Diagnostic Efficacy and Tolerability of Molded Plastic Nasopharyngeal Swab (FinSwab) Compared to Flocked Nylon Swab in Detection of SARS-CoV-2 and Other Respiratory Viruses.

The supply of testing equipment is vital in controlling the spread of SARS-CoV-2. We compared the diagnostic efficacy and tolerability of molded plastic (FinSwab; Valukumpu, Finland) versus flocked nylon (FLOQSwab; Copan, Italy) nasopharyngeal swabs in a clinical setting. Adults (n = 112) with suspected symptomatic COVID-19 infection underwent nasopharyngeal sampling with FinSwab and FLOQSwab from the same nostril at a drive-in coronavirus testing station. In a subset of 36 patients the samples were collected in a randomized order to evaluate the discomfort associated with sampling. SARS-CoV-2 and 16 other respiratory viruses, as well as human ß-actin mRNA were analyzed by using reverse transcriptase PCR (RT-PCR) assays. Among the 112 patients (mean age, 38 [standard deviation (SD), 14] years) ß-actin mRNA was found in all samples. There was no difference in the ß-actin mRNA cycle threshold (CT) values between FinSwab (mean, 22.3; SD, 3.61) and FLOQSwab (mean, 22.1; SD, 3.50; P = 0.46) swabs. There were 31 virus-positive cases (26 rhinovirus, 4 SARS-CoV-2, and 1 coronavirus-OC43), 24 of which were positive in both swabs; 3 rhinovirus positives were only found in the FinSwab, and similarly 4 rhinovirus positives were only found in the FLOQSwab. Rhinovirus CT values were similar between swab types. Of the 36 patients, 22 (61%) tolerated the sampling with the FinSwab better than with the FLOQSwab (P = 0.065). The molded plastic nasopharyngeal swab (FinSwab) was comparable to the standard flocked swab in terms of efficacy for respiratory virus detection and tolerability of sampling. IMPORTANCE We demonstrate that a molded plastic swab is a valid alternative to conventional brush-like swabs in collection of a nasopharyngeal sample for virus diagnostics.

Herpes simplex virus 1 induces egress channels through marginalized host chromatin.

Lytic infection with herpes simplex virus type 1 (HSV-1) induces profound modification of the cell nucleus including formation of a viral replication compartment and chromatin marginalization into the nuclear periphery. We used three-dimensional soft X-ray tomography, combined with cryogenic fluorescence, confocal and electron microscopy, to analyse the transformation of peripheral chromatin during HSV-1 infection. Our data showed an increased presence of low-density gaps in the marginalized chromatin at late infection. Advanced data analysis indicated the formation of virus-nucleocapsid-sized (or wider) channels extending through the compacted chromatin of the host. Importantly, confocal and electron microscopy analysis showed that these gaps frequently contained viral nucleocapsids. These results demonstrated that HSV-1 infection induces the formation of channels penetrating the compacted layer of cellular chromatin and allowing for the passage of progeny viruses to the nuclear envelope, their site of nuclear egress.

Cathepsins are involved in virus-induced cell death in ICP4 and Us3 deletion mutant herpes simplex virus type 1-infected monocytic cells.

We have studied cell death and its mechanisms in herpes simplex virus type 1 (HSV-1)-infected monocytic cells. The HSV-1 ICP4 and Us3 deletion mutant, d120 caused both apoptosis and necroptosis in d120-infected monocytic cells. At a late time point of infection the number of apoptotic cells was increased significantly in d120-infected cells when compared with uninfected or parental HSV-1 (KOS)-infected cells. Necroptosis inhibitor treatment increased the number of viable cells among the d120-infected cells, indicating that cell death in d120-infected cells was, in part, because of necroptosis. Moreover, lysosomal membrane permeabilization and cathepsin B and H activities were increased significantly in d120-infected cells. Inhibition of cathepsin B and S activities with specific cathepsin inhibitors led to increased cell viability, and inhibition of cathepsin L activity resulted in a decreased number of apoptotic cells. This indicates that cathepsins B, L and S may act as cell-death mediators in d120-infected monocytic cells. In addition, caspase 3 activity was increased significantly in d120-infected cells. However, the caspase 3 inhibitor treatment did not decrease the number of apoptotic cells. In contrast, inhibition of cathepsin L activity by cathepsin L-specific inhibitor clearly decreased caspase 3 activity and the number of apoptotic cells in d120-infected cells. This might suggest that, in d120-infected monocytic cells, cathepsin L activates caspase 3 and thus mediates d120-induced apoptosis. Taken together, these findings suggest that d120-induced cell death is both apoptotic and necroptotic.

Herpes simplex virus type 1 Us3 gene deletion influences toll-like receptor responses in cultured monocytic cells.

BACKGROUND: Toll-like receptors have a key role in innate immune response to microbial infection. The toll-like receptor (TLR) family consists of ten identified human TLRs, of which TLR2 and TLR9 have been shown to initiate innate responses to herpes simplex virus type 1 (HSV-1) and TLR3 has been shown to be involved in defence against severe HSV-1 infections of the central nervous system. However, no significant activation of the TLR3 pathways has been observed in wild type HSV-1 infections. In this work, we have studied the TLR responses and effects on TLR gene expression by HSV-1 with Us3 and ICP4 gene deletions, which also subject infected cells to apoptosis in human monocytic (U937) cell cultures. RESULTS: U937 human monocytic cells were infected with the Us3 and ICP4 deletion herpes simplex virus (d120), its parental virus HSV-1 (KOS), the Us3 deletion virus (R7041), its rescue virus (R7306) or wild type HSV-1 (F). The mRNA expression of TLR2, TLR3, TLR4, TLR9 and type I interferons (IFN) were analyzed by quantitative real-time PCR. The intracellular expression of TLR3 and type I IFN inducible myxovirus resistance protein A (MxA) protein as well as the level of apoptosis were analyzed by flow cytometry. We observed that the mRNA expression of TLR3 and type I IFNs were significantly increased in d120, R7041 and HSV-1 (F)-infected U937 cells. Moreover, the intracellular expression of TLR3 and MxA were significantly increased in d120 and R7041-infected cells. We observed activation of IRF-3 in infections with d120 and R7041. The TLR4 mRNA expression level was significantly decreased in d120 and R7041-infected cells but increased in HSV-1 (KOS)-infected cells in comparison with uninfected cells. No significant difference in TLR2 or TLR9 mRNA expression levels was seen. Both the R7041 and d120 viruses were able to induce apoptosis in U937 cell cultures. CONCLUSION: The levels of TLR3 and type I IFN mRNA were increased in d120, R7041 and HSV-1 (F)-infected cells when compared with uninfected cells. Also IRF-3 was activated in cells infected with the Us3 gene deletion viruses d120 and R7041. This is consistent with activation of TLR3 signaling in the cells. The intracellular TLR3 and type I IFN inducible MxA protein levels were increased in d120 and R7041-infected cells but not in cells infected with the corresponding parental or rescue viruses, suggesting that the HSV-1 Us3 gene is involved in control of TLR3 responses in U937 cells.

The cysteine protease inhibitors cystatins inhibit herpes simplex virus type 1-induced apoptosis and virus yield in HEp-2 cells.

The role of cystatins in herpes simplex virus (HSV)-induced apoptosis and viral replication has been studied. Human epithelial (HEp-2) cells infected with wild-type HSV-1 (F), with a deletion virus lacking the anti-apoptotic gene Us3 (R7041) or with a deletion virus lacking the anti-apoptotic genes Us3 and ICP4 (d120) were treated with cystatin A, C or D. Cells and culture media were studied at different time points for replicating HSV-1 and for apoptosis. Cystatins C and D inhibited the yield of replicative HSV-1 significantly in HEp-2 cells. In addition, cystatin D inhibited R7041 and d120 virus-induced apoptosis. Moreover, cystatin A inhibited R7041-induced apoptosis. These inhibitory effects of cystatins on virus replication and apoptosis are likely to be separate functions. Cystatin D treatment decreased cellular cathepsin B activity in HSV-1 infection, suggesting that cathepsin B is involved in virus-induced apoptosis.

Antisense RNA directed to the human papillomavirus type 16 E7 mRNA from herpes simplex virus type 1 derived vectors is expressed in CaSki cells and downregulates E7 mRNA.

BACKGROUND: Human papillomavirus (HPV) infection is known to be the most important etiologic factor of cervical cancer. There is no HPV specific therapy available for treatment of invasive squamous cell carcinoma of the cervix and its precursor lesions. The present study elucidates the potential to use herpes simplex virus (HSV) derived vectors for expression of antisense RNA to HPV -16 E7 oncogene. RESULTS: We have constructed replication competent, nonneuroinvasive HSV-1 vectors, deleted of the gamma134.5 gene. The vectors express RNA antisense to the first 100 nucleotides of the HPV-16 E7 gene. We assayed the ability of the antisense E7 vectors R5225 (tk-) and R5226 (tk+), to produce antisense RNA, as well as the consequent effects on E7 mRNA and protein levels in HPV-16 positive CaSki cells. Anti-E7 RNA was expressed by both constructs in a dose-dependent manner. Expression of HPV-16 E7 mRNA was downregulated effectively in CaSki cells infected with the tk- recombinant R5225 or with R5226. The tk+ recombinant R5226 was effective in downregulating E7 protein expression. CONCLUSION: We have shown that anti-E7 RNA expressed from an HSV vector could efficiently downregulate HPV-16 E7 mRNA and E7 protein expression in CaSki cells. We conclude that HSV vectors may become a useful tool for gene therapy of HPV infections.