Variation-tolerant capture and multiplex detection of nucleic acids: application to detection of microbes.

In contrast to ordinary PCRs, which have a limited multiplex capacity and often return false-negative results due to target variation or inhibition, our new detection strategy, VOCMA (variation-tolerant capture multiplex assay), allows variation-tolerant, target-specific capture and detection of many nucleic acids in one test. Here we demonstrate the use of a single-tube, dual-step amplification strategy that overcomes the usual limitations of PCR multiplexing, allowing at least a 22-plex format with retained sensitivity. Variation tolerance was achieved using long primers and probes designed to withstand variation at known sites and a judicious mix of degeneration and universal bases. We tested VOCMA in situations where enrichment from a large sample volume with high sensitivity and multiplexity is important (sepsis; streptococci, enterococci, and staphylococci, several enterobacteria, candida, and the most important antibiotic resistance genes) and where variation tolerance and high multiplexity is important (gastroenteritis; astrovirus, adenovirus, rotavirus, norovirus genogroups I and II, and sapovirus, as well as enteroviruses, which are not associated with gastroenteritis). Detection sensitivities of 10 to 1,000 copies per reaction were achieved for many targets. VOCMA is a highly multiplex, variation-tolerant, general purpose nucleic acid detection concept. It is a specific and sensitive method for simultaneous detection of nucleic acids from viruses, bacteria, fungi, and protozoa, as well as host nucleic acid, in the same test. It can be run on an ordinary PCR and a Luminex machine and is suitable for both clinical diagnoses and microbial surveillance.

Hybridization properties of long nucleic acid probes for detection of variable target sequences, and development of a hybridization prediction algorithm.

One of the main problems in nucleic acid-based techniques for detection of infectious agents, such as influenza viruses, is that of nucleic acid sequence variation. DNA probes, 70-nt long, some including the nucleotide analog deoxyribose-Inosine (dInosine), were analyzed for hybridization tolerance to different amounts and distributions of mismatching bases, e.g. synonymous mutations, in target DNA. Microsphere-linked 70-mer probes were hybridized in 3M TMAC buffer to biotinylated single-stranded (ss) DNA for subsequent analysis in a Luminex® system. When mismatches interrupted contiguous matching stretches of 6 nt or longer, it had a strong impact on hybridization. Contiguous matching stretches are more important than the same number of matching nucleotides separated by mismatches into several regions. dInosine, but not 5-nitroindole, substitutions at mismatching positions stabilized hybridization remarkably well, comparable to N (4-fold) wobbles in the same positions. In contrast to shorter probes, 70-nt probes with judiciously placed dInosine substitutions and/or wobble positions were remarkably mismatch tolerant, with preserved specificity. An algorithm, NucZip, was constructed to model the nucleation and zipping phases of hybridization, integrating both local and distant binding contributions. It predicted hybridization more exactly than previous algorithms, and has the potential to guide the design of variation-tolerant yet specific probes.

Rational recombinant XMRV antigen preparation and bead coupling for multiplex serology in a suspension array.

Diagnosis of infectious diseases often requires demonstration of antibodies to the microbe (serology). A large set of antigens, covering viruses, bacteria, fungi and parasites may be needed. Recombinant proteins have a prime role in serological tests. Suspension arrays offer high throughput for simultaneous measurement of many different antibodies. We here describe a rational process for preparation, purification and coupling to beads of recombinant proteins prepared in Escherichia coli derivate Origami B, to be used in a serological Luminex suspension array. All six Gag and Env proteins (p10, p12, p15, p30, gp70 and p15E), from the xenotropic murine leukemia virus-related virus (XMRV), were prepared, allowing the creation of a multiepitope XMRV antibody assay. The procedure is generic and allows production of protein antigens ready for serological testing in a few working days. Instability and aggregation problems were circumvented by expression of viral proteins fused to a carrier protein (thioredoxin A; TrxA), purification via inclusion body formation, urea solubilization, His tag affinity chromatography and direct covalent coupling to microspheres without removal of the elution buffer. The yield of one preparation (2-10mg fusion protein per 100ml culture) was enough for 20-100 coupling reactions, sufficing for tests of many tens of thousands of sera. False serological positivity due to antibodies binding to TrxA and to traces of E. coli proteins remaining in the preparation could be reduced by preabsorption of sera with free TrxA and E. coli extract. The recombinant antigens were evaluated using anti-XMRV antibodies. Although hybrid proteins expressed in E. coli in this way will not have the entire tertiary structure and posttranslational modifications of the native proteins, they contain a large subset of the epitopes associated with them. The described strategy is simple, quick, efficient and cheap. It should be applicable for suspension array serology in general.

Low Temperature and Low UV Indexes Correlated with Peaks of Influenza Virus Activity in Northern Europe during 2010⁻2018.

With the increasing pace of global warming, it is important to understand the role of meteorological factors in influenza virus (IV) epidemics. In this study, we investigated the impact of temperature, UV index, humidity, wind speed, atmospheric pressure, and precipitation on IV activity in Norway, Sweden, Finland, Estonia, Latvia and Lithuania during 2010⁻2018. Both correlation and machine learning analyses revealed that low temperature and UV indexes were the most predictive meteorological factors for IV epidemics in Northern Europe. Our in vitro experiments confirmed that low temperature and UV radiation preserved IV infectivity. Associations between these meteorological factors and IV activity could improve surveillance and promote development of accurate predictive models for future influenza outbreaks in the region.

Novel activities of safe-in-human broad-spectrum antiviral agents.

According to the WHO, there is an urgent need for better control of viral diseases. Re-positioning existing safe-in-human antiviral agents from one viral disease to another could play a pivotal role in this process. Here, we reviewed all approved, investigational and experimental antiviral agents, which are safe in man, and identified 59 compounds that target at least three viral diseases. We tested 55 of these compounds against eight different RNA and DNA viruses. We found novel activities for dalbavancin against echovirus 1, ezetimibe against human immunodeficiency virus 1 and Zika virus, as well as azacitidine, cyclosporine, minocycline, oritavancin and ritonavir against Rift valley fever virus. Thus, the spectrum of antiviral activities of existing antiviral agents could be expanded towards other viral diseases.

Development of single-tube nested real-time PCR assays with long internally quenched probes for detection of norovirus genogroup II.

The high sequence variation of RNA viruses necessitates use of degenerate primers and probes or multiple primers and probes in molecular diagnostic assays. We showed previously that PCR amplification in two rounds, first with long target-specific primers and then with short generic primers, followed by detection using long probes, can tolerate sequence variation. Here we demonstrate that long primers and probes of up to 56 nucleotides can also be applied in real-time PCR for the detection of norovirus genogroup II with improved sensitivity. Probe design (method of incorporating quenchers, use of Zen internal quencher or traditional quenchers) greatly affects the sensitivity of the real-time PCR assays.

Detection of rotavirus using padlock probes and rolling circle amplification.

Rotavirus infections are one of the most common reasons for hospitalizations due to gastrointestinal diseases. Rotavirus is often diagnosed by latex agglutination assay, chromatography immunoassay, or by electron microscopy, which are all quite insensitive. Reverse transcription polymerase chain reaction, on the other hand, is very sensitive to variations at the genomic level. We developed a novel assay based on a set of 58 different padlock probes with a detection limit of 1,000 copies. Twenty-two patient samples were analyzed and the assay showed high concordance with a PCR-based assay. In summary, we present a new assay for sensitive and variation tolerant detection of rotavirus.

No evidence for xenotropic murine leukemia-related virus infection in Sweden using internally controlled multiepitope suspension array serology.

Many syndromes have a large number of differential diagnoses, a situation which calls for multiplex diagnostic systems. Myalgic encephalomyelitis (ME), also named chronic fatigue syndrome (CFS), is a common disease of unknown etiology. A mouse retrovirus, xenotropic murine leukemia-related virus (XMRV), was found in ME/CFS patients and blood donors, but this was not corroborated. However, the paucity of serological investigations on XMRV in humans prompted us to develop a serological assay which cover many aspects of XMRV antigenicity. It is a novel suspension array method, using a multiplex IgG assay with nine recombinant proteins from the env and gag genes of XMRV and 38 peptides based on known epitopes of vertebrate gammaretroviruses. IgG antibodies were sought in 520 blood donors and 85 ME/CFS patients and in positive- and negative-control sera from animals. We found no differences in seroreactivity between blood donors and ME/CFS patients for any of the antigens. This did not support an association between ME/CFS and XMRV infection. The multiplex serological system had several advantages: (i) biotinylated protein G allowed us to run both human and animal sera, which is essential because of a lack of XMRV-positive humans; (ii) a novel quality control was a pan-peptide positive-control rabbit serum; and (iii) synthetic XMRV Gag peptides with degenerate positions covering most of the variation of murine leukemia-like viruses did not give higher background than nondegenerate analogs. The principle may be used for creation of variant tolerant peptide serologies. Thus, our system allows rational large-scale serological assays with built-in quality control.

Phylogeny-directed search for murine leukemia virus-like retroviruses in vertebrate genomes and in patients suffering from myalgic encephalomyelitis/chronic fatigue syndrome and prostate cancer.

Gammaretrovirus-like sequences occur in most vertebrate genomes. Murine Leukemia Virus (MLV) like retroviruses (MLLVs) are a subset, which may be pathogenic and spread cross-species. Retroviruses highly similar to MLLVs (xenotropic murine retrovirus related virus (XMRV) and Human Mouse retrovirus-like RetroViruses (HMRVs)) reported from patients suffering from prostate cancer (PC) and myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) raise the possibility that also humans have been infected. Structurally intact, potentially infectious MLLVs occur in the genomes of some mammals, especially mouse. Mouse MLLVs contain three major groups. One, MERV G3, contained MLVs and XMRV/HMRV. Its presence in mouse DNA, and the abundance of xenotropic MLVs in biologicals, is a source of false positivity. Theoretically, XMRV/HMRV could be one of several MLLV transspecies infections. MLLV pathobiology and diversity indicate optimal strategies for investigating XMRV/HMRV in humans and raise ethical concerns. The alternatives that XMRV/HMRV may give a hard-to-detect "stealth" infection, or that XMRV/HMRV never reached humans, have to be considered.

Murine gammaretrovirus group G3 was not found in Swedish patients with myalgic encephalomyelitis/chronic fatigue syndrome and fibromyalgia.

BACKGROUND: The recent report of gammaretroviruses of probable murine origin in humans, called xenotropic murine retrovirus related virus (XMRV) and human murine leukemia virus related virus (HMRV), necessitated a bioinformatic search for this virus in genomes of the mouse and other vertebrates, and by PCR in humans. RESULTS: Three major groups of murine endogenous gammaretroviruses were identified. The third group encompassed both exogenous and endogenous Murine Leukemia Viruses (MLVs), and most XMRV/HMRV sequences reported from patients suffering from myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS). Two sensitive real-time PCRs for this group were developed. The predicted and observed amplification range for these and three published XMRV/HMRV PCRs demonstrated conspicuous differences between some of them, partly explainable by a recombinatorial origin of XMRV. Three reverse transcription real-time PCRs (RTQPCRs), directed against conserved and not overlapping stretches of env, gag and integrase (INT) sequences of XMRV/HMRV were used on human samples. White blood cells from 78 patients suffering from ME/CFS, of which 30 patients also fulfilled the diagnostic criteria for fibromyalgia (ME/CFS/FM) and in 7 patients with fibromyalgia (FM) only, all from the Gothenburg area of Sweden. As controls we analyzed 168 sera from Uppsala blood donors. We controlled for presence and amplifiability of nucleic acid and for mouse DNA contamination. To score as positive, a sample had to react with several of the XMRV/HMRV PCRs. None of the samples gave PCR reactions which fulfilled the positivity criteria. CONCLUSIONS: XMRV/HMRV like proviruses occur in the third murine gammaretrovirus group, characterized here. PCRs developed by us, and others, approximately cover this group, except for the INT RTQPCR, which is rather strictly XMRV specific. Using such PCRs, XMRV/HMRV could not be detected in PBMC and plasma samples from Swedish patients suffering from ME/CFS/FM, and in sera from Swedish blood donors.

Cellular splicing and transcription regulatory protein p32 represses adenovirus major late transcription and causes hyperphosphorylation of RNA polymerase II.

The cellular protein p32 is a multifunctional protein, which has been shown to interact with a large number of cellular and viral proteins and to regulate several important activities like transcription and RNA splicing. We have previously shown that p32 regulates RNA splicing by binding and inhibiting the essential SR protein ASF/SF2. To determine whether p32 also functions as a regulator of splicing in virus-infected cells, we constructed a recombinant adenovirus expressing p32 under the transcriptional control of an inducible promoter. Much to our surprise the results showed that p32 overexpression effectively blocked mRNA and protein expression from the adenovirus major late transcription unit (MLTU). Interestingly, the p32-mediated inhibition of MLTU transcription was accompanied by an approximately 4.5-fold increase in Ser 5 phosphorylation and an approximately 2-fold increase in Ser 2 phosphorylation of the carboxy-terminal domain (CTD). Further, in p32-overexpressing cells the efficiency of RNA polymerase elongation was reduced approximately twofold, resulting in a decrease in the number of polymerase molecules that reached the end of the major late L1 transcription unit. We further show that p32 stimulates CTD phosphorylation in vitro. The inhibitory effect of p32 on MLTU transcription appears to require the CAAT box element in the major late promoter, suggesting that p32 may become tethered to the MLTU via an interaction with the CAAT box binding transcription factor.