Avian influenza A (H5N1) viruses cause severe disease in humans, but the basis for their virulence remains unclear. In vitro and animal studies indicate that high and disseminated viral replication is important for disease pathogenesis. Laboratory experiments suggest that virus-induced cytokine dysregulation may contribute to disease severity. To assess the relevance of these findings for human disease, we performed virological and immunological studies in 18 individuals with H5N1 and 8 individuals infected with human influenza virus subtypes. Influenza H5N1 infection in humans is characterized by high pharyngeal virus loads and frequent detection of viral RNA in rectum and blood. Viral RNA in blood was present only in fatal H5N1 cases and was associated with higher pharyngeal viral loads. We observed low peripheral blood T-lymphocyte counts and high chemokine and cytokine levels in H5N1-infected individuals, particularly in those who died, and these correlated with pharyngeal viral loads. Genetic characterization of H5N1 viruses revealed mutations in the viral polymerase complex associated with mammalian adaptation and virulence. Our observations indicate that high viral load, and the resulting intense inflammatory responses, are central to influenza H5N1 pathogenesis. The focus of clinical management should be on preventing this intense cytokine response, by early diagnosis and effective antiviral treatment.

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Fatal outcome of human influenza A (H5N1) is associated with high viral load and hypercytokinemia

Three human coronaviruses are known to exist: human coronavirus 229E (HCoV-229E), HCoV-OC43 and severe acute respiratory syndrome (SARS)-associated coronavirus (SARS-CoV). Here we report the identification of a fourth human coronavirus, HCoV-NL63, using a new method of virus discovery. The virus was isolated from a 7-month-old child suffering from bronchiolitis and conjunctivitis. The complete genome sequence indicates that this virus is not a recombinant, but rather a new group 1 coronavirus. The in vitro host cell range of HCoV-NL63 is notable because it replicates on tertiary monkey kidney cells and the monkey kidney LLC-MK2 cell line. The viral genome contains distinctive features, including a unique N-terminal fragment within the spike protein. Screening of clinical specimens from individuals suffering from respiratory illness identified seven additional HCoV-NL63-infected individuals, indicating that the virus was widely spread within the human population.

/pdf/identification-of-a-new-human-coronavirus-8kcwqjc0nn.pdf

Identification of a new human coronavirus

Our purpose was to identify an experimental procedure using PCR that provides a reliable genotype at a microsatellite locus using only a few picograms of template DNA. Under these circumstances, it is possible (i) that one allele of a heterozygous individual will not be detected and (ii) that PCR-generated alleles or 'false alleles' will arise. A mathematical model has been developed to account for stochastic events when pipetting template DNA in a very dilute DNA extract and computer simulations have been performed. Laboratory experiments were also carried out using DNA extracted from a bear feces sample to determine if experimental results correlate with the mathematical model. The results of 150 typing experiments are consistent with the proposed model. Based on this model and the level of observed false alleles, an experimental procedure using the multiple tubes approach is proposed to obtain reliable genotypes with a confidence level of 99%. This multiple tubes procedure should be systematically used when genotyping nuclear loci of ancient or forensic samples, museum specimens and hair or feces of free ranging animals.

Reliable Genotyping of Samples with Very Low DNA Quantities Using PCR

Although commercial kits are available for automated DNA extraction, ‘artisanal’ protocols are not. In this study, we present a silica-based method that is sensitive, inexpensive and compliant with automation. The effectiveness of this protocol has now been tested on more than 5000 animal specimens with highly positive results.

/pdf/an-inexpensive-automation-friendly-protocol-for-recovering-4i11lp843n.pdf

An inexpensive, automation-friendly protocol for recovering high-quality DNA

Noninvasive sampling allows genetic studies of free-ranging animals without the need to capture or even observe them, and thus allows questions to be addressed that cannot be answered using conventional methods. Initially, this sampling strategy promised to exploit fully the existing DNA-based technology for studies in ethology, conservation biology and population genetics. However, recent work now indicates the need for a more cautious approach, which includes quantifying the genotyping error rate. Despite this, many of the difficulties of noninvasive sampling will probably be overcome with improved methodology.

https://www.cell.com/trends/ecology-evolution/pdf/S0169-5347(99)01637-7.pdf

Noninvasive genetic sampling: look before you leap

We have developed a simple, rapid, and reliable protocol for the small-scale purification of DNA and RNA from, e.g., human serum and urine. The method is based on the lysing and nuclease-inactivating properties of the chaotropic agent guanidinium thiocyanate together with the nucleic acid-binding properties of silica particles or diatoms in the presence of this agent. By using size-fractionated silica particles, nucleic acids (covalently closed circular, relaxed circular, and linear double-stranded DNA; single-stranded DNA; and rRNA) could be purified from 12 different specimens in less than 1 h and were recovered in the initial reaction vessel. Purified DNA (although significantly sheared) was a good substrate for restriction endonucleases and DNA ligase and was recovered with high yields (usually over 50%) from the picogram to the microgram level. Copurified rRNA was recovered almost undegraded. Substituting size-fractionated silica particles for diatoms (the fossilized cell walls of unicellular algae) allowed for the purification of microgram amounts of genomic DNA, plasmid DNA, and rRNA from cell-rich sources, as exemplified for pathogenic gram-negative bacteria. In this paper, we show representative experiments illustrating some characteristics of the procedure which may have wide application in clinical microbiology.

Rapid and simple method for purification of nucleic acids.

silica particles, nucleic acids (covalently closed circular, relaxed circular, andlinear double-stranded DNA;single-stranded DNA; and rRNA)could bepurified from12different specimens inless than1handwere recovered intheinitial reaction vessel. Purified DNA (although significantly sheared) was a goodsubstrate forrestriction endonucleases and DNA ligase andwas recovered withhighyields (usually over50%)fromthepicogram tothemicrogram level. Copurified rRNAwas recovered almost undegraded. Substituting size-fractionated silica particles fordiatoms (the fossilized cell walls ofunicellular algae) allowed forthepurification ofmicrogram amountsofgenomic DNA,plasmid DNA,andrRNAfromcell-rich sources,asexemplified forpathogenic gram-negative bacteria. Inthis paper,we showrepresentative experiments illustrating some characteristics oftheprocedure whichmay havewideapplication inclinical microbiology. Theuse ofnucleic acid(NA)probes inthedetection of humanpathogens hasbecomeincreasingly important (for reviews, seereferences 18and24).Thepurification ofNA fromspecimens like serum, urine, or bacterial cultures, however, hasbeenlaborious andtime-consuming. Moreover,themany stepsinvolved inthepurification ofNA from suchspecimens byclassical procedures (involving detergent-mediated lysis, proteinase treatment, extractions with organic solvents, andethanol precipitation) increase therisk oftransmission ofNA fromsample tosample. Whenthe extremely sensitive polymerase chain reaction (PCR)(21, 22)or thetranscription-based amplification system(13)is usedforthedetection ofafewNA molecules ofapathogen, thetransmission ofNA mighteasily leadtofalse-positive results. Thebinding ofDNA inthepresenceofthechaotropic agentsNator NaCl04tosilica or glass particles iswell known(16, 26,29)buthasnot,toourknowledge, resulted in methods forNA purification directly fromclinical specimens like humanserum orurine. TheNA-binding properties of fossilized cellwallsofunicellular algae (hereafter called diatoms) orsilica orglass particles havenotbeenevaluated forthechaotropes guanidinium thiocyanate (GuSCN)or guanidinium hydrochloride (GuHCI). GuSCN hasbeen showntobea powerful agentinthepurification anddetectionofbothDNA andRNA because ofitspotential tolyse cells combined withitspotential toinactivate nucleases (6-8, 10,15,25,28). Guanidinium hydrochloride hasalsobeen successfully usedinNA purification frommammaliancells buthasappeared tobelesseffective thanGuSCN with respect toRNaseinhibition (4-6, 9,28). Inthis paper,we describe a methodfortherapid purification ofNA (covalently closed circular, relaxedcircular, andlinear double-stranded DNA;single-stranded DNA;and rRNA)directly fromhumanserum andurine. Themethodisbasedon our observation that, inthe

M. M. M. Salimans

Papers

Rapid and simple method for purification of nucleic acids.

RapidandSimple MethodforPurification ofNucleic Acids