A Novel High-Throughput Method for Molecular Detection of Human Pathogenic Viruses Using a Nanofluidic Real-Time PCR System

Human enteric viruses are recognized as the main causes of food- and waterborne diseases worldwide. Sensitive and quantitative detection of human enteric viruses is typically achieved through quantitative RT-PCR (RT-qPCR). A nanofluidic real-time PCR system was used to develop novel high-throughput methods for qualitative molecular detection (RT-qPCR array) and quantification of human pathogenic viruses by digital RT-PCR (RT-dPCR). The performance of high-throughput PCR methods was investigated for detecting 19 human pathogenic viruses and two main process controls used in food virology. The conventional real-time PCR system was compared to the RT-dPCR and RT-qPCR array. Based on the number of genome copies calculated by spectrophotometry, sensitivity was found to be slightly better with RT-qPCR than with RT-dPCR for 14 viruses by a factor range of from 0.3 to 1.6 log₁₀. Conversely, sensitivity was better with RT-dPCR than with RT-qPCR for seven viruses by a factor range of from 0.10 to 1.40 log₁₀. Interestingly, the number of genome copies determined by RT-dPCR was always from 1 to 2 log₁₀ lower than the expected copy number calculated by RT-qPCR standard curve. The sensitivity of the RT-qPCR and RT-qPCR array assays was found to be similar for two viruses, and better with RT-qPCR than with RT-qPCR array for eighteen viruses by a factor range of from 0.7 to 3.0 log₁₀. Conversely, sensitivity was only 0.30 log₁₀ better with the RT-qPCR array than with conventional RT-qPCR assays for norovirus GIV detection. Finally, the RT-qPCR array and RT-dPCR assays were successfully used together to screen clinical samples and quantify pathogenic viruses. Additionally, this method made it possible to identify co-infection in clinical samples. In conclusion, given the rapidity and potential for large numbers of viral targets, this nanofluidic RT-qPCR assay should have a major impact on human pathogenic virus surveillance and outbreak investigations and is likely to be of benefit to public health.     

New tools for the study and direct surveillance of viral pathogens in water

Half a century ago scientists attempted the detection of poliovirus in water. Since then other enteric viruses responsible for gastroenteritis and hepatitis have replaced enteroviruses as the main target for detection. However, most viral outbreaks are restricted to norovirus and hepatitis A virus, making them the main targets in water. The inclusion of virus analysis in regulatory standards for viruses in water samples must overcome several shortcomings such as the technical difficulties and high costs of virus monitoring, the lack of harmonised and standardised assays and the challenge posed by the ever-changing nature of viruses. However, new tools are nowadays available for the study and direct surveillance of viral pathogens in water that may contribute to fulfil these requirements.     

Accurate Strand-Specific Quantification of Viral RNA

The presence of full-length complements of viral genomic RNA is a hallmark of RNA virus replication within an infected cell. As such, methods for detecting and measuring specific strands of viral RNA in infected cells and tissues are important in the study of RNA viruses. Strand-specific quantitative real-time PCR (ssqPCR) assays are increasingly being used for this purpose, but the accuracy of these assays depends on the assumption that the amount of cDNA measured during the quantitative PCR (qPCR) step accurately reflects amounts of a specific viral RNA strand present in the RT reaction. To specifically test this assumption, we developed multiple ssqPCR assays for the positive-strand RNA virus o''nyong-nyong (ONNV) that were based upon the most prevalent ssqPCR assay design types in the literature. We then compared various parameters of the ONNV-specific assays. We found that an assay employing standard unmodified virus-specific primers failed to discern the difference between cDNAs generated from virus specific primers and those generated through false priming. Further, we were unable to accurately measure levels of ONNV (−) strand RNA with this assay when higher levels of cDNA generated from the (+) strand were present. Taken together, these results suggest that assays of this type do not accurately quantify levels of the anti-genomic strand present during RNA virus infectious cycles. However, an assay permitting the use of a tag-specific primer was able to distinguish cDNAs transcribed from ONNV (−) strand RNA from other cDNAs present, thus allowing accurate quantification of the anti-genomic strand. We also report the sensitivities of two different detection strategies and chemistries, SYBR® Green and DNA hydrolysis probes, used with our tagged ONNV-specific ssqPCR assays. Finally, we describe development, design and validation of ssqPCR assays for chikungunya virus (CHIKV), the recent cause of large outbreaks of disease in the Indian Ocean region.     

Human enteric viruses–potential indicators for enhanced monitoring of recreational water quality

Recreational waters contaminated with human fecal pollution are a public health concern, and ensuring the safety of recreational waters for public use is a priority of both the Environmental Protection Agency (EPA) and the Centers for Disease Control and Prevention (CDC). Current recreational water standards rely on fecal indicator bacteria (FIB) levels as indicators of human disease risk. However present evidence indicates that levels of FIB do not always correspond to the presence of other potentially harmful organisms, such as viruses. Thus, enteric viruses are currently tested as water quality indicators, but have yet to be successfully implemented in routine monitoring of water quality. This study utilized enteric viruses as possible alternative indicators of water quality to examine 18 different fresh and offshore recreational waters on O‘ahu, Hawai‘i, by using newly established laboratory techniques including highly optimized PCR, real time PCR, and viral infectivity assays. All sample sites were detected positive for human enteric viruses by PCR including enterovirus, norovirus genogroups I and II, and male specific FRNA coliphage. A six time-point seasonal study of enteric virus presence indicated significant variation in virus detection between the rainy and dry seasons. Quantitative PCR detected the presence of norovirus genogroup II at levels at which disease risk may occur, and there was no correlation found between enteric virus presence and FIB counts. Under the present laboratory conditions, no infectious viruses were detected from the samples PCR-positive for enteric viruses. These data emphasize both the need for additional indicators for improved monitoring of water quality, and the feasibility of using enteric viruses as these indicators. Electronic Supplementary MaterialSupplementary material is available for this article at 10.1007/s12250-015-3644-x and is accessible for authorized users.     

Exploring the diversity of plant DNA viruses and their satellites using vector-enabled metagenomics on whiteflies

Current knowledge of plant virus diversity is biased towards agents of visible and economically important diseases. Less is known about viruses that have not caused major diseases in crops, or viruses from native vegetation, which are a reservoir of biodiversity that can contribute to viral emergence. Discovery of these plant viruses is hindered by the traditional approach of sampling individual symptomatic plants. Since many damaging plant viruses are transmitted by insect vectors, we have developed "vector-enabled metagenomics" (VEM) to investigate the diversity of plant viruses. VEM involves sampling of insect vectors (in this case, whiteflies) from plants, followed by purification of viral particles and metagenomic sequencing. The VEM approach exploits the natural ability of highly mobile adult whiteflies to integrate viruses from many plants over time and space, and leverages the capability of metagenomics for discovering novel viruses. This study utilized VEM to describe the DNA viral community from whiteflies (Bemisia tabaci) collected from two important agricultural regions in Florida, USA. VEM successfully characterized the active and abundant viruses that produce disease symptoms in crops, as well as the less abundant viruses infecting adjacent native vegetation. PCR assays designed from the metagenomic sequences enabled the complete sequencing of four novel begomovirus genome components, as well as the first discovery of plant virus satellites in North America. One of the novel begomoviruses was subsequently identified in symptomatic Chenopodium ambrosiodes from the same field site, validating VEM as an effective method for proactive monitoring of plant viruses without a priori knowledge of the pathogens. This study demonstrates the power of VEM for describing the circulating viral community in a given region, which will enhance our understanding of plant viral diversity, and facilitate emerging plant virus surveillance and management of viral diseases.     

Microfluidic Quantitative PCR for Simultaneous Quantification of Multiple Viruses in Environmental Water Samples

To secure food and water safety, quantitative information on multiple pathogens is important. In this study, we developed a microfluidic quantitative PCR (MFQPCR) system to simultaneously quantify 11 major human viral pathogens, including adenovirus, Aichi virus, astrovirus, enterovirus, human norovirus, rotavirus, sapovirus, and hepatitis A and E viruses. Murine norovirus and mengovirus were also quantified in our MFQPCR system as a sample processing control and an internal amplification control, respectively. River water contaminated with effluents from a wastewater treatment plant in Sapporo, Japan, was collected and used to validate our MFQPCR system for multiple viruses. High-throughput quantitative information was obtained with a quantification limit of 2 copies/μl of cDNA/DNA. Using this MFQPCR system, we could simultaneously quantify multiple viral pathogens in environmental water samples. The viral quantities obtained using MFQPCR were similar to those determined by conventional quantitative PCR. Thus, the MFQPCR system developed in this study can provide direct and quantitative information for viral pathogens, which is essential for risk assessments.     

Mutations that decrease DNA binding of the processivity factor of the herpes simplex virus DNA polymerase reduce viral yield, alter the kinetics of viral DNA replication, and decrease the fidelity of DNA replication

The processivity subunit of the herpes simplex virus DNA polymerase, UL42, is essential for viral replication and possesses both Pol- and DNA-binding activities. Previous studies demonstrated that the substitution of alanine for each of four arginine residues, which reside on the positively charged surface of UL42, resulted in decreased DNA binding affinity and a decreased ability to synthesize long-chain DNA by the polymerase. In this study, the effects of each substitution on the production of viral progeny, viral DNA replication, and DNA replication fidelity were examined. Each substitution mutant was able to complement the replication of a UL42 null mutant in transient complementation assays and to support the replication of plasmid DNA containing herpes simplex virus type 1 (HSV-1) origin sequences in transient DNA replication assays. Mutant viruses containing each substitution and a lacZ insertion in a nonessential region of the genome were constructed and characterized. In single-cycle growth assays, the mutants produced significantly less progeny virus than the control virus containing wild-type UL42. Real-time PCR assays revealed that these UL42 mutants synthesized less viral DNA during the early phase of infection. Interestingly, during the late phase of infection, the mutant viruses synthesized larger amounts of viral DNA than the control virus. The frequencies of mutations of the virus-borne lacZ gene increased significantly in the substitution mutants compared to those observed for the control virus. These results demonstrate that the reduced DNA binding of UL42 is associated with significant effects on virus yields, viral DNA replication, and replication fidelity. Thus, a processivity factor can influence replication fidelity in mammalian cells.     

Potency estimation of measles, mumps and rubella trivalent vaccines with quantitative PCR infectivity assay.

The quantitative PCR infectivity assay is a combination of virus propagation and quantitative PCR. Previously [Schalk JAC, van den Elzen C, Ovelgonne H, Baas C, Jongen PMJM. Estimation of the number of infectious measles viruses in live virus vaccines using quantitative real-time PCR. J Virol Methods 2004;117:179-87.], we used this assay to estimate the titer of infectious measles virus in trivalent, live, measles, mumps, rubella vaccines (MMR). Here we describe the further improvement and development of the assay for simultaneous potency estimation of measles, mumps and rubella viruses. The potency of measles and mumps virus is estimated within one assay after 1 day of cell culture. The potency of rubella virus is estimated in a separate assay after 2 days of cell culture. Compared to conventional CCID50 and plaque assays, the quantitative PCR infectivity assay has the advantage in being fast because the assay is not dependent on the formation of cytopathic effect. Furthermore assay design is simplified: serological neutralization can be omitted because PCR is virus-specific and, under the conditions used, the individual components of trivalent measles, mumps, rubella vaccines do not interfere with each other. The assay was validated and compared to the performance of a plaque assay.     

A comparison of bats and rodents as reservoirs of zoonotic viruses: are bats special?

Bats are the natural reservoirs of a number of high-impact viral zoonoses. We present a quantitative analysis to address the hypothesis that bats are unique in their propensity to host zoonotic viruses based on a comparison with rodents, another important host order. We found that bats indeed host more zoonotic viruses per species than rodents, and we identified life-history and ecological factors that promote zoonotic viral richness. More zoonotic viruses are hosted by species whose distributions overlap with a greater number of other species in the same taxonomic order (sympatry). Specifically in bats, there was evidence for increased zoonotic viral richness in species with smaller litters (one young), greater longevity and more litters per year. Furthermore, our results point to a new hypothesis to explain in part why bats host more zoonotic viruses per species: the stronger effect of sympatry in bats and more viruses shared between bat species suggests that interspecific transmission is more prevalent among bats than among rodents. Although bats host more zoonotic viruses per species, the total number of zoonotic viruses identified in bats (61) was lower than in rodents (68), a result of there being approximately twice the number of rodent species as bat species. Therefore, rodents should still be a serious concern as reservoirs of emerging viruses. These findings shed light on disease emergence and perpetuation mechanisms and may help lead to a predictive framework for identifying future emerging infectious virus reservoirs.     

Sequence analysis of the human virome in febrile and afebrile children

Unexplained fever (UF) is a common problem in children under 3 years old. Although virus infection is suspected to be the cause of most of these fevers, a comprehensive analysis of viruses in samples from children with fever and healthy controls is important for establishing a relationship between viruses and UF. We used unbiased, deep sequencing to analyze 176 nasopharyngeal swabs (NP) and plasma samples from children with UF and afebrile controls, generating an average of 4.6 million sequences per sample. An analysis pipeline was developed to detect viral sequences, which resulted in the identification of sequences from 25 viral genera. These genera included expected pathogens, such as adenoviruses, enteroviruses, and roseoloviruses, plus viruses with unknown pathogenicity. Viruses that were unexpected in NP and plasma samples, such as the astrovirus MLB-2, were also detected. Sequencing allowed identification of virus subtype for some viruses, including roseoloviruses. Highly sensitive PCR assays detected low levels of viruses that were not detected in approximately 5 million sequences, but greater sequencing depth improved sensitivity. On average NP and plasma samples from febrile children contained 1.5- to 5-fold more viral sequences, respectively, than samples from afebrile children. Samples from febrile children contained a broader range of viral genera and contained multiple viral genera more frequently than samples from children without fever. Differences between febrile and afebrile groups were most striking in the plasma samples, where detection of viral sequence may be associated with a disseminated infection. These data indicate that virus infection is associated with UF. Further studies are important in order to establish the range of viral pathogens associated with fever and to understand of the role of viral infection in fever. Ultimately these studies may improve the medical treatment of children with UF by helping avoid antibiotic therapy for children with viral infections.     

采用基因微阵列技术对常见呼吸道病毒进行检测的初步研究

建立一种高通量的基因微阵列检测技术,对常见呼吸道病毒感染进行监控.根据公开发表的8个病毒科38种常见呼吸道病毒的序列,计算其保守区域,设计病毒的特异性检测探针,制备呼吸道病毒检测基因微阵列.利用随机引物PCR方法标记样品中的病毒靶序列,标记产物与基因微阵列上的探针杂交,清洗、扫描后进行结果分析.采用流感病毒、麻疹病毒、腮腺炎病毒和风疹病毒作为报告病毒,并对80例上呼吸道感染患者的咽拭子标本进行验证测试.初步结果表明,该呼吸道病毒微阵列基因芯片检测是可行的,在利用基因微阵列技术对病毒监控方面进行了有益的尝试,得到了有经验的信息.     

Development,standardization and assessment of PCR systems for purity testing of avian viral vaccines

The European Pharmacopoeia (Ph. Eur.) requires avian viral vaccines to be free of adventitious agents. Purity testing is an essential quality requirement of immunological veterinary medicinal products (IVMPs) and testing for extraneous agents includes monitoring for many different viruses. Conventional virus detection methods include serology or virus culture, however, molecular tests have become a valid alternative testing method.Nucleic acid testing (NAT) is fast, highly sensitive and has a higher degree of discrimination than conventional approaches. These advantages have led to the development and standardization of polymerase chain reaction (PCR) assays for the detection of avian leucosis virus, avian orthoreovirus, infectious bursal disease virus, infectious bronchitis virus, Newcastle disease virus, infectious laryngotracheitis virus, influenza A virus, Marek's disease virus, turkey rhinotracheitis virus, egg drop syndrome virus, chicken anaemia virus, avian adenovirus and avian encephalomyelitis virus. This paper reviews the development, standardization and assessment of PCR for extraneous agent testing in IVMPs with examples from an Official Medicines Control Laboratory (OMCL).     

Cost-effective Method for Microbial Source Tracking Using Specific Human and Animal Viruses

Microbial contamination of the environment represents a significant health risk. Classical bacterial fecal indicators have shown to have significant limitations, viruses are more resistant to many inactivation processes and standard fecal indicators do not inform on the source of contamination. The development of cost-effective methods for the concentration of viruses from water and molecular assays facilitates the applicability of viruses as indicators of fecal contamination and as microbial source tracking (MST) tools. Adenoviruses and polyomaviruses are DNA viruses infecting specific vertebrate species including humans and are persistently excreted in feces and/or urine in all geographical areas studied. In previous studies, we suggested the quantification of human adenoviruses (HAdV) and JC polyomaviruses (JCPyV) by quantitative PCR (qPCR) as an index of human fecal contamination. Recently, we have developed qPCR assays for the specific quantification of porcine adenoviruses (PAdV) and bovine polyomaviruses (BPyV) as animal fecal markers of contamination with sensitivities of 1-10 genome copies per test tube. In this study, we present the procedure to be followed to identify the source of contamination in water samples using these tools. As example of representative results, analysis of viruses in ground water presenting high levels of nitrates is shown.Detection of viruses in low or moderately polluted waters requires the concentration of the viruses from at least several liters of water into a much smaller volume, a procedure that usually includes two concentration steps in series. This somewhat cumbersome procedure and the variability observed in viral recoveries significantly hamper the simultaneous processing of a large number of water samples.In order to eliminate the bottleneck caused by the two-step procedures we have applied a one-step protocol developed in previous studies and applicable to a diversity of water matrices. The procedure includes: acidification of ten-liter water samples, flocculation by skimmed milk, gravity sedimentation of the flocculated materials, collection of the precipitate and centrifugation, resuspension of the precipitate in 10 ml phosphate buffer. The viral concentrate is used for the extraction of viral nucleic acids and the specific adenoviruses and polyomaviruses of interest are quantified by qPCR. High number of samples may be simultaneously analyzed using this low-cost concentration method.The procedure has been applied to the analysis of bathing waters, seawater and river water and in this study, we present results analyzing groundwater samples. This high-throughput quantitative method is reliable, straightforward, and cost-effective.     

Analytical Sensitivity Comparison between Singleplex Real-Time PCR and a Multiplex PCR Platform for Detecting Respiratory Viruses

Multiplex PCR methods are attractive to clinical laboratories wanting to broaden their detection of respiratory viral pathogens in clinical specimens. However, multiplexed assays must be well optimized to retain or improve upon the analytic sensitivity of their singleplex counterparts. In this experiment, the lower limit of detection (LOD) of singleplex real-time PCR assays targeting respiratory viruses is compared to an equivalent panel on a multiplex PCR platform, the GenMark eSensor RVP. LODs were measured for each singleplex real-time PCR assay and expressed as the lowest copy number detected 95–100% of the time, depending on the assay. The GenMark eSensor RVP LODs were obtained by converting the TCID₅₀/mL concentrations reported in the package insert to copies/μL using qPCR. Analytical sensitivity between the two methods varied from 1.2–1280.8 copies/μL (0.08–3.11 log differences) for all 12 assays compared. Assays targeting influenza A/H3N2, influenza A/H1N1pdm09, influenza B, and human parainfluenza 1 and 2 were most comparable (1.2–8.4 copies/μL, <1 log difference). Largest differences in LOD were demonstrated for assays targeting adenovirus group E, respiratory syncytial virus subtype A, and a generic assay for all influenza A viruses regardless of subtype (319.4–1280.8 copies/μL, 2.50–3.11 log difference). The multiplex PCR platform, the GenMark eSensor RVP, demonstrated improved analytical sensitivity for detecting influenza A/H3 viruses, influenza B virus, human parainfluenza virus 2, and human rhinovirus (1.6–94.8 copies/μL, 0.20–1.98 logs). Broader detection of influenza A/H3 viruses was demonstrated by the GenMark eSensor RVP. The relationship between TCID₅₀/mL concentrations and the corresponding copy number related to various ATCC cultures is also reported.     

Respiratory virus multiplex RT-PCR assay sensitivities and influence factors in hospitalized children with lower respiratory tract infections

Multiplex RT-PCR assays have been widely used tools for detection and differentiation of a panel of respiratory viral pathogens. In this study, we evaluated the Qiagen ResPlex II V2.0 kit and explored factors influencing its sensitivity. Nasopharyngeal swab (NPS) specimens were prospectively collected from pediatric inpatients with lower respiratory tract infections at the time of admission in the Shenzhen Children’s Hospital from May 2009 to April 2010. Total nucleic acids were extracted using the EZ1 system (Qiagen, Germany) and 17 respiratory viruses and genotypes including influenza A virus (FluA), FluB, parainfluenza virus 1 (PIV1), PIV2, PIV3, PIV4, respiratory syncytial virus (RSV), human metapneumovirus (hMPV), rhinoviruses (RhV), enteroviruses (EnV), human bocaviruses (hBoV), adenoviruses (AdV), four coronaviruses (229E, OC43, NL63 and HKU1), and FluA 2009 pandemic H1N1(H1N1-p) were detected and identified by the ResPlex II kit. In parallel, 16 real-time TaqMan quantitative RT-PCR assays were used to quantitatively detect each virus except for RhV. Influenza and parainfluenza viral cultures were also performed. Among the total 438 NPS specimens collected during the study period, one or more viral pathogens were detected in 274 (62.6%) and 201(45.9%) specimens by monoplex TaqMan RT-PCR and multiplex ResPlex, respectively. When results from monoplex PCR or cell culture were used as the reference standard, the multiplex PCR possessed specificities of 92.9–100.0%. The sensitivity of multiplex PCR for PIV3, hMPV, PIV1 and BoV were 73.1%, 70%, 66.7% and 55.6%, respectively, while low sensitivities (11.1%–40.0%) were observed for FluA, EnV, OC43, RSV and H1N1. Among the seven viruses/genotypes detected with higher frequencies, multiplex PCR sensitivities were correlated significantly with viral loads determined by the TaqMan RT-PCR in FluA, H1N1-p and RSV (p=0.011?0.000). The Qiagen ResPlex II multiplex RT-PCR kit possesses excellent specificity for simultaneous detection of 17 viral pathogens in NPS specimens in pediatric inpatients at the time of admission. The sensitivity of multiplex RT-PCR was influenced by viral loads, specimen process methods, primer and probe design and amplification condition.     

猪源胰酶中特异病毒和非特异病毒安全检测简

目的：对猪源胰酶样品进行病毒检测,以评价其病毒安全性。方法：非特异性病毒检测采用致细胞病变、血凝吸附试验和形态学检测方法;特异性病毒检测包括猪繁殖与呼吸障碍综合征病毒（PRRSV）、猪瘟病毒（CSFV）、猪圆环病毒（PCV）、猪细小病毒（PPV）、猪口蹄疫病毒（FMDV）和猪伪狂犬病病毒（PRV）特异性核酸检测,以及CSFV、PPV、PRV特定性抗原蛋白直接免疫荧光检测。结果：受检样品非特异性病毒检测中,未见可观察到的细胞病变产生和病毒粒子,对豚鼠、鸡和人的0型红细胞无凝集现象。特异性病毒检测中,PRRSV、CSFV、PCV、PPV、FMDV和PRV核酸检测均为阴性,CSFV、PPV、PRV免疫荧光检测均为阴性。结论：猪源胰酶样品经非特异性病毒检测和特异性病毒检测均无可检出的病毒存在。     

Demonstration of coinfection with and recombination by caprine arthritis-encephalitis virus and maedi-visna virus in naturally infected goats

Recombination of different strains and subtypes is a hallmark of lentivirus infections, particularly for human immunodeficiency virus, and contributes significantly to viral diversity and evolution both within individual hosts and within populations. Recombinant viruses are generated in individuals coinfected or superinfected with more than one lentiviral strain or subtype. This, however, has never been described in vivo for the prototype lentivirus maedi-visna virus of sheep and its closely related caprine counterpart, the caprine arthritis-encephalitis virus. Cross-species infections occur in animals living under natural conditions, which suggests that dual infections with small-ruminant lentiviruses (SRLVs) are possible. In this paper we describe the first documented case of coinfection and viral recombination in two naturally infected goats. DNA fragments encompassing a variable region of the envelope glycoprotein were obtained from these two animals by end-limiting dilution PCR of peripheral blood mononuclear cells or infected cocultures. Genetic analyses, including nucleotide sequencing and heteroduplex mobility assays, showed that these goats harbored two distinct populations of SRLVs. Phylogenetic analysis permitted us to assign these sequences to the maedi-visna virus group (SRLV group A) or the caprine arthritis-encephalitis virus group (SRLV group B). SimPlot analysis showed clear evidence of A/B recombination within the env gene segment of a virus detected in one of the two goats. This case provides conclusive evidence that coinfection by different strains of SRLVs of groups A and B can indeed occur and that these viruses actually recombine in vivo.