation factor 1alpha (EF-1alpha) genes were used as internal controls. Using real-time RT-PCR, the virus was successfully detected in pectoral fin, spleen, and head kidney tissue samples. The dissociation curves for each amplicon showed a single melting peak at 83, 81.5, and 84 degrees C for IPNV NS, trout beta-actin, and EF-1alpha genes, respectively. The amplicon size and nucleotide sequence was used to confirm the specificity of the products. Using a dilution series of in vitro transcribed RNA, IPNV was reliably detected down to 10 RNA copies and had a dynamic range up to 10(7) RNA copies. A time course assay, using immersion challenged samples, revealed that the virus could be detected in pectoral fin, spleen, and head kidney as early as 24h post-challenge. The average viral load in all three tissues increased over time, reaching its highest level at 21 days post-challenge, which was followed by a slight decrease at 28 days post-challenge. IPNV load in pectoral fin tissue was comparable to the viral load in spleen and head kidney tissues, indicating that pectoral fin could be used for the detection and quantification of IPNV. The development of a non-lethal detection method will be useful for the detection of IPNV and potentially other viruses of finfish in farmed and wild fish.     

Comparison of RNA and cDNA transfection methods for rescue of infectious bursal disease virus

Specific alterations in the genetic material of RNA viruses rely on a technique known as reverse genetics. Transfection of cells with the altered generic material is a critical step of this procedure. In this report we have compared RNA and cDNA transfection methods for the efficiency of transient protein expression and rescue of (recombinant) infectious bursal disease virus (IBDV). Quantitative expression analysis of the secreted alkaline phosphatase reporter protein, and qualitative expression levels of an IBDV protein showed both that cDNA transfection results in a much higher level of protein expression than RNA transfection. Because the rescue of a crippled variant of IBDV was achieved consistently using the cDNA transfection method, but failed when we used the RNA transfection method, we favor the cDNA transfection method for the rescue of (recombinant) IBDV from cloned cDNA.     

Use of whole blood specimens for routine clinical quantitation of hepatitis C virus RNA does not increase assay sensitivity

The measurement of hepatitis C virus (HCV) RNA levels in the blood has, in the last few years, become a critical component in the therapy of patients with HCV infections. Initially, extraction methods for serum and plasma were used, but a newer method that uses Catrimox-14 as the extraction agent for whole blood has been reported. Because the whole blood extraction method may yield higher virus levels if significant levels of virus are present in the white blood cells (WBC), the method was evaluated for use in our clinical diagnostic laboratory despite its higher reagent costs and more time-consuming methodology. RNA was simultaneously extracted from 39 clinical samples by four different methods: Catrimox-14-Trizol extraction from whole blood, Trizol extraction from whole blood, Trizol extraction from serum, and a commercial serum extraction method, the EZNA total RNA kit. In addition, in an effort to quantitate the amount of HCV RNA virus in the WBC, Trizol extraction from isolated WBC was also performed. Quantitative results for samples from which RNA was extracted by all four methods were essentially the same; the Catrimox-14-Trizol method did not yield increased virus levels. Insignificant levels of virus were found in the WBC. The results did not demonstrate a clinical usefulness for the Catrimox-14-Trizol method.     

Quantification of human immunodeficiency virus in plasma by RNA PCR, viral culture, and p24 antigen detection.

A semiquantitative PCR technique for detecting human immunodeficiency virus type 1 (HIV-1) RNA in plasma was compared with quantitative viral culture and p24 antigen detection in plasma. Ninety-three samples from 20 symptomatic, 10 asymptomatic, and 10 seronegative individuals were tested. For most of the seropositive patients, consecutives samples were examined. Viral RNA was extracted from plasma by the method described by Boom et al. (R. Boom, C.J. A. Sol, M. M. M. Salimans, C.L. Jansen, P. M. E. Wertheim-van Dillen, and J. van der Noordaa, J. Clin. Microbiol. 28:495-503, 1990). The RNA PCR was the most sensitive method (100 and 74% sensitivity for symptomatic and asymptomatic patients, respectively) and produced less divergent results with the consecutive samples from individual patients compared with the other techniques. All samples positive by viral culture or p24 antigen assay were also positive in the RNA PCR. For each of the three assays, the number of positive results obtained correlated with the disease stage. The estimated mean number of HIV-1 RNA copies was significantly higher in symptomatic patients (22,750 copies per ml) than in asymptomatic patients (1,820 copies per ml). It was also higher in samples positive for viral culture than in culture-negative samples. No close correlation was found between the amount of HIV-1 RNA and the amount of p24 antigen or the titer of infectious virus in plasma or between this titer and the level of p24 antigen. The plasma RNA PCR may be a useful additional marker of disease progression and may be valuable for monitoring the effects of antiviral therapy.