Correlates of infectiousness
The role that individuals with asymptomatic or mildly symptomatic severe acute respiratory syndrome coronavirus 2 have in transmission of the virus is not well ...understood. Jones
et al.
investigated viral load in patients, comparing those showing few, if any, symptoms with hospitalized cases. Approximately 400,000 individuals, mostly from Berlin, were tested from February 2020 to March 2021 and about 6% tested positive. Of the 25,381 positive subjects, about 8% showed very high viral loads. People became infectious within 2 days of infection, and in hospitalized individuals, about 4 days elapsed from the start of virus shedding to the time of peak viral load, which occurred 1 to 3 days before the onset of symptoms. Overall, viral load was highly variable, but was about 10-fold higher in persons infected with the B.1.1.7 variant. Children had slightly lower viral loads than adults, although this difference may not be clinically significant.
Science
, abi5273, this issue p.
eabi5273
Analysis of thousands of people who tested positive in Germany reveals that many were asymptomatic and a minority exhibited high viral loads.
INTRODUCTION
Although post facto studies have revealed the importance of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) transmission from presymptomatic, asymptomatic, and mildly symptomatic (PAMS) cases, the virological basis of their infectiousness remains largely unquantified. The reasons for the rapid spread of variant lineages of concern, such as B.1.1.7, have yet to be fully determined.
RATIONALE
Viral load (viral RNA concentration) in patient samples and the rate of isolation success of virus from clinical specimens in cell culture are the clinical parameters most directly relevant to infectiousness and hence to transmission. To increase our understanding of the infectiousness of SARS-CoV-2, especially in PAMS cases and those infected with the B.1.1.7 variant, we analyzed viral load data from 25,381 German cases, including 9519 hospitalized patients, 6110 PAMS cases from walk-in test centers, 1533 B.1.1.7 variant infections, and the viral load time series of 4434 (mainly hospitalized) patients. Viral load results were then combined with estimated cell culture isolation probabilities, producing a clinical proxy estimate of infectiousness.
RESULTS
PAMS subjects had, at the first positive test, viral loads and estimated infectiousness only slightly less than hospitalized patients. Similarly, children were found to have mean viral loads only slightly lower (0.5 log
10
units
or less) than those of adults and ~78% of the adult peak cell culture isolation probability. Eight percent of first-positive viral loads were 10
9
copies per swab or higher, across a wide age range (mean 37.6 years, standard deviation 13.4 years), representing a likely highly infectious minority, one-third of whom were PAMS. Relative to non-B.1.1.7 cases, patients with the B.1.1.7 variant had viral loads that were higher by a factor of 10 and estimated cell culture infectivity that was higher by a factor of 2.6. Similar ranges of viral loads from B.1.1.7 and B.1.177 samples were shown to be capable of causing infection in Caco-2 cell culture. A time-course analysis estimates that a peak viral load of 10
8.1
copies per swab is reached 4.3 days after onset of shedding and shows that, across the course of infection, hospitalized patients have slightly higher viral loads than nonhospitalized cases, who in turn have viral loads slightly higher than PAMS cases. Higher viral loads are observed in first-positive tests of PAMS subjects, likely as a result of systematic earlier testing. Mean culture isolation probability declines to 0.5 at 5 days after peak viral load and to 0.3 at 10 days after peak viral load. We estimate a rate of viral load decline of 0.17 log
10
units per day, which, combined with reported estimates of incubation time and time to loss of successful cell culture isolation, suggests that viral load peaks 1 to 3 days before onset of symptoms (in symptomatic cases).
CONCLUSION
PAMS subjects who test positive at walk-in test centers can be expected to be approximately as infectious as hospitalized patients. The level of expected infectious viral shedding of PAMS people is of high importance because they are circulating in the community at the time of detection of infection. Although viral load and cell culture infectivity cannot be translated directly to transmission probability, it is likely that the rapid spread of the B.1.1.7 variant is partly attributable to higher viral load in these cases. Easily measured virological parameters can be used, for example, to estimate transmission risk from different groups (by age, gender, clinical status, etc.), to quantify variance, to show differences in virus variants, to highlight and quantify overdispersion, and to inform quarantine, containment, and elimination strategies.
Viral load and cell culture infectivity in 25,381 SARS-CoV-2 infections.
(
A
) Viral loads in presymptomatic, asymptomatic, and mildly symptomatic cases (PAMS; red), hospitalized patients (blue), and other subjects (black). (
B
) Expected first-positive viral load and cell culture isolation probability, colored as in (A). (
C
) Temporal estimation with lines representing patients, colored as in (A). (
D
) As in (C), but colored by age.
Two elementary parameters for quantifying viral infection and shedding are viral load and whether samples yield a replicating virus isolate in cell culture. We examined 25,381 cases of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in Germany, including 6110 from test centers attended by presymptomatic, asymptomatic, and mildly symptomatic (PAMS) subjects, 9519 who were hospitalized, and 1533 B.1.1.7 lineage infections. The viral load of the youngest subjects was lower than that of the older subjects by 0.5 (or fewer) log
10
units, and they displayed an estimated ~78% of the peak cell culture replication probability; in part this was due to smaller swab sizes and unlikely to be clinically relevant. Viral loads above 10
9
copies per swab were found in 8% of subjects, one-third of whom were PAMS, with a mean age of 37.6 years. We estimate 4.3 days from onset of shedding to peak viral load (10
8.1
RNA copies per swab) and peak cell culture isolation probability (0.75). B.1.1.7 subjects had mean log
10
viral load 1.05 higher than that of non-B.1.1.7 subjects, and the estimated cell culture replication probability of B.1.1.7 subjects was higher by a factor of 2.6.
The ongoing outbreak of the recently emerged novel coronavirus (2019-nCoV) poses a challenge for public health laboratories as virus isolates are unavailable while there is growing evidence that the ...outbreak is more widespread than initially thought, and international spread through travellers does already occur.
We aimed to develop and deploy robust diagnostic methodology for use in public health laboratory settings without having virus material available.
Here we present a validated diagnostic workflow for 2019-nCoV, its design relying on close genetic relatedness of 2019-nCoV with SARS coronavirus, making use of synthetic nucleic acid technology.
The workflow reliably detects 2019-nCoV, and further discriminates 2019-nCoV from SARS-CoV. Through coordination between academic and public laboratories, we confirmed assay exclusivity based on 297 original clinical specimens containing a full spectrum of human respiratory viruses. Control material is made available through European Virus Archive - Global (EVAg), a European Union infrastructure project.
The present study demonstrates the enormous response capacity achieved through coordination of academic and public laboratories in national and European research networks.
To estimate the seroprevalence and temporal course of SARS-CoV-2 neutralizing antibodies, we embedded a multi-tiered seroprevalence survey within an ongoing community-based cohort study in Bonn, ...Germany. We first assessed anti-SARS-CoV-2 immunoglobulin G levels with an immunoassay, followed by confirmatory testing of borderline and positive test results with a recombinant spike-based immunofluorescence assay and a plaque reduction neutralization test (PRNT). Those with a borderline or positive immunoassay result were retested after 4 to 5 months. At baseline, 4771 persons participated (88% response rate). Between April 24
and June 30
, 2020, seroprevalence was 0.97% (95% CI: 0.72-1.30) by immunoassay and 0.36% (95% CI: 0.21-0.61) when considering only those with two additional positive confirmatory tests. Importantly, about 20% of PRNT+ individuals lost their neutralizing antibodies within five months. Here, we show that neutralizing antibodies are detectable in only one third of those with a positive immunoassay result, and wane relatively quickly.
Ebola virus (EBOV) causes a severe hemorrhagic fever in humans and non-human primates. While no licensed therapeutics are available, recently there has been tremendous progress in developing ...antivirals. Targeting the ribonucleoprotein complex (RNP) proteins, which facilitate genome replication and transcription, and particularly the polymerase L, is a promising antiviral approach since these processes are essential for the virus life cycle. However, until now little is known about L in terms of its structure and function, and in particular the catalytic center of the RNA-dependent RNA polymerase (RdRp) of L, which is one of the most promising molecular targets, has never been experimentally characterized.
Using multiple sequence alignments with other negative sense single-stranded RNA viruses we identified the putative catalytic center of the EBOV RdRp. An L protein with mutations in this center was then generated and characterized using various life cycle modelling systems. These systems are based on minigenomes, i.e. miniature versions of the viral genome, in which the viral genes are exchanged against a reporter gene. When such minigenomes are coexpressed with RNP proteins in mammalian cells, the RNP proteins recognize them as authentic templates for replication and transcription, resulting in reporter activity reflecting these processes. Replication-competent minigenome systems indicated that our L catalytic domain mutant was impaired in genome replication and/or transcription, and by using replication-deficient minigenome systems, as well as a novel RT-qPCR-based genome replication assay, we showed that it indeed no longer supported either of these processes. However, it still showed similar expression to wild-type L, and retained its ability to be incorporated into inclusion bodies, which are the sites of EBOV genome replication.
We have experimentally defined the catalytic center of the EBOV RdRp, and thus a promising antiviral target regulating an essential aspect of the EBOV life cycle.
Antigen point-of-care tests (AgPOCTs) can accelerate SARS-CoV-2 testing. As some AgPOCTs have become available, interest is growing in their utility and performance. Here we aimed to compare the ...analytical sensitivity and specificity of seven commercially available AgPOCT devices.
In a single-centre, laboratory evaluation study, we compared AgPOCT products from seven suppliers: the Abbott Panbio COVID-19 Ag Rapid Test, the RapiGEN BIOCREDIT COVID-19 Ag, the Healgen Coronavirus Ag Rapid Test Cassette (Swab), the Coris BioConcept COVID-19 Ag Respi-Strip, the R-Biopharm RIDA QUICK SARS-CoV-2 Antigen, the nal von minden NADAL COVID-19 Ag Test, and the Roche-SD Biosensor SARS-CoV Rapid Antigen Test. Tests were evaluated on recombinant SARS-CoV-2 nucleoprotein, cultured endemic and emerging coronaviruses, stored respiratory samples with known SARS-CoV-2 viral loads, stored samples from patients with respiratory pathogens other than SARS-CoV-2, and self-sampled swabs from healthy volunteers. We estimated analytical sensitivity in terms of approximate viral concentrations (quantified by real-time RT-PCR) that yielded positive AgPOCT results, and specificity in terms of propensity to generate false-positive results.
In 138 clinical samples with quantified SARS-CoV-2 viral load, the 95% limit of detection (concentration at which 95% of test results were positive) in six of seven AgPOCT products ranged between 2·07 × 106 and 2·86 × 107 copies per swab, with an outlier (RapiGEN) at 1·57 × 1010 copies per swab. The assays showed no cross-reactivity towards cell culture or tissue culture supernatants containing any of the four endemic human coronaviruses (HCoV‑229E, HCoV‑NL63, HCoV‑OC43, or HCoV‑HKU1) or MERS-CoV, with the exception of the Healgen assay in one repeat test on HCoV-HKU1 supernatant. SARS-CoV was cross-detected by all assays. Cumulative specificities among stored clinical samples with non-SARS-CoV-2 infections (n=100) and self-samples from healthy volunteers (n=35; cumulative sample n=135) ranged between 98·5% (95% CI 94·2–99·7) and 100·0% (97·2–100·0) in five products, with two outliers at 94·8% (89·2–97·7; R-Biopharm) and 88·9% (82·1–93·4; Healgen). False-positive results did not appear to be associated with any specific respiratory pathogen.
The sensitivity range of most AgPOCTs overlaps with SARS-CoV-2 viral loads typically observed in the first week of symptoms, which marks the infectious period in most patients. The AgPOCTs with limit of detections that approximate virus concentrations at which patients are infectious might enable shortcuts in decision making in various areas of health care and public health.
EU's Horizon 2020 research and innovation programme, German Ministry of Research, German Federal Ministry for Economic Affairs and Energy, German Ministry of Health, and Bill & Melinda Gates Foundation.
Work with infectious Ebola virus is restricted to biosafety level (BSL) 4 laboratories. To overcome this limitation, life cycle modeling systems, which recapitulate part or all of the virus life ...cycle under BSL-1 or -2 conditions, have been developed. The tetracistronic transcription and replication-competent virus-like particle (trVLP) system is currently the most advanced of these systems and is particularly useful for drug screening. However, previous versions have used luciferase reporters, limiting the types of screening assays that can be performed. Here we describe the generation and optimization of a green fluorescent protein-expressing tetracistronic trVLP system, enabling high-content imaging and flow cytometry approaches.Summary: Transcription and replication-competent virus-like particle (trVLP) systems are powerful tools to model the life cycle of highly pathogenic Ebola viruses. Here we describe the generation of a novel, GFP-based trVLP system that allows high content imaging and flow cytometry approaches.
Hepatitis A virus (HAV) is a common human pathogen found exclusively in primates. In a molecular and serologic study of 64 alpacas in Bolivia, we detected RNA of distinct HAV in ≈9% of animals and ...HAV antibodies in ≈64%. Complete-genome analysis suggests a long association of HAV with alpacas.
The Ebola virus (EBOV) matrix protein VP40 drives budding of virions and encodes 2 overlapping late domain motifs at amino acid positions 7-13 (PTAPPEY). However, these motifs are not absolutely ...essential for replication in cell culture, and recently a potential third late domain motif (YPx(6)I) was proposed at amino acid positions 18-26 of VP40. To analyze the importance of this motif in viral budding, we used a transcription and replication-competent virus-like particle system. Using this system, we show that this motif does not contribute to EBOV budding or particle propagation.
Sequences for Lloviu virus (LLOV), a putative novel filovirus, were first identified in Miniopterus schreibersii bats in Spain following a massive bat die-off in 2002, and also recently found in bats ...in Hungary. However, until now it is unclear if these sequences correspond to a fully functional, infectious virus, and whether it will show a pathogenic phenotype like African filoviruses, such as ebola- and marburgviruses, or be apathogenic for humans, like the Asian filovirus Reston virus. Since no infectious virus has been recovered, the only opportunity to study infectious LLOV is to use a reverse genetics-based full-length clone system to de novo generate LLOV. As a first step in this process, and to investigate whether the identified sequences indeed correspond to functional viral proteins, we have developed life cycle modelling systems for LLOV, which allow us to study genome replication and transcription as well as entry of this virus. We show that all LLOV proteins fulfill their canonical role in the virus life cycle as expected based on the well-studied related filovirus Ebola virus. Further, we have analysed the intergenus-compatibility of proteins that have to act in concert to facilitate the virus life cycle. We show that some but not all proteins from LLOV and Ebola virus are compatible with each other, emphasizing the close relationship of these viruses, and informing future studies of filovirus biology with respect to the generation of genus-chimeric proteins in order to probe virus protein-protein interactions on a functional level.
Ebola virus (EBOV) causes severe outbreaks of viral hemorrhagic fever in humans. While virus-host interactions are promising targets for antivirals, there is only limited knowledge regarding the ...interactions of EBOV with cellular host factors. Recently, we performed a genome-wide siRNA screen that identified the nuclear RNA export factor 1 (NXF1) as an important host factor for the EBOV life cycle. NXF1 is a major component of the nuclear mRNA export pathway that is usurped by many viruses whose life cycles include nuclear stages. However, the role of NXF1 in the life cycle of EBOV, a virus replicating in cytoplasmic inclusion bodies, remains unknown. In order to better understand the role of NXF1 in the EBOV life cycle, we performed a combination of co-immunoprecipitation and double immunofluorescence assays to characterize the interactions of NXF1 with viral proteins and RNAs. Additionally, using siRNA-mediated knockdown of NXF1 together with functional assays, we analyzed the role of NXF1 in individual aspects of the virus life cycle. With this approach we identified the EBOV nucleoprotein (NP) as a viral interaction partner of NXF1. Further studies revealed that NP interacts with the RNA-binding domain of NXF1 and competes with RNA for this interaction. Co-localization studies showed that RNA binding-deficient, but not wildtype NXF1, accumulates in NP-derived inclusion bodies, and knockdown experiments demonstrated that NXF1 is necessary for viral protein expression, but not for viral RNA synthesis. Finally, our results showed that NXF1 interacts with viral mRNAs, but not with viral genomic RNAs. Based on these results we suggest a model whereby NXF1 is recruited into inclusion bodies to promote the export of viral mRNA:NXF1 complexes from these sites. This would represent a novel function for NXF1 in the life cycle of cytoplasmically replicating viruses, and may provide a basis for new therapeutic approaches against EBOV, and possibly other emerging viruses.
PDF
