Autologous Antibody Capture to Enrich Immunogenic Viruses for Viral Discovery

Discovery of new viruses has been boosted by novel deep sequencing technologies. Currently, many viruses can be identified by sequencing without knowledge of the pathogenicity of the virus. However, attributing the presence of a virus in patient material to a disease in the patient can be a challenge. One approach to meet this challenge is identification of viral sequences based on enrichment by autologous patient antibody capture. This method facilitates identification of viruses that have provoked an immune response within the patient and may increase the sensitivity of the current virus discovery techniques. To demonstrate the utility of this method, virus discovery deep sequencing (VIDISCA-454) was performed on clinical samples from 19 patients: 13 with a known respiratory viral infection and 6 with a known gastrointestinal viral infection. Patient sera was collected from one to several months after the acute infection phase. Input and antibody capture material was sequenced and enrichment was assessed. In 18 of the 19 patients, viral reads from immunogenic viruses were enriched by antibody capture (ranging between 1.5x to 343x in respiratory material, and 1.4x to 53x in stool). Enriched reads were also determined in an identity independent manner by using a novel algorithm Xcompare. In 16 of the 19 patients, 21% to 100% of the enriched reads were derived from infecting viruses. In conclusion, the technique provides a novel approach to specifically identify immunogenic viral sequences among the bulk of sequences which are usually encountered during virus discovery metagenomics.     

Assembly and characterization of pandemic influenza A H1N1 genome in nasopharyngeal swabs using high-throughput pyrosequencing

De novo high-throughput pyrosequencing was used to detect and characterize 2009 pandemic influenza A (H1N1) virus directly in nasopharyngeal swabs in the context of the microbial community. Data were generated with a prior sequence independent amplification by 454 pyrosequencing on GS-FLX platform (Roche). Influenza A assembled reads allowed near full-length genome reconstruction with the simultaneous analysis of site-specific heterogeneity. The molecular approach applied proved to be a powerful tool to characterize the new pandemic H1N1 influenza virus in clinical samples. This approach could be of great value in identifying possibly new reassortants that may occur in the near future.     

Mycoplasma detection by PCR analysis

Summary The polymerase chain reaction (PCR) method was used to detect mycoplasma contamination in a panel of 42 continuous cell lines. According to the microbiological cultivation assay on agar, 29 cell lines were chronically infected and 13 cell lines were negative. Sets of outer and inner primers (nested double-step PCR) were applied which anneal to DNA sequences coding for conserved regions of the 16S rRNA. These oligonucleotides allow for the amplification of DNA regions found in at least 25 mycoplasma species (including the ones most commonly found in cell cultures), but do not cross-hybridize with DNA from eukaryotic cells. Mycoplasma-positive cell lines showed distinctive bands in ethidium bromide-stained gels, both after the first round of amplification as well as after the second PCR; all agar-negative cell lines were also unambiguously negative in the PCR assay. Thus, neither false-positive nor false-negative results occurred. Provided that the proper PCR working conditions are scrupulously observed, the PCR amplification has several outstanding advantages: high sensitivity, specificity, reliability, objectivity, speed, and simplicity.     

Self-collected mid-turbinate swabs for the detection of respiratory viruses in adults with acute respiratory illnesses

Background

The gold standard for respiratory virus testing is a nasopharyngeal (NP) swab, which is collected by a healthcare worker. Midturbinate (MT) swabs are an alternative due to their ease of collection and possible self-collection by patients. The objective of this study was to compare the respiratory virus isolation of flocked MT swabs compared to flocked NP swabs.

Methods

Beginning in October 2008, healthy adults aged 18 to 69 years were recruited into a cohort and followed up for symptoms of influenza. They were asked to have NP and MT swabs taken as soon as possible after the onset of a fever or two or more respiratory symptoms with an acute onset. The swabs were tested for viral respiratory infections using Seeplex® RV12 multiplex PCR detection kit. Seventy six pairs of simultaneous NP and MT swabs were collected from 38 symptomatic subjects. Twenty nine (38%) of these pairs were positive by either NP or MT swabs or both. Sixty nine (91%) of the pair results were concordant. Two samples (3%) for hCV OC43/HKU1 and 1 sample (1%) for rhinovirus A/B were positive by NP but negative by MT. One sample each for hCV 229E/NL63, hCV OC43/HKU1, respiratory syncytial virus A, and influenza B were positive by MT but negative by NP.

Conclusions

Flocked MT swabs are sensitive for the diagnosis of multiple respiratory viruses. Given the ease of MT collection and similar results between the two swabs, it is likely that MT swabs should be the preferred method of respiratory cell collection for outpatient studies. In light of this data, larger studies should be performed to ensure that this still holds true and data should also be collected on the patient preference of collection methods.     

Rapid simultaneous detection and identification of six species Candida using polymerase chain reaction and reverse line hybridization assay

The aim of this study was to develop and evaluate PCR based reverse line blot (RLB) hybridization assay for rapid detection of the most common Candida isolates from clinical specimens. A pair of universal primers targeting the ITS2 region of the gene from 28S rRNA to 5.8S rRNA was designed for PCR amplification of DNA from 6 Candida species (C. albicans, C. tropicalis, C. krusei, C. glabrata, C. parapsilosis, C. dubliniensis), the reverse primer was biotin labeled. PCR products, which were 302-441 bp length, were hybridized with 6 specific oligonucleotides probes immobilized on a nylon membrane. These 6 probes proved specific (they hybridized with only their target molecules). The assay was shown to be sensitive in detecting yeast to a concentration of 10 CFU/ml. This method was used to test 100 isolates and 200 vaginal swabs. The results agreed with those of culture for all but 3 of 100 isolates. Sequencing was performed on these 3 samples and confirmed that the culture results were inaccurate. Our results show the PCR-RLB positive rate (49%) is higher than culture (39%) and smear microscopic screening (27%) (P<0.05). In conclusion, the PCR/RLB developed in this study is specific and offers increased sensitivity compared to culture for the detection of Candida species in swab specimens. Moreover, the improved detection of cases of polycandidal candidiasis is advantageous.     

A Novel Virus Causes Scale Drop Disease in Lates calcarifer

From 1992 onwards, outbreaks of a previously unknown illness have been reported in Asian seabass (Lates calcarifer) kept in maricultures in Southeast Asia. The most striking symptom of this emerging disease is the loss of scales. It was referred to as scale drop syndrome, but the etiology remained enigmatic. By using a next-generation virus discovery technique, VIDISCA-454, sequences of an unknown virus were detected in serum of diseased fish. The near complete genome sequence of the virus was determined, which shows a unique genome organization, and low levels of identity to known members of the Iridoviridae. Based on homology of a series of putatively encoded proteins, the virus is a novel member of the Megalocytivirus genus of the Iridoviridae family. The virus was isolated and propagated in cell culture, where it caused a cytopathogenic effect in infected Asian seabass kidney and brain cells. Electron microscopy revealed icosahedral virions of about 140 nm, characteristic for the Iridoviridae. In vitro cultured virus induced scale drop syndrome in Asian seabass in vivo and the virus could be reisolated from these infected fish. These findings show that the virus is the causative agent for the scale drop syndrome, as each of Koch’s postulates is fulfilled. We have named the virus Scale Drop Disease Virus. Vaccines prepared from BEI- and formalin inactivated virus, as well as from E. coli produced major capsid protein provide efficacious protection against scale drop disease.