The SARS-CoV-2 viral load in COVID-19 patients is lower on face mask filters than on nasopharyngeal swabs.

Face masks and personal respirators are used to curb the transmission of SARS-CoV-2 in respiratory droplets; filters embedded in some personal protective equipment could be used as a non-invasive sample source for applications, including at-home testing, but information is needed about whether filters are suited to capture viral particles for SARS-CoV-2 detection. In this study, we generated inactivated virus-laden aerosols of 0.3-2 microns in diameter (0.9 µm mean diameter by mass) and dispersed the aerosolized viral particles onto electrostatic face mask filters. The limit of detection for inactivated coronaviruses SARS-CoV-2 and HCoV-NL63 extracted from filters was between 10 to 100 copies/filter for both viruses. Testing for SARS-CoV-2, using face mask filters and nasopharyngeal swabs collected from hospitalized COVID-19-patients, showed that filter samples offered reduced sensitivity (8.5% compared to nasopharyngeal swabs). The low concordance of SARS-CoV-2 detection between filters and nasopharyngeal swabs indicated that number of viral particles collected on the face mask filter was below the limit of detection for all patients but those with the highest viral loads. This indicated face masks are unsuitable to replace diagnostic nasopharyngeal swabs in COVID-19 diagnosis. The ability to detect nucleic acids on face mask filters may, however, find other uses worth future investigation.

An exploration of the gut and environmental resistome in a community in northern Vietnam in relation to antibiotic use.

Background: Antibiotic resistance is a major global public health threat. Antibiotic use can directly impact the antibiotic resistant genes (ARGs) profile of the human intestinal microbiome and consequently the environment through shedding. Methods: We determined the resistome of human feces, animal stools, human food and environmental (rain, well, and irrigative water) samples (n = 304) in 40 households within a community cohort and related the data to antibiotic consumption. Metagenomic DNA was isolated and qPCR was used to determine presence of mobile colistin resistance (mcr) genes, genes encoding extended-spectrum ß-lactamases (ESBL), carbapenemases and quinolone resistance genes. Results: Nearly 40 % (39.5%, 120/304) of samples contained ESBL genes (most frequent were CTX-M-9 (23.7% [72/304]), CTX-M-1 (18.8% [57/304]). Quinolone resistance genes (qnrS) were detected in all human and 91% (41/45) of animal stool samples. Mcr-1 and mcr-3 were predominantly detected in human feces at 88% (82/93) and 55% (51/93) and animal feces at 93% (42/45) and 51% (23/45), respectively. Mcr-2, mrc-4 and mcr-5 were not detected in human feces, and only sporadically (< 6%) in other samples. Carbapenemase-encoding genes were most common in water (15% [14/91]) and cooked food (13% [10/75]) samples, while their prevalence in human and animal stools was lower at 4% in both human (4/93) and animal (2/45) samples. We did not find an association between recent antibiotic consumption and ARGs in human stools. Principal component analysis showed that the resistome differs between ecosystems with a strong separation of ARGs profiles of human and animal stools on the one hand versus cooked food and water samples on the other. Conclusions: Our study indicated that ARGs were abundant in human and animal stools in a rural Vietnamese community, including ARGs targeting last resort antibiotics. The resistomes of animal and human stools were similar as opposed to the resistomes from water and food sources. No association between antibiotic use and ARG profiles was found in a setting of high background rates of AMR.

Detection of the plasmid-mediated colistin-resistance gene mcr-1 in faecal metagenomes of Dutch travellers.

BACKGROUND: Recently, the first plasmid-mediated colistin-resistance gene, mcr-1, was reported. Colistin is increasingly used as an antibiotic of last resort for the treatment of infections caused by carbapenem-resistant bacteria, which have been rapidly disseminating worldwide in recent years. OBJECTIVES: The reported carriage rate of mcr-1 in humans remains sporadic thus far, except for those reported in Chinese populations. We aimed to determine its presence in the faecal metagenomes of healthy Dutch travellers between 2010 and 2012. METHODS: Faecal metagenomic DNA of pre- and post-travel samples from 122 healthy Dutch long-distance travellers was screened for the presence of mcr-1 using a TaqMan quantitative PCR assay, which was designed in this study. All positive samples were confirmed by sequencing of the amplicons. RESULTS: The mcr-1 gene was detected in 6 (4.9%, 95% CI = 2.1%-10.5%) of 122 healthy Dutch long-distance travellers after they had visited destinations in South(-east) Asia or southern Africa between 2011 and 2012. One of these participants was already found to be positive before travel. CONCLUSIONS: Our study highlights the potential of PCR-based targeted metagenomics as an unbiased and sensitive method to screen for the carriage of the mcr-1 gene and suggests that mcr-1 is widespread in various parts of the world. The observation that one participant was found to be positive before travel suggests that mcr-1 may already have disseminated to the microbiomes of Dutch residents at a low prevalence, warranting a more extensive investigation of its prevalence in the general population and possible sources.

Efficacy of IFN-λ1 to protect human airway epithelial cells against human rhinovirus 1B infection.

Impaired interferon (IFN) production has been observed in various obstructive respiratory diseases. This contributes to enhanced sensitivity towards viral infections triggering acute exacerbations. To compensate for this impaired host IFN response, there is need to explore new therapeutic strategies, like exogenous administration of IFNs as prophylactic treatment. In the present study, we examined the protective potential of IFN-λ1 and compared it with the previously established protecting effect of IFN-ß. A549 cells and human primary bronchial epithelial cells were first treated with either IFN-ß (500 IU/ml) or IFN-λ1 (500 ng/ml) for 18 h. For infection, two approaches were adopted: i) Continuous scenario: after pre-treatment, cells were infected immediately for 24 h with human rhinovirus 1B (HRV1B) in IFN-containing medium, or were cultured for another 72 h in IFN-containing medium, and then infected for 24 h with HRV1B, ii) Pre-treatment scenario: IFN-containing medium was replaced after 18 h and cells were infected for 4 h either immediately after pre-treatment or after additional culturing for 72 h in IFN-free medium. The protective effect was evaluated in terms of reduction in the number of viral copies/infectious progeny, and enhanced expression of IFN-stimulated genes (ISGs). In both cell types and in both approaches, IFN-λ1 and IFN-ß treatment resulted in pronounced and long-lasting antiviral effects exemplified by significantly reduced viral copy numbers and diminished infectious progeny. This was associated with strong up-regulation of multiple ISGs. However, in contrast to the IFN-ß induced expression of ISGs, which decreased over time, expression of ISGs induced by IFN-λ1 was sustained or even increased over time. Here we demonstrate that the protective potential of IFN-λ1 is comparable to IFN-ß. Yet, the long-lasting induction of ISGs by IFN-λ1 and most likely less incitement of side effects due to more localized expression of its receptors could make it an even more promising candidate for prophylactic treatment than IFN-ß.

Interferon-ß induces a long-lasting antiviral state in human respiratory epithelial cells.

OBJECTIVES: Interferon-ß (IFNß) induces strong antiviral effects and is therefore an attractive agent to prevent or reduce the incidence of virus-mediated exacerbations in asthmatic or chronic obstructive pulmonary disease (COPD) patients. We therefore investigated the effects of prophylactic IFNß on respiratory epithelial cells infected with rhinovirus (RV). METHODS: A549 cells and primary bronchial epithelial cells (PBECs) were exposed for 18 h to IFNß. Then, IFNß was either removed or maintained in the supernatant for the rest of the experiment and cells were infected with RV-1B at t = 0 or 72 h after the initial exposure to IFNß. RESULTS: Viral RNA levels were decreased in both cell types. Furthermore, both viral RNA and infectious virus levels in the supernatant of infected A549 cells were still significantly reduced at 72 h after removal of IFNß. This pronounced antiviral pre-treatment effect was associated with increased expression of the antiviral genes IFN-stimulated protein of MR15000 (ISG15) and Myxovirus resistance 1 (Mx1) and the effect was maintained even when IFNß levels in the supernatant of A549 cells were undetectable. CONCLUSIONS: These data show that IFNß has not only a strong, but also a long-lasting protective effect against RV infection of respiratory epithelium.

Rapid identification of bacteria by real-time amplification and sequencing of the 16S rRNA gene.

To allow rapid identification of bacteria in pure cultures and blood culture bottles, an assay was developed which is based on real-time amplification and sequencing of bacterial 16 S rRNA genes. In principle, this assay allows identification of bacteria from pure cultures within 6.5 h, and from blood cultures within approximately 7 h.

The Epstein-Barr virus BILF1 gene encodes a G protein-coupled receptor that inhibits phosphorylation of RNA-dependent protein kinase.

Epstein-Barr virus (EBV) infection is associated with many lymphoproliferative diseases, such as infectious mononucleosis and Burkitt's lymphoma. Consequently, EBV is one of the most extensively studied herpesviruses. Surprisingly, a putative G protein-coupled receptor (GPCR) gene of EBV, BILF1, has hitherto escaped attention, yet BILF1-like genes are conserved among all known lymphocryptovirus species, suggesting that they play a pivotal role in viral infection. To determine the function of EBV BILF1, the activity of this gene and its products was studied. BILF1-specific mRNA was detected in various EBV-positive cell types and found to be expressed predominantly during the immediate early and early phases of infection in vitro. Interestingly, in COS-7 cells transfected with BILF1 expression constructs, a decrease in forskolin-induced CRE-mediated transcription was measured, as well as an increase in NF-kappaB-mediated transcription. In contrast, CRE-mediated transcription was increased in EBV-positive Burkitt's lymphoma cells as well as EBV-positive lymphoblastoid B cells transfected with BILF1, whereas NF-kappaB-mediated transcription levels remained unaffected in these cells. All observed activities were sensitive to treatment with pertussis toxin, indicating that the BILF1-encoded protein mediates these activities by coupling to G proteins of the G(i/o) class. Finally, reduced levels of phosphorylated RNA-dependent antiviral protein kinase were observed in COS-7 and Burkitt's lymphoma cells transfected with BILF1. Neither of the observed effects required a ligand to interact with the BILF1 gene product, suggesting that BILF1 encodes a constitutively active GPCR capable of modulating various intracellular signaling pathways.

The r131 gene of rat cytomegalovirus encodes a proinflammatory CC chemokine homolog which is essential for the production of infectious virus in the salivary glands.

Rat cytomegalovirus (RCMV) possesses two adjacent genes, r131 and r129, which have the potential to encode CC chemokine homologs. Interestingly, the amino acid sequences encoded by both genes show similarity to the sequence of the murine CMV (MCMV) MCK-2 protein, which is encoded by the m131/129 gene. In order to study the significance of the r131 gene in the pathogenesis of RCMV infection, we generated two different virus strains in which the r131 open reading frame is disrupted. Replication of these null mutant strains, designated RCMVdeltar131a and RCMVdeltar131b, was evaluated in vitro and in vivo. Both strains were found to replicate with a similar efficiency as wild-type (WT) RCMV in vitro. However, in contrast to WT virus, neither RCMVdeltar131a nor RCMVdeltar131b established a high-titer infection in the salivary glands of immunocompromised rats. Furthermore, in a local, rat footpad infection model, both recombinant viruses induced a significantly lower amount of paw swelling than did WT RCMV. Also, a higher number of infiltrating macrophages was observed in paws infected with WT RCMV than in those infected with the recombinants. Taken together, these results suggest that r131 (i) promotes inflammation at initial sites of inoculation and, subsequently, efficient virus dissemination to or infection of the salivary glands and (ii) might be involved in the persistence of virus infection, at least in the spleen. In addition, our data indicate that r131 represents the functional homolog of the MCMV m131/129 gene.

The rat cytomegalovirus homologue of parvoviral rep genes, r127, encodes a nuclear protein with single- and double-stranded DNA-binding activity that is dispensable for virus replication.

An intriguing feature of the rat cytomegalovirus (RCMV) genome is open reading frame (ORF) r127, which shows similarity to the rep genes of parvoviruses as well as the U94 genes of human herpesvirus type 6A (HHV-6A) and 6B (HHV-6B). Counterparts of these genes have not been found in other herpesviruses. Here, it is shown that the r127 gene is transcribed during the early and late phases of virus replication in vitro as an unspliced 1.1 kb transcript containing the complete r127 ORF. Transcripts of r127 were also detected in various organs of RCMV-infected rats at 1 week post-infection (p.i.), but only in the salivary gland at 4 months p.i. Using rabbit polyclonal antibodies raised against the r127-encoded protein (pr127), pr127 was found to be expressed as early as 12 h p.i. within the nuclei of RCMV-infected cells in vitro. Expression of pr127 was also observed within the nuclei of cells in various organs of RCMV-infected rats at 3 weeks p.i. Moreover, pr127 was demonstrated to bind single- as well as double-stranded DNA. Finally, an RCMV r127 deletion mutant (RCMVDeltar127) was generated, in which the r127 ORF was disrupted. This deletion mutant, however, was shown to replicate with a similar efficiency as wild-type RCMV (wt RCMV), both in vitro and in vivo. Taken together, it is concluded that the RCMV r127 gene encodes a nuclear protein with single- and double-stranded DNA-binding activity that is dispensable for virus replication, not only in vitro, but also during the acute phase of infection in vivo.

Mutational analysis of the R33-encoded G protein-coupled receptor of rat cytomegalovirus: identification of amino acid residues critical for cellular localization and ligand-independent signalling.

The rat cytomegalovirus (RCMV) R33 gene encodes a G protein-coupled receptor (GPCR), pR33, which possesses agonist-independent, constitutive signalling activity. To characterize this activity further, we generated a series of point and deletion mutants of pR33. Both expression of and signalling by the mutants was evaluated. Several point mutants were generated that contained modifications in the NRY motif. This motif, at aa 130-132 of pR33, is the counterpart of the common DRY motif of GPCRs, which is known to be involved in G protein coupling. We found that mutation of the asparagine residue within the NRY motif of pR33 (N(130)) to aspartic acid resulted in a mutant (N(130)D) with similar signalling characteristics to the wild-type (WT) protein, indicating that N(130) is not the determinant of constitutive activity of pR33. Interestingly, a mutant carrying an alanine at aa 130 (N(130)A) was severely impaired in G(q/11)-mediated, constitutive activation of phospholipase C, whereas it displayed similar levels of activity to pR33 in G(i/0)-mediated signalling. Another protein that contained a modified NRY motif, R(131)A, did not show constitutive activity, whereas mutants Y(132)F and Y(132)A displayed similar activities to the WT receptor. This indicated that residue R(131) is critical for pR33 function in vitro, whereas Y(132) is not. Finally, we identified two consecutive arginines within the C-terminal tails of both pR33 and its homologue from human CMV, pUL33, which are important for correct cell-surface expression of these receptors.

The rat cytomegalovirus R78 G protein-coupled receptor gene is required for production of infectious virus in the spleen.

The rat cytomegalovirus (RCMV) R33 and R78 genes are conserved within members of the subfamily Betaherpesvirinae and encode proteins (pR33 and pR78, respectively) that show sequence similarity with G protein-coupled receptors. Previously, the biological relevance of these genes was demonstrated by the finding that R33- and R78-deleted RCMV strains are severely attenuated in vivo. In addition, R78-deleted strains were found to replicate less efficiently in cell culture. To monitor of the expression of R33- and R78-encoded proteins, recombinant RCMV strains, designated RCMV33G and RCMV78G, were generated. These recombinants expressed enhanced green fluorescent protein (EGFP)-tagged versions of pR33 and pR78 instead of native pR33 and pR78, respectively. Here it is reported that, although RCMV33G replicates as efficiently as wt virus in rat embryo fibroblast cultures, strain RCMV78G produces virus titres that are 3- to 4-fold lower than wt RCMV in the culture medium. This result indicates that the pR78-EGFP protein, as expressed by RCMV78G, does not completely functionally replace its native counterpart (pR78) in vitro. Interestingly, in infected rats, infectious RCMV33G was produced in significantly lower amounts than infectious wt RCMV, as well as RCMV78G, in the salivary glands. Conversely, although RCMV33G replicated to similar levels as wt virus in the spleen, both RCMV78G and an R78 knock-out strain (RCMV Delta R78a) replicated poorly in this organ. Together, these data indicate that R78 is crucial for the production of infectious RCMV in the spleen of infected rats.

The rat cytomegalovirus R32 gene encodes a virion-associated protein that elicits a strong humoral immune response in infected rats.

A gene of rat cytomegalovirus (RCMV), designated R32, has been identified that encodes a homologue of the human cytomegalovirus (HCMV) pp150 (ppUL32) major tegument phosphoprotein. The R32 ORF has the capacity to encode a 667 amino acid polypeptide (pR32) with a calculated molecular mass of 73 kDa. The predicted amino acid sequence of pR32 shows similarity to that of polypeptides predicted to be encoded by the HCMV UL32, murine cytomegalovirus M32 and human herpesvirus types 6 and 7 U11 genes. The R32 gene is transcribed as a 2.5 kb mRNA during the late phase of RCMV infection in rat embryo fibroblasts in vitro. To study expression of the pR32 protein in vitro and in vivo, a rabbit polyclonal antiserum was raised against a recombinant protein that comprised amino acids 252-522 of pR32. By using this antiserum, pR32 could be detected predominantly in the cytoplasm of RCMV-infected fibroblasts at 24 and 48 h post-infection in vitro. The pR32 protein was also detected within virions isolated from the culture medium of RCMV-infected cells. Expression of pR32 in vivo was observed within the cytoplasm of salivary gland epithelial cells of RCMV-infected rats. In addition, recombinant pR32 was found to react with sera from rats that were previously infected with RCMV, whereas reactivity was not seen with sera from mock-infected rats. Together, these findings indicate that RCMV pR32 represents the homologue of HCMV ppUL32, both in primary structure and in function.