The role of sub-genomic RNA in discordant results from RT-PCR tests for COVID-19.

Reverse transcription-PCR (RT-PCR) is the standard method of diagnosing COVID-19. An inconclusive test result occurs when one RT-PCR target is positive for SARS-CoV-2 and one RT-PCR target is negative within the same sample. An inconclusive result generally requires retesting. One reason why a sample may yield an inconclusive result is that one target is at a higher concentration than another target. It was hypothesized that concentration differences across targets may be due to the transcription of sub-genomic RNA, as this would result in an increase in the concentration of gene targets near the 3’ end of the SARS-CoV-2 genome. A panel of six digital droplet (dd)PCR assays was designed to quantitate the ORF1, E-gene, and N-gene of SARS-CoV-2. This panel was used to quantify viral cultures of SARS-CoV-2 that were harvested during the eclipse phase and at peak infectivity in such a way as to maximize gene-to-gene copy ratios. Eleven clinical nasopharyngeal swabs were also tested with this panel. In culture, infected cells showed higher N-gene/ORF1 copy ratios than culture supernatants. Both the highest specific infectivity (copies/pfu) and the highest differences between gene targets were observed at 6 hours post-infection (eclipse phase) in infected cells. The same trends in the relative abundance of copies across different targets observed in infected cells was observed in clinical samples, though trends were more pronounced in infected cells. This study showed that a greater copy number of N-gene relative to E-gene and ORF1 transcripts could potentially explain inconclusive results for some RT-PCR tests on low viral load samples. The use of N-gene RT-PCR target(s) as opposed to ORF1 targets for routine testing is supported by this data.

Impact of the H275Y and I223V Mutations in the Neuraminidase of the 2009 Pandemic Influenza Virus In Vitro and Evaluating Experimental Reproducibility

The 2009 pandemic H1N1 (H1N1pdm09) influenza virus is naturally susceptible to neuraminidase (NA) inhibitors, but mutations in the NA protein can cause oseltamivir resistance. The H275Y and I223V amino acid substitutions in the NA of the H1N1pdm09 influenza strain have been separately observed in patients exhibiting oseltamivir-resistance. Here, we apply mathematical modelling techniques to compare the fitness of the wild-type H1N1pdm09 strain relative to each of these two mutants. We find that both the H275Y and I223V mutations in the H1N1pdm09 background significantly lengthen the duration of the eclipse phase (by 2.5 h and 3.6 h, respectively), consistent with these NA mutations delaying the release of viral progeny from newly infected cells. Cells infected by H1N1pdm09 virus carrying the I223V mutation display a disadvantageous, shorter infectious lifespan (17 h shorter) than those infected with the wild-type or MUT-H275Y strains. In terms of compensating traits, the H275Y mutation in the H1N1pdm09 background results in increased virus infectiousness, as we reported previously, whereas the I223V exhibits none, leaving it overall less fit than both its wild-type counterpart and the MUT-H275Y strain. Using computer simulated competition experiments, we determine that in the presence of oseltamivir at doses even below standard therapy, both the MUT-H275Y and MUT-I223V dominate their wild-type counterpart in all aspects, and the MUT-H275Y outcompetes the MUT-I223V. The H275Y mutation should therefore be more commonly observed than the I223V mutation in circulating H1N1pdm09 strains, assuming both mutations have a similar impact or no significant impact on between-host transmission. We also show that mathematical modelling offers a relatively inexpensive and reliable means to quantify inter-experimental variability and assess the reproducibility of results.

Impact of the H275Y and I223V Mutations in the Neuraminidase of the 2009 Pandemic Influenza Virus In Vitro and Evaluating Experimental Reproducibility

The 2009 pandemic H1N1 (H1N1pdm09) influenza virus is naturally susceptible to neuraminidase (NA) inhibitors, but mutations in the NA protein can cause oseltamivir resistance. The H275Y and I223V amino acid substitutions in the NA of the H1N1pdm09 influenza strain have been separately observed in patients exhibiting oseltamivir-resistance. Here, we apply mathematical modelling techniques to compare the fitness of the wild-type H1N1pdm09 strain relative to each of these two mutants. We find that both the H275Y and I223V mutations in the H1N1pdm09 background significantly lengthen the duration of the eclipse phase (by 2.5 h and 3.6 h, respectively), consistent with these NA mutations delaying the release of viral progeny from newly infected cells. Cells infected by H1N1pdm09 virus carrying the I223V mutation display a disadvantageous, shorter infectious lifespan (17 h shorter) than those infected with the wild-type or MUT-H275Y strains. In terms of compensating traits, the H275Y mutation in the H1N1pdm09 background results in increased virus infectiousness, as we reported previously, whereas the I223V exhibits none, leaving it overall less fit than both its wild-type counterpart and the MUT-H275Y strain. Using computer simulated competition experiments, we determine that in the presence of oseltamivir at doses even below standard therapy, both the MUT-H275Y and MUT-I223V dominate their wild-type counterpart in all aspects, and the MUT-H275Y outcompetes the MUT-I223V. The H275Y mutation should therefore be more commonly observed than the I223V mutation in circulating H1N1pdm09 strains, assuming both mutations have a similar impact or no significant impact on between-host transmission. We also show that mathematical modelling offers a relatively inexpensive and reliable means to quantify inter-experimental variability and assess the reproducibility of results.

Dips and eclipses in the X-ray binary Swift J1858.6–0814 observed with NICER

We present the discovery of eclipses in the X-ray light curves of the X-ray binary Swift J1858.6–0814. From these, we find an orbital period of $P=76841.3_{-1.4}^{+1.3}$ s (≈21.3 hours) and an eclipse duration of $t_{\rm ec}=4098_{-18}^{+17}$ s (≈1.14 hours). We also find several absorption dips during the pre-eclipse phase. From the eclipse duration to orbital period ratio, the inclination of the binary orbit is constrained to i > 70○. The most likely range for the companion mass suggests that the inclination is likely to be closer to this value than 90○. The eclipses are also consistent with earlier data, in which strong variability (‘flares’) and the long orbital period prevent clear detection of the period or eclipses. We also find that the bright flares occurred preferentially in the post-eclipse phase of the orbit, likely due to increased thickness at the disc-accretion stream interface preventing flares being visible during the pre-eclipse phase. This supports the notion that variable obscuration is responsible for the unusually strong variability in Swift J1858.6–0814.

RNase R, a new virulence determinant of Streptococcus pneumoniae

ABSTRACTPneumococcal infections have increasingly high mortality rates despite the availability of vaccines and antibiotics. The increase of bacterial resistance to antibiotics urges the discovery of new alternative therapeutics. Therefore, the identification of new virulence determinants, and the understanding of the molecular mechanisms behind pathogenesis and pneumococcal-host interactions has become of paramount importance in the search of new targets for drug development. The exoribonuclease RNase R has been involved in virulence in a growing number of pathogens. In this work, we have used Galleria mellonella as an infection model to demonstrate that the presence of ribonuclease R increases the pneumococcus virulence. Although the absence of RNase R does not affect exponential growth, the ability of the RNase R deleted strain to replicate in the hemolymph is compromised. Larvae infected with the RNase R mutant strain show an increased expression level of antimicrobial peptides, and have a lower bacterial load in the haemolymph in the later stages of infection, leading to a higher survival rate. Interestingly RNase R carrying pneumococci suffer a sudden drop in bacterial numbers immediately after infection, resembling the eclipse phase observed after intravenous inoculation in mice. Together our results suggest that RNase R might be involved in the ability of pneumococci to evade the host immune response, probably by interfering with internalisation and/or replication inside the larval hemocytes.

Quantification of Ebola virus replication kinetics in vitro

Mathematical modelling has successfully been used to provide quantitative descriptions of many viral infections, but for the Ebola virus, which requires biosafety level 4 facilities for experimentation, modelling can play a crucial role. Ebola virus modelling efforts have primarily focused on in vivo virus kinetics, e.g., in animal models, to aid the development of antivirals and vaccines. But, thus far, these studies have not yielded a detailed specification of the infection cycle, which could provide a foundational description of the virus kinetics and thus a deeper understanding of their clinical manifestation. Here, we obtain a diverse experimental data set of the Ebola virus infection in vitro, and then make use of Bayesian inference methods to fully identify parameters in a mathematical model of the infection. Our results provide insights into the distribution of time an infected cell spends in the eclipse phase (the period between infection and the start of virus production), as well as the rate at which infectious virions lose infectivity. We suggest how these results can be used in future models to describe co-infection with defective interfering particles, which are an emerging alternative therapeutic.

In-host Modelling of COVID-19 in Humans

ABSTRACTCOVID-19 pandemic has underlined the impact of emergent pathogens as a major threat for human health. The development of quantitative approaches to advance comprehension of the current outbreak is urgently needed to tackle this severe disease. In this work, several mathematical models are proposed to represent SARS-CoV-2 dynamics in infected patients. Considering different starting times of infection, parameters sets that represent infectivity of SARS-CoV-2 are computed and compared with other viral infections that can also cause pandemics.Based on the target cell model, SARS-CoV-2 infecting time between susceptible cells (mean of 30 days approximately) is much slower than those reported for Ebola (about 3 times slower) and influenza (60 times slower). The within-host reproductive number for SARS-CoV-2 is consistent to the values of influenza infection (1.7-5.35). The best model to fit the data was including immune responses, which suggest a slow cell response peaking between 5 to 10 days post onset of symptoms. The model with eclipse phase, time in a latent phase before becoming productively infected cells, was not supported. Interestingly, both, the target cell model and the model with immune responses, predict that virus may replicate very slowly in the first days after infection, and it could be below detection levels during the first 4 days post infection. A quantitative comprehension of SARS-CoV-2 dynamics and the estimation of standard parameters of viral infections is the key contribution of this pioneering work.

Spectral evidence of an accretion disc in wind-fed X-ray pulsar Vela X-1 during an unusual spin-up period

ABSTRACT In classical supergiant X-ray binaries (SgXBs), the Bondi–Hoyle–Lyttleton wind accretion was usually assumed, and the angular momentum transport to the accretors is inefficient. The observed spin-up/spin-down behaviour of the neutron star in SgXBs is not well understood. In this paper, we report an extended low state of Vela X-1 (at orbital phases 0.16–0.2), lasting for at least 30 ks, observed with Chandra during the onset of an unusual spin-up period. During this low state, the continuum fluxes dropped by a factor of 10 compared to the preceding flare period, and the continuum pulsation almost disappeared. Meanwhile, the Fe K α fluxes of the low state were similar to the preceding flare period, leading to an Fe K α equivalent width (EW) of 0.6 keV, as high as the Fe K α EW during the eclipse phase of Vela X-1. Both the pulsation cessation and the high Fe K α EW indicate an axisymmetric structure with a column density larger than 1024 cm−2 on a spatial scale of the accretion radius of Vela X-1. These phenomena are consistent with the existence of an accretion disc that leads to the following spin-up of Vela X-1. It indicates that disc accretion, although not always, does occur in classical wind-fed SgXBs.

The Posthuman in the Schismatrix Stories and Eclipse Phase

This chapter examines exemplary texts within science fiction, Bruce Sterling’s Schismatrix stories and the TRPG, Eclipse Phase (EP). It begins by looking at SF as a mythic form of storytelling, one rooted in modernity. It uses literary theorist Istvan Csicsery-Ronay’s ideas in The Seven Beauties of Science Fiction (2008) to argue that tropes within SF are often highly fantastic, the cyborg transforming into posthuman tropes far beyond simple human-machine interfaces. In Sterling’s stories we see this dynamic in extreme forms of posthuman imaginings. With Sterling, he represents both transhuman and posthuman possibilities, some as extreme as biological space stations. In EP, players can choose a variety of characters to play that go beyond normative human forms into the strange and exotic. This provides a template for posthuman subjectivity.