Highly sensitive techniques, such as PCR, have greatly improved the detection of respiratory viruses. However, the sensitivity of PCR tests also complicates clinical interpretation, as the presence of small amounts of viral targets may not necessarily have clinical relevance. We performed a prospective case-control study in asymptomatic and symptomatic young children. PCR detection of 14 respiratory viruses was performed in nasal washes, and results were quantified in copies per milliliter. A total of 141 cases and 157 controls were included. In 72% of the cases and 28% of the controls, at least one virus was identified. When stratified for age, at least one virus was identified in 47% of the controls younger than 1 year old. Rhinovirus (RV) was frequently detected in both symptomatic and asymptomatic individuals. Receiver operating characteristic analysis for quantitative rhinovirus detection showed that cutoff values for clinical relevance are feasible for RV. In contrast to rhinovirus, respiratory syncytial virus (RSV) was rarely detected in controls, suggesting that a positive RSV test result is almost always of clinical relevance, independent of viral quantity. In conclusion, our study shows that asymptomatic carriage of a respiratory virus occurs frequently in young children. However, significant differences in the amount of virus present were observed between cases and controls. This suggests that defining cutoff levels should be feasible and represents the next necessary step for diagnosing viral respiratory infections using molecular tests.

In 2005, a human bocavirus was discovered in children with respiratory tract illnesses. Attempts to culture this virus on conventional cell lines has failed thus far. We investigated whether the virus can replicate on pseudostratified human airway epithelium. This cell culture system mimics the human airway environment and facilitates culturing of various respiratory agents. The cells were inoculated with human bocavirus-positive nasopharyngeal washes from children, and virus replication was monitored by measuring apical release of the virus via real-time PCR. Furthermore, we identified different viral mRNAs in the infected cells. All mRNAs were transcribed from a single promoter but varied due to alternative splicing and alternative polyadenylation, similar to what has been described for bovine parvovirus and minute virus of canines, the other two members of the Bocavirus genus. Thus, transcription of human bocavirus displays strong homology to the transcription of the other bocaviruses. In conclusion, we report here for the first time that human bocavirus can be propagated in an in vitro culture system and present a detailed map of the set of mRNAs that are produced by the virus.

eThe human airway epithelium (HAE) represents the entry port of many human respiratory viruses, including human coronaviruses (HCoVs). Nowadays, four HCoVs, HCoV-229E, HCoV-OC43, HCoV-HKU1, and HCoV-NL63, are known to be circulating worldwide, causing upper and lower respiratory tract infections in nonhospitalized and hospitalized children. Studies of the fundamental aspects of these HCoV infections at the primary entry port, such as cell tropism, are seriously hampered by the lack of a universal culture system or suitable animal models. To expand the knowledge on fundamental virus-host interactions for all four HCoVs at the site of primary infection, we used pseudostratified HAE cell cultures to isolate and characterize representative clinical HCoV strains directly from nasopharyngeal material. Ten contemporary isolates were obtained, representing HCoV-229E (n ‫؍‬ 1), HCoV-NL63 (n ‫؍‬ 1), HCoV-HKU1 (n ‫؍‬ 4), and HCoV-OC43 (n ‫؍‬ 4). For each strain, we analyzed the replication kinetics and progeny virus release on HAE cell cultures derived from different donors. Surprisingly, by visualizing HCoV infection by confocal microscopy, we observed that HCoV-229E employs a target cell tropism for nonciliated cells, whereas HCoV-OC43, HCoV-HKU1, and HCoV-NL63 all infect ciliated cells. Collectively, the data demonstrate that HAE cell cultures, which morphologically and functionally resemble human airways in vivo, represent a robust universal culture system for isolating and comparing all contemporary HCoV strains.

In 5–40% of respiratory infections in children, the diagnostics remain negative, suggesting that the patients might be infected with a yet unknown pathogen. Virus discovery cDNA-AFLP (VIDISCA) is a virus discovery method based on recognition of restriction enzyme cleavage sites, ligation of adaptors and subsequent amplification by PCR. However, direct discovery of unknown pathogens in nasopharyngeal swabs is difficult due to the high concentration of ribosomal RNA (rRNA) that acts as competitor. In the current study we optimized VIDISCA by adjusting the reverse transcription enzymes and decreasing rRNA amplification in the reverse transcription, using hexamer oligonucleotides that do not anneal to rRNA. Residual cDNA synthesis on rRNA templates was further reduced with oligonucleotides that anneal to rRNA but can not be extended due to 3′-dideoxy-C6-modification. With these modifications >90% reduction of rRNA amplification was established. Further improvement of the VIDISCA sensitivity was obtained by high throughput sequencing (VIDISCA-454). Eighteen nasopharyngeal swabs were analysed, all containing known respiratory viruses. We could identify the proper virus in the majority of samples tested (11/18). The median load in the VIDISCA-454 positive samples was 7.2 E5 viral genome copies/ml (ranging from 1.4 E3–7.7 E6). Our results show that optimization of VIDISCA and subsequent high-throughput-sequencing enhances sensitivity drastically and provides the opportunity to perform virus discovery directly in patient material.

Hepatitis C virus (HCV) genotype 4 (HCV-4) infection is considered to be difficult to treat and has become increasingly prevalent in European countriesHepatitis C virus (HCV) affects an estimated 170 million people worldwide. HCV infection persists in 50 to 85% of those infected and can, over decades, lead to cirrhosis and hepatocellular carcinoma (11). The HCV genome displays considerable sequence divergence, and HCV variants have been classified into seven major genotypes. Genotypes 1, 2, 3, 4, and 6 are further subdivided into numerous subtypes (subtypes a, b, c, etc.) (27). In the absence of complete genome sequences, the designation of a subtype is based mainly on consensus regions in the core/E1 and NS5B regions of the HCV genome (27). The HCV genotype distribution depends on the geographical region and the mode of transmission. As the distribution of HCV genotypes can change over time, genotyping provides a powerful tool that may be used to investigate the spread of HCV within a community (18).In Europe, North America, and Australia, most HCV-infected patients (Ͼ80%) are infected with genotype 1, 2, or 3 (10). HCV genotype 4 (HCV-4) is the most common genotype in the Middle East and in northern and central Africa, accounting for more than 20% of all chronic HCV infections worldwide (28). In Egypt, the country with the highest prevalence of HCV in the world, more than 90% of patients are infected with . HCV-4 is considered difficult to treat and has a sustained virological response rate of approximately 60% (28), where the rates are 40 to 50% for genotype 1 and 80 to 90% for genotypes 2 and 3 (17).Recent studies emphasize that the prevalence of HCV-4 in Europe has increased in the past few decades due to the immigration of HCV carriers and the subsequent spread of HCV-4 in European populations at risk for HCV infection (2,5,16,24,25,30). In southern Europe, HCV-4 is responsible for 10 to 24% of chronic HCV infections. In The Netherlands, HCV-4 accounts for an estimated 10% of chronic HCV infections (6, 32). Currently, the development of new genotypespecific antiviral agents is focused mainly on HCV genotype 1. The emergence of HCV-4 may require agents specific for HCV-4 to improve the response rates and decrease the future burden of HCV-4 disease. The population of the region around Amsterdam, The Netherlands, comprises many ethnicities and diverse groups at risk for HCV infection, providing

This multiplex showed excellent specificities for all 14 respiratory viruses and sensitivity was high except for clinical samples with low levels of enterovirus.

Hepatitis C virus (HCV) infection is a leading cause of chronic liver disease, cirrhosis, and hepatocellular carcinoma in humans and afflicts more than 58 million people worldwide. The HCV envelope E1 and E2 glycoproteins are essential for viral entry and comprise the primary antigenic target for neutralizing antibody responses. The molecular mechanisms of E1E2 assembly, as well as how the E1E2 heterodimer binds broadly neutralizing antibodies, remain elusive. Here, we present the cryo–electron microscopy structure of the membrane-extracted full-length E1E2 heterodimer in complex with three broadly neutralizing antibodies—AR4A, AT1209, and IGH505—at ~3.5-angstrom resolution. We resolve the interface between the E1 and E2 ectodomains and deliver a blueprint for the rational design of vaccine immunogens and antiviral drugs.

Since its initial identification in St. Petersburg, Russia, the recombinant hepatitis C virus (HCV) 2k/1b has been isolated from several countries throughout Eurasia. The 2k/1b strain is the only recombinant HCV to have spread widely, raising questions about the epidemiological background in which it first appeared. In order to further understand the circumstances by which HCV recombinants might be formed and spread, we estimated the date of the recombination event that generated the 2k/1b strain using a Bayesian phylogenetic approach. Our study incorporates newly isolated 2k/1b strains from Amsterdam, The Netherlands, and has employed a hierarchical Bayesian framework to combine information from different genomic regions. We estimate that 2k/1b originated sometime between 1923 and 1956, substantially before the first detection of the strain in 1999. The timescale and the geographic spread of 2k/1b suggest that it originated in the former Soviet Union at about the time that the world's first centralized national blood transfusion and storage service was being established. We also reconstructed the epidemic history of 2k/1b using coalescent theory-based methods, matching patterns previously reported for other epidemic HCV subtypes. This study demonstrates the practicality of jointly estimating dates of recombination from flanking regions of the breakpoint and further illustrates that rare genetic-exchange events can be particularly informative about the underlying epidemiological processes. Hepatitis C virus (HCV) infection presents a major global health burden, with the WHO estimating that 170 million chronic carriers are at risk of developing severe clinical outcomes such as cirrhosis and hepatic cellular carcinoma (56, 71). The virus belongs to the single-stranded positive-sense RNA virus family Flaviviridae and is characterized by considerable genetic diversity. HCV diversity is classified into six main genotypes (genotypes 1 to 6), each of which is further divided into numerous subtypes, and the virus exhibits nucleotide sequence divergences of 30 and 20% at the genotype and subtype levels, respectively (58). The high genomic heterogeneity of HCV is a result of both its high rate of evolution and its long-term association with human populations (60). Although there is no indication for a zoonotic virus reservoir, a related virus has recently been discovered in dogs (22).The greatest diversity of HCV is found in West and Central Africa and in Southeast Asia, where the virus appears to have persisted endemically for at least several centuries (49, 60). The current distribution of HCV genotypes and subtypes is geographically structured, reflecting differences in the rates and routes of transmission of the various subtypes and genotypes. Epidemic strains, exemplified by subtypes 1a, 1b, and 3a, are characterized by high prevalence, low genetic diversity, and a global distribution and are typically associated with transmission via infected blood products and injecting drug use (IDU) during the 20th century (13, 4...