Longitudinal Detection of Twenty DNA and RNA Viruses in Allogeneic Hematopoietic Stem Cell Transplant Recipients Plasma.

Metagenomics revealed novel and routinely overlooked viruses, representing sources of unrecognized infections after allogeneic hematopoietic stem cell transplantation (allo-HSCT). We aim to describe DNA and RNA virus prevalence and kinetics in allo-HSCT recipients' plasma for one year post HSCT. We included 109 adult patients with first allo-HSCT from 1 March 2017 to 31 January 2019 in this observational cohort study. Seventeen DNA and three RNA viral species were screened with qualitative and/or quantitative r(RT)-PCR assays using plasma samples collected at 0, 1, 3, 6, and 12 months post HSCT. TTV infected 97% of patients, followed by HPgV-1 (prevalence: 26-36%). TTV (median 3.29 × 105 copies/mL) and HPgV-1 (median 1.18 × 106 copies/mL) viral loads peaked at month 3. At least one Polyomaviridae virus (BKPyV, JCPyV, MCPyV, HPyV6/7) was detected in >10% of patients. HPyV6 and HPyV7 prevalence reached 27% and 12% at month 3; CMV prevalence reached 27%. HSV, VZV, EBV, HHV-7, HAdV and B19V prevalence remained <5%. HPyV9, TSPyV, HBoV, EV and HPg-V2 were never detected. At month 3, 72% of patients had co-infections. TTV and HPgV-1 infections were highly prevalent. BKPyV, MCPyV and HPyV6/7 were frequently detected relative to classical culprits. Further investigation is needed into associations between these viral infections and immune reconstitution or clinical outcomes.

Fecal Components Modulate Human Astrovirus Infectivity in Cells and Reconstituted Intestinal Tissues.

Human astroviruses (HAstV) are among the most common causative agents of viral gastroenteritis, especially in children, and extraintestinal manifestations have also been described. These viruses are transmitted by the fecal-oral route, implying that stool composition and the gut microbiota may impact their ability to remain infectious. For some enteric viruses, individual bacterial envelope components and other polysaccharide-containing molecules, which are abundant in stools, have been shown to enhance capsid stability. However, the role of the complex stool environment and, most importantly, the role of interindividual differences have been poorly studied. We used HAstV as a model to investigate how the stool environment in itself, its interindividual variability, and some specific stool components could affect HAstV stability and infectivity. Using two different HAstV genotypes, we found that stools as a whole modulate astrovirus infectivity not only in an individual-dependent manner but also in a manner that depends on the viral genotype. A virus-protective effect was observed after incubation with various Gram-positive and Gram-negative bacteria as well as with bacterial components, such as lipopolysaccharide and peptidoglycan. These results were further confirmed in human intestinal tissues, a more physiologically relevant system. Astrovirus infectivity was also preserved by mucin, a major component of intestinal mucus. We further confirmed that these components stabilize the viral capsid. These results show that although HAstV benefits from the stabilizing effect of fecal components, the complexity and variability of the stool composition and the multiple potential interactions may explain the interindividual differences in viral transmission observed in real life.IMPORTANCE To ensure transmission, enteric viruses must maintain their infectivity during the various environmental challenges that they face in transit within and between hosts. Increased knowledge of the factors affecting enteric virus survival may help to control their transmission. This study reveals that specific fecal bacterial components preserve classic human astrovirus infectivity by stabilizing viral particles. However, the outcomes of stool-virus interactions are very variable, ranging from protection to a reduction of viral infectivity, depending on the viral genotype and the individual from whom the stool has been collected. We show that the transmissibility of enteric viruses is dependent on the intestinal contents of the infected individual and highlight the complex multiple interactions that could explain the stochastic nature of enteric virus transmission in humans.

Novel human astroviruses in pediatric respiratory samples: A one-year survey in a Swiss tertiary care hospital.

Although classical human astroviruses (HAstV) are known to be a leading cause of viral gastroenteritis, the pathogenesis and clinical manifestations of novel HAstV remain largely unknown. There is mounting evidence that, in contrast to classical astroviruses, novel HAstV exhibit tropism for the upper respiratory tract. This one-year period prevalence screened all available clinical nasopharyngeal swab samples collected from pediatric patients aged ≤5 years for novel and classical HAstV using real-time reverse transcription polymerase chain reaction. A total of 205 samples were tested; two novel HAstV cases were detected for a prevalence of 1.3%, with viral loads suggesting active upper respiratory tract replication. No classical HAstV was detected.

Usutu virus in cerebrospinal fluid: A 2-year survey in a Tertiary Care Hospital, Geneva, Switzerland.

In 2009, the Usutu virus (USUV) was first reported as a cause of human neuroinvasive disorders. In Switzerland, USUV has been detected in wild birds with a seroprevalence of up to 6.59% in captive specimens sampled from zoo enclosures. This study investigates the clinical prevalence of USUV in human acute neuroinvasive disorders in Switzerland. Two hundred and fifty-eight cerebrospinal fluid samples collected between 2015 and 2017 for routine clinical care in a tertiary level hospital (Geneva) were tested for USUV by rRT-PCR. No samples were found positive, suggesting the absence, or the extremely low circulation of USUV in Western Switzerland.

Novel and classical human astroviruses in stool and cerebrospinal fluid: comprehensive screening in a tertiary care hospital, Switzerland.

Classical human astroviruses (HAstV) are the third most common cause of non-bacterial acute gastroenteritis. Due to the lack of routine molecular assays, novel HAstV are underdiagnosed and the magnitude of their contribution to clinical disease remains unknown. To better understand their prevalence and the susceptible patient profile, we conducted a comprehensive screening of novel and classical HAstV in stool and cerebrospinal fluid (CSF) samples collected for clinical care in a tertiary care hospital using a specially designed rRT-PCR panel for the detection of novel (MLB1-3 and VA1-4) and classical HAstV. Of the 654 stool samples, 20 were positive for HAstV, and the novel (n=10; 3 MLB1, 4 MLB2; 3 VA2) and classical (n=10) serotypes were equally prevalent. None of the 105 CSF samples were positive. Investigating the patient profile, we found a higher prevalence (P=0.0002) of both novel and classical HAstV in pediatric stool samples (3.4% and 3%, respectively) compared with adult stool samples (0.5% and 0.7%, respectively). Furthermore, all novel and classical HAstV-positive pediatric subjects were ≤four years old, demonstrating similar susceptible populations. Forty-five percent of positive patients were immunocompromised (novel: 40%, classical: 50%). A comparison of novel and classical HAstV-positive cases showed a lower viral load for novel HAstV (P=0.0007) with significantly more upper respiratory symptoms (70% of subjects; P=0.02); this observation may suggest a unique pathogenic pathway. This study confirms the clinical and epidemiological relevance of novel HAstV and identifies a target population in which routine screening may yield clinically valuable information.

Metagenomics analysis of red blood cell and fresh-frozen plasma units.

BACKGROUND: Although the risk of transmitting infectious agents by blood transfusion is dramatically reduced after donor selection, leukoreduction, and laboratory testing, some could still be present in donor's blood. A description of metagenomes in blood products eligible for transfusion represents relevant information to evaluate the risk of pathogen transmission by transfusion. STUDY DESIGN AND METHODS: Detection of viruses, bacteria, and fungi genomes was made by high-throughput sequencing (HTS) of 600 manufactured blood products eligible for transfusion: 300 red blood cell (RBC) and 300 fresh-frozen plasma (FFP) units. RESULTS: Anelloviruses and human pegivirus, frequent in the blood of healthy individuals, were found. Human papillomavirus type 27 and Merkel cell polyomavirus, present on the skin, were also detected. Unexpectedly, astrovirus MLB2 was identified and characterized in a FFP unit. The presence of astrovirus MLB2 was confirmed in donor's blood and corresponded to an asymptomatic acute viremia. Sequences of bacteria and fungi were also detected; they are likely the result of environmental contamination. CONCLUSION: This study demonstrates that HTS is a promising tool for detecting common and less frequent infectious pathogens in blood products.

Astrovirus MLB2, a New Gastroenteric Virus Associated with Meningitis and Disseminated Infection.

Next-generation sequencing has identified novel astroviruses for which a pathogenic role is not clearly defined. We identified astrovirus MLB2 infection in an immunocompetent case-patient and an immunocompromised patient who experienced diverse clinical manifestations, notably, meningitis and disseminated infection. The initial case-patient was identified by next-generation sequencing, which revealed astrovirus MLB2 RNA in cerebrospinal fluid, plasma, urine, and anal swab specimens. We then used specific real-time reverse transcription PCR to screen 943 fecal and 424 cerebrospinal fluid samples from hospitalized patients and identified a second case of meningitis, with positive results for the agent in the patient's feces and plasma. This screening revealed 5 additional positive fecal samples: 1 from an infant with acute diarrhea and 4 from children who had received transplants. Our findings demonstrate that astrovirus MLB2, which is highly prevalent in feces, can disseminate outside the digestive tract and is an unrecognized cause of central nervous system infection.

Molecular Epidemiology of Human Rhinoviruses and Enteroviruses Highlights Their Diversity in Sub-Saharan Africa.

Human rhinoviruses (HRVs) and enteroviruses (HEVs) belong to the Enterovirus genus and are the most frequent cause of infection worldwide, but data on their molecular epidemiology in Africa are scarce. To understand HRV and HEV molecular epidemiology in this setting, we enrolled febrile pediatric patients participating in a large prospective cohort assessing the causes of fever in Tanzanian children. Naso/oropharyngeal swabs were systematically collected and tested by real-time RT-PCR for HRV and HEV. Viruses from positive samples were sequenced and phylogenetic analyses were then applied to highlight the HRV and HEV types as well as recombinant or divergent strains. Thirty-eight percent (378/1005) of the enrolled children harboured an HRV or HEV infection. Although some types were predominant, many distinct types were co-circulating, including a vaccinal poliovirus, HEV-A71 and HEV-D68. Three HRV-A recombinants were identified: HRV-A36/HRV-A67, HRV-A12/HRV-A67 and HRV-A96/HRV-A61. Four divergent HRV strains were also identified: one HRV-B strain and three HRV-C strains. This is the first prospective study focused on HRV and HEV molecular epidemiology in sub-Saharan Africa. This systematic and thorough large screening with careful clinical data management confirms the wide genomic diversity of these viruses, brings new insights about their evolution and provides data about associated symptoms.

Comprehensive human virus screening using high-throughput sequencing with a user-friendly representation of bioinformatics analysis: a pilot study.

High-throughput sequencing (HTS) provides the means to analyze clinical specimens in unprecedented molecular detail. While this technology has been successfully applied to virus discovery and other related areas of research, HTS methodology has yet to be exploited for use in a clinical setting for routine diagnostics. Here, a bioinformatics pipeline (ezVIR) was designed to process HTS data from any of the standard platforms and to evaluate the entire spectrum of known human viruses at once, providing results that are easy to interpret and customizable. The pipeline works by identifying the most likely viruses present in the specimen given the sequencing data. Additionally, ezVIR can generate optional reports for strain typing, can create genome coverage histograms, and can perform cross-contamination analysis for specimens prepared in series. In this pilot study, the pipeline was challenged using HTS data from 20 clinical specimens representative of those most often collected and analyzed in daily practice. The specimens (5 cerebrospinal fluid, 7 bronchoalveolar lavage fluid, 5 plasma, 2 serum, and 1 nasopharyngeal aspirate) were originally found to be positive for a diverse range of DNA or RNA viruses by routine molecular diagnostics. The ezVIR pipeline correctly identified 14 of 14 specimens containing viruses with genomes of <40,000 bp, and 4 of 6 specimens positive for large-genome viruses. Although further validation is needed to evaluate sensitivity and to define detection cutoffs, results obtained in this pilot study indicate that the overall detection success rate, coupled with the ease of interpreting the analysis reports, makes it worth considering using HTS for clinical diagnostics.

Identification of site-specific adaptations conferring increased neural cell tropism during human enterovirus 71 infection.

Enterovirus 71 (EV71) is one of the most virulent enteroviruses, but the specific molecular features that enhance its ability to disseminate in humans remain unknown. We analyzed the genomic features of EV71 in an immunocompromised host with disseminated disease according to the different sites of infection. Comparison of five full-length genomes sequenced directly from respiratory, gastrointestinal, nervous system, and blood specimens revealed three nucleotide changes that occurred within a five-day period: a non-conservative amino acid change in VP1 located within the BC loop (L97R), a region considered as an immunogenic site and possibly important in poliovirus host adaptation; a conservative amino acid substitution in protein 2B (A38V); and a silent mutation in protein 3D (L175). Infectious clones were constructed using both BrCr (lineage A) and the clinical strain (lineage C) backgrounds containing either one or both non-synonymous mutations. In vitro cell tropism and competition assays revealed that the VP197 Leu to Arg substitution within the BC loop conferred a replicative advantage in SH-SY5Y cells of neuroblastoma origin. Interestingly, this mutation was frequently associated in vitro with a second non-conservative mutation (E167G or E167A) in the VP1 EF loop in neuroblastoma cells. Comparative models of these EV71 VP1 variants were built to determine how the substitutions might affect VP1 structure and/or interactions with host cells and suggest that, while no significant structural changes were observed, the substitutions may alter interactions with host cell receptors. Taken together, our results show that the VP1 BC loop region of EV71 plays a critical role in cell tropism independent of EV71 lineage and, thus, may have contributed to dissemination and neurotropism in the immunocompromised patient.

Critical analysis of rhinovirus RNA load quantification by real-time reverse transcription-PCR.

Rhinoviruses are the most frequent cause of human respiratory infections, and quantitative rhinovirus diagnostic tools are needed for clinical investigations. Although results obtained by real-time reverse-transcription PCR (RT-PCR) assays are frequently converted to viral RNA loads, this presents several limitations regarding accurate virus RNA quantification, particularly given the need to reliably quantify all known rhinovirus genotypes with a single assay. Using an internal extraction control and serial dilutions of an in vitro-transcribed rhinovirus RNA reference standard, we validated a quantitative one-step real-time PCR assay. We then used chimeric rhinovirus genomes with 5'-untranslated regions (5'UTRs) originating from the three rhinovirus species and from one enterovirus to estimate the impact of the 5'UTR diversity. Respiratory specimens from infected patients were then also analyzed. The assay quantification ability ranged from 4.10 to 9.10 log RNA copies/ml, with an estimated error margin of ±10%. This variation was mainly linked to target variability and interassay variability. Taken together, our results indicate that our assay can reliably estimate rhinovirus RNA load, provided that the appropriate error margin is used. In contrast, due to the lack of a universal rhinovirus RNA standard and the variability related to sample collection procedures, accurate absolute rhinovirus RNA quantification in respiratory specimens is currently hardly feasible.

Experimental human rhinovirus and enterovirus interspecies recombination.

Human rhinoviruses (HRVs) and enteroviruses (HEVs), two important human pathogens, are non-enveloped, positive-sense RNA viruses of the genus Enterovirus within the family Picornaviridae. Intraspecies recombination is known as a driving force for enterovirus and, to a lesser extent, rhinovirus evolution. Interspecies recombination is much less frequent among circulating strains, and supporting evidence for such recombination is limited to ancestral events, as shown by recent phylogenetic analyses reporting ancient HRV-A/HRV-C, HEV-A/HEV-C and HEV-A/HEV-D recombination mainly at the 5'-untranslated region (5' UTR)-polyprotein junction. In this study, chimeric genomes were artificially generated using the 5' UTR from two different clinical HRV-C strains (HRV-Ca and HRV-Cc), an HRV-B strain (HRV-B37) and an HEV-A strain (HEV-A71), and the remaining part of the genome from an HRV-A strain (HRV-A16). Whilst the chimeric viruses were easily propagated in cell culture, the wild-type HRV-A16 retained a replication advantage, both individually and in competition experiments. Assessment of protein synthesis ability did not show a correlation between translation and replication efficiencies. These results reflect the interchangeability of the 5' UTR, including its functional RNA structural elements implicated in both genome translation and replication among different enterovirus species. The 5' UTR-polyprotein junction therefore represents a theoretic interspecies recombination breakpoint. This recombination potential is probably restricted by the need for co-infection opportunities and the requirement for the progeny chimera to outcompete the parental genomes' fitness, explaining the rare occurrence of such events in vivo.

New respiratory enterovirus and recombinant rhinoviruses among circulating picornaviruses.

Rhinoviruses and enteroviruses are leading causes of respiratory infections. To evaluate genotypic diversity and identify forces shaping picornavirus evolution, we screened persons with respiratory illnesses by using rhinovirus-specific or generic real-time PCR assays. We then sequenced the 5 untranslated region, capsid protein VP1, and protease precursor 3CD regions of virus-positive samples. Subsequent phylogenetic analysis identified the large genotypic diversity of rhinoviruses circulating in humans. We identified and completed the genome sequence of a new enterovirus genotype associated with respiratory symptoms and acute otitis media, confirming the close relationship between rhinoviruses and enteroviruses and the need to detect both viruses in respiratory specimens. Finally, we identified recombinants among circulating rhinoviruses and mapped their recombination sites, thereby demonstrating that rhinoviruses can recombine in their natural host. This study clarifies the diversity and explains the reasons for evolution of these viruses.

New molecular detection tools adapted to emerging rhinoviruses and enteroviruses.

Human rhinoviruses (HRV), and to a lesser extent human enteroviruses (HEV), are important respiratory pathogens. Like other RNA viruses, these picornaviruses have an intrinsic propensity to variability. This results in a large number of different serotypes as well as the incessant discovery of new genotypes. This large and growing diversity not only complicates the design of real-time PCR assays but also renders immunofluorescence unfeasible for broad HRV and HEV detection or quantification in cells. In this study, we used the 5' untranslated region, the most conserved part of the genome, as a target for the development of both a real-time PCR assay (Panenterhino/Ge/08) and a peptide nucleic acid-based hybridization oligoprobe (Panenterhino/Ge/08 PNA probe) designed to detect all HRV and HEV species members according to publicly available sequences. The reverse transcription-PCR assay has been validated, using not only plasmid and viral stocks but also quantified RNA transcripts and around 1,000 clinical specimens. These new generic detection PCR assays overcame the variability of circulating strains and lowered the risk of missing emerging and divergent HRV and HEV. An additional real-time PCR assay (Entero/Ge/08) was also designed specifically to provide sensitive and targeted detection of HEV in cerebrospinal fluid. In addition to the generic probe, we developed specific probes for the detection of HRV-A and HRV-B in cells. This investigation provides a comprehensive toolbox for accurate molecular identification of the different HEV and HRV circulating in humans.