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The advent of whole genome sequencing has revealed that common laboratory strains of human cytomegalovirus (HCMV) have major genetic deficiencies resulting from serial passage in fibroblasts. In particular, tropism for epithelial and endothelial cells is lost due to mutations disrupting genes UL128, UL130, or UL131A, which encode subunits of a virion-associated pentameric complex (PC) important for viral entry into these cells but not for entry into fibroblasts. The endothelial cell-adapted strain TB40/E has a relatively intact genome and has emerged as a laboratory strain that closely resembles wild-type virus. However, several heterogeneous TB40/E stocks and cloned variants exist that display a range of sequence and tropism properties. Here, we report the use of PacBio sequencing to elucidate the genetic changes that occurred, both at the consensus level and within subpopulations, upon passaging a TB40/E stock on ARPE-19 epithelial cells. The long-read data also facilitated examination of the linkage between mutations. Consistent with inefficient ARPE-19 cell entry, at least 83% of viral genomes present before adaptation contained changes impacting PC subunits. In contrast, and consistent with the importance of the PC for entry into endothelial and epithelial cells, genomes after adaptation lacked these or additional mutations impacting PC subunits. The sequence data also revealed six single noncoding substitutions in the inverted repeat regions, single nonsynonymous substitutions in genes UL26, UL69, US28, and UL122, and a frameshift truncating gene UL141. Among the changes affecting protein-coding regions, only the one in UL122 was strongly selected. This change, resulting in a D390H substitution in the encoded protein IE2, has been previously implicated in rendering another viral protein, UL84, essential for viral replication in fibroblasts. This finding suggests that IE2, and perhaps its interactions with UL84, have important functions unique to HCMV replication in epithelial cells.

We present an extension of the Minimum Information about any (x) Sequence (MIxS) standard for reporting sequences of uncultivated virus genomes. Minimum Information about an Uncultivated Virus Genome (MIUViG) standards were developed within the Genomic Standards Consortium framework and include virus origin, genome quality, genome annotation, taxonomic classification, biogeographic distribution and in silico host prediction. Community-wide adoption of MIUViG standards, which complement the Minimum Information about a Single Amplified Genome (MISAG) and Metagenome-Assembled Genome (MIMAG) standards for uncultivated bacteria and archaea, will improve the reporting of uncultivated virus genomes in public databases. In turn, this should enable more robust comparative studies and a systematic exploration of the global virosphere.

We present an extension of the Minimum Information about any (x) Sequence (MIxS) standard for reporting sequences of uncultivated virus genomes. Minimum Information about an Uncultivated Virus Genome (MIUViG) standards were developed within the Genomic Standards Consortium framework and include virus origin, genome quality, genome annotation, taxonomic classification, biogeographic distribution and in silico host prediction. Community-wide adoption of MIUViG standards, which complement the Minimum Information about a Single Amplified Genome (MISAG) and Metagenome-Assembled Genome (MIMAG) standards for uncultivated bacteria and archaea, will improve the reporting of uncultivated virus genomes in public databases. In turn, this should enable more robust comparative studies and a systematic exploration of the global virosphere.

The emergence of congenital Zika virus (ZIKV) disease, with its devastating effects on the fetus, has prompted development of vaccines and examination of how ZIKV breaches the maternal-fetal barrier. Infection of placental and decidual tissue explants has demonstrated cell types at the uterine-placental interface susceptible to infection and suggests routes for transmission across the placenta and amniochorionic membrane. ZIKV replicates in proliferating Hofbauer cells within chorionic villi in placentas from severe congenital infection. Explants of anchoring villi recapitulate placental architecture and early-stage development and suggest infected Hofbauer cells disseminate virus to fetal blood vessels. ZIKV infection of explants represents a surrogate human model for evaluating protection against transmission by antibodies in vaccine recipients and passive immune formulations and novel therapeutics.

Poliovirus (PV) is the prototypical picornavirus. It is a non-enveloped RNA virus with a small (~7.5-kb) genome of positive polarity. It has long served as a model to study RNA virus biology, pathogenesis, and evolution. cDNA clones of several strains are available, and infectious virus can be produced by the transfection of in vitro transcribed viral genomes into an appropriate host cell. PV infects many human and non-human primate cell lines including HeLa and HeLa S3 cells, and can grow to high titer in culture. Protocols for the production, propagation, quantification, and purification of PV are presented.

A high prevalence and diversity of avian influenza (AI) viruses were detected in a population of wild mallards sampled during summer 2011 in California, providing an opportunity to compare results obtained before and after virus culture. We tested cloacal swab samples prior to culture by matrix real-time PCR, and by amplifying and sequencing a 640bp portion of the hemagglutinin (HA) gene. Each sample was also inoculated into embryonated chicken eggs, and full genome sequences were determined for cultured viruses. While low matrix Ct values were a good predictor of virus isolation from eggs, samples with high or undetectable Ct values also yielded isolates. Furthermore, a single passage in eggs altered the occurrence and detection of viral strains, and mixed infections (different HA subtypes) were detected less frequently after culture. There is no gold standard or perfect reference comparison for surveillance of unknown viruses, and true negatives are difficult to distinguish from false negatives. This study showed that sequencing samples prior to culture increases the detection of mixed infections and enhances the identification of viral strains and sequences that may have changed or even disappeared during culture.

Severe acute respiratory syndrome-associated coronavirus (SARS-CoV) emerged as the causal agent of an endemic atypical pneumonia, infecting thousands of people worldwide. Although a number of promising potential vaccines and therapeutic agents for SARS-CoV have been described, no effective antiviral drug against SARS-CoV is currently available. The intricate, sequential nature of the viral entry process provides multiple valid targets for drug development. Here, we describe a rapid and safe cell-based high-throughput screening system, dual envelope pseudovirion (DEP) assay, for specifically screening inhibitors of viral entry. The assay system employs a novel dual envelope strategy, using lentiviral pseudovirions as targets whose entry is driven by the SARS-CoV Spike glycoprotein. A second, unrelated viral envelope is used as an internal control to reduce the number of false positives. As an example of the power of this assay a class of inhibitors is reported with the potential to inhibit SARS-CoV at two steps of the replication cycle, viral entry and particle assembly. This assay system can be easily adapted to screen entry inhibitors against other viruses with the careful selection of matching partner virus envelopes.

Severe acute respiratory syndrome-associated coronavirus (SARS-CoV) emerged as the causal agent of an endemic atypical pneumonia, infecting thousands of people worldwide. Although a number of promising potential vaccines and therapeutic agents for SARS-CoV have been described, no effective antiviral drug against SARS-CoV is currently available. The intricate, sequential nature of the viral entry process provides multiple valid targets for drug development. Here, we describe a rapid and safe cell-based high-throughput screening system, dual envelope pseudovirion (DEP) assay, for specifically screening inhibitors of viral entry. The assay system employs a novel dual envelope strategy, using lentiviral pseudovirions as targets whose entry is driven by the SARS-CoV Spike glycoprotein. A second, unrelated viral envelope is used as an internal control to reduce the number of false positives. As an example of the power of this assay a class of inhibitors is reported with the potential to inhibit SARS-CoV at two steps of the replication cycle, viral entry and particle assembly. This assay system can be easily adapted to screen entry inhibitors against other viruses with the careful selection of matching partner virus envelopes.

The performance of 4 laboratory methods for diagnosis of viral respiratory tract infections (RTI) in older adults was evaluated. Seventy-four nasopharyngeal (NP) swab specimens were obtained from 60 patients with RTI at a long-term care facility over 2 respiratory seasons. Sixteen specimens were positive for a respiratory virus by at least 1 method. Multiplex reverse transcriptase polymerase chain reaction (RT-PCR) by the Luminex xTAG Respiratory Viral Panel (RVP) detected 16 (100%) of the positive specimens, RVP of 24-h culture supernatant detected 8 (50%), direct fluorescent antibody testing detected 4 (25%), rapid culture detected 2 (12.5%), and rapid antigen testing detected none. For a comparison group, RVP was performed on NP swabs from 20 outpatient children with RTI. The mean fluorescence intensity by RVP was significantly lower for positive adult patients than pediatric patients (P = 0.0373). Our data suggest that older adult patients shed lower titers of viruses, necessitating a highly sensitive assay such as RT-PCR to reliably detect respiratory viral pathogens.

The performance of 4 laboratory methods for diagnosis of viral respiratory tract infections (RTI) in older adults was evaluated. Seventy-four nasopharyngeal (NP) swab specimens were obtained from 60 patients with RTI at a long-term care facility over 2 respiratory seasons. Sixteen specimens were positive for a respiratory virus by at least 1 method. Multiplex reverse transcriptase polymerase chain reaction (RT-PCR) by the Luminex xTAG Respiratory Viral Panel (RVP) detected 16 (100%) of the positive specimens, RVP of 24-h culture supernatant detected 8 (50%), direct fluorescent antibody testing detected 4 (25%), rapid culture detected 2 (12.5%), and rapid antigen testing detected none. For a comparison group, RVP was performed on NP swabs from 20 outpatient children with RTI. The mean fluorescence intensity by RVP was significantly lower for positive adult patients than pediatric patients (P = 0.0373). Our data suggest that older adult patients shed lower titers of viruses, necessitating a highly sensitive assay such as RT-PCR to reliably detect respiratory viral pathogens.

BackgroundBetween 50% and 80% of asthma exacerbations are associated with viral respiratory tract infections (RTIs), yet the influence of viral pathogen diversity on asthma outcomes is poorly understood because of the limited scope and throughput of conventional viral detection methods.MethodsWe investigated the capability of the Virochip, a DNA microarray-based viral detection platform, to characterize viral diversity in RTIs in adults with and without asthma.ResultsThe Virochip detected viruses in a higher proportion of samples (65%) than did culture isolation (17%) while exhibiting high concordance (98%) with and comparable sensitivity (97%) and specificity (98%) to pathogen-specific polymerase chain reaction. A similar spectrum of viruses was identified in the RTIs of each patient subgroup; however, unexpected diversity among human coronaviruses (HCoVs) and human rhinoviruses (HRVs) was revealed. All but one of the HCoVs corresponded to the newly recognized HCoV-NL63 and HCoV-HKU1 viruses, and >20 different serotypes of HRVs were detected, including a set of 5 divergent isolates that formed a distinct genetic subgroup.ConclusionsThe Virochip can detect both known and novel variants of viral pathogens present in RTIs. Given the diversity detected here, larger-scale studies will be necessary to determine whether particular substrains of viruses confer an elevated risk of asthma exacerbation.

BackgroundBetween 50% and 80% of asthma exacerbations are associated with viral respiratory tract infections (RTIs), yet the influence of viral pathogen diversity on asthma outcomes is poorly understood because of the limited scope and throughput of conventional viral detection methods.MethodsWe investigated the capability of the Virochip, a DNA microarray-based viral detection platform, to characterize viral diversity in RTIs in adults with and without asthma.ResultsThe Virochip detected viruses in a higher proportion of samples (65%) than did culture isolation (17%) while exhibiting high concordance (98%) with and comparable sensitivity (97%) and specificity (98%) to pathogen-specific polymerase chain reaction. A similar spectrum of viruses was identified in the RTIs of each patient subgroup; however, unexpected diversity among human coronaviruses (HCoVs) and human rhinoviruses (HRVs) was revealed. All but one of the HCoVs corresponded to the newly recognized HCoV-NL63 and HCoV-HKU1 viruses, and >20 different serotypes of HRVs were detected, including a set of 5 divergent isolates that formed a distinct genetic subgroup.ConclusionsThe Virochip can detect both known and novel variants of viral pathogens present in RTIs. Given the diversity detected here, larger-scale studies will be necessary to determine whether particular substrains of viruses confer an elevated risk of asthma exacerbation.

We have compared the biochemical capacity of cultured cells from a variety of animal sources to replicate the genetic information of two small RNA viruses. Mengovirus is shown to infect and kill cultured cells from many animal species regardless of whether the virus replicated well, moderately, or poorly in any cell type. The production of infectious viral RNA, and of viral protein by any cell type was proportional to the yield of intact virus produced by that cell type. The virus yield per cell was compared in a variety of cells employing two small RNA viruses (mengovirus and bovine enterovirus 1) capable of adsorbing to, penetrating, and infecting all the cells tested. The yields varied over a range of more than a thousandfold with both viruses, but the patterns of cell susceptibility were markedly different for each virus. Both viruses caused profound inhibition of host cell protein synthesis even in cells which replicated the virus very inefficiently. Possible biochemical explanations for such highly specific host cell restriction are discussed. © 1967.

We have compared the biochemical capacity of cultured cells from a variety of animal sources to replicate the genetic information of two small RNA viruses. Mengovirus is shown to infect and kill cultured cells from many animal species regardless of whether the virus replicated well, moderately, or poorly in any cell type. The production of infectious viral RNA, and of viral protein by any cell type was proportional to the yield of intact virus produced by that cell type. The virus yield per cell was compared in a variety of cells employing two small RNA viruses (mengovirus and bovine enterovirus 1) capable of adsorbing to, penetrating, and infecting all the cells tested. The yields varied over a range of more than a thousandfold with both viruses, but the patterns of cell susceptibility were markedly different for each virus. Both viruses caused profound inhibition of host cell protein synthesis even in cells which replicated the virus very inefficiently. Possible biochemical explanations for such highly specific host cell restriction are discussed. © 1967.