Jennifer L. Welch

There is a critical need for an inactivation method that completely inactivates pathogens at the time of sample collection but maintains the nucleic acid quality required for diagnostic PCR testing. This inactivation method is needed to alleviate concerns about transmission potential, reduce shipping complications and cost, and allow testing in lower containment laboratories to improve disease diagnostics by improving turn-around time. This study evaluated a panel of ten surrogate viruses that represent highly pathogenic animal diseases. These results showed that a commercial (PrimeStore®) molecular transport media (PSMTM) completely inactivated all viruses tested by >99.99% as determined by infectivity and serial passage assays. However, detection of viral nucleic acid by qRT-PCR was comparable in PSMTM and control-treated conditions. These results were consistent when viruses were evaluated in the presence of biological material such as sera and cloacal swabs to mimic diagnostic sample conditions for non-avian and avian viruses, respectively. The results of this study may be utilized by diagnostic testing laboratories for highly pathogenic agents affecting animal and human populations. These results may be used to revise guidance for select agent diagnostic testing and shipment of infectious substances. Contribution to the field Active surveillance and confirmatory testing efforts are in place to protect animals in the United States from certain highly contagious diseases and to limit financial impacts to consumers and producers when the food supply is disrupted. Confirmatory testing typically utilizes nucleic acid detection to identify active infection. Testing is required to be completed in high containment facilities due to the elevated pathogenicity and impact potential of animal diseases. The requirement for testing in high containment facilities limits the ability for regional and state laboratories to test for Tier 1 select agents. Shipment of diagnostic samples is costly, as well as time and temperature sensitive to avoid deterioration of sample quality needed for testing. These constraints lengthen response time and testing turn-around time. Here, we showed that a commercial (PrimeStore®) molecular transport media (PSMTM) completely inactivated all viruses tested without affecting nucleic acid detection/integrity. These data suggest that highly contagious agents are effectively inactivated by PSMTM without compromising the nucleic acid needed for diagnostic testing. These data provide support that this inactivation method can be utilized during sample collection to reduce constraints in disease diagnostics and in reagent sharing among international laboratories.

Air pollution particulate matter (PM) is associated with SARS-CoV-2 infection and severity, although mechanistic studies are lacking. We tested whether airway surface liquid (ASL) from primary human airway epithelial cells is antiviral against SARS-CoV-2 and human alphacoronavirus 229E (CoV-229E) (responsible for common colds), and whether PM (urban, indoor air pollution [IAP], volcanic ash) affected ASL antiviral activity. ASL inactivated SARS-CoV-2 and CoV-229E. Independently, urban PM also decreased SARS-CoV-2 and CoV-229E infection, and IAP PM decreased CoV-229E infection. However, in combination, urban PM impaired ASL’s antiviral activity against both viruses, and the same effect occurred for IAP PM and ash against SARS-CoV-2, suggesting that PM may enhance SARS-CoV-2 infection.

Although extracellular vesicle (EV) surface electrostatic properties (measured as zeta potential, ζ-potential) have been reported by many investigators, the biophysical implications of charge and EV origin remains uncertain. Here, we compared the ζ-potential of human blood EVs (BEVs) and semen EVs (SEVs) from 26 donors that were HIV-infected (HIV+, n = 13) or HIV uninfected (HIV-, n = 13). We found that, compared to BEVs that bear neutral surface charge, SEVs were significantly more negatively charged, even when BEVs and SEVs were from the same individual. Comparison of BEVs and SEVs from HIV- and HIV+ groups revealed subtle HIV-induced alteration in the ζ-potential of EVs, with the effect being more significant in SEVs (∆ζ-potential = −8.82 mV, p-value = 0.0062) than BEVs (∆ζ-potential = −1.4 mV, p-value = 0.0462). These observations were validated by differences in the isoelectric point (IEP) of EVs, which was in the order of HIV + SEV ≤ HIV-SEV ≪ HIV + BEV ≤ HIV-BEV. Functionally, the rate and efficiency of SEV internalization by the human cervical epithelial cell line, primary peripheral blood lymphocytes, and primary blood-derived monocytes were significantly higher than those of BEVs. Mechanistically, removal of sialic acids from the surface of EVs using neuraminidase treatment significantly decreased SEV’s surface charge, concomitant with a substantial reduction in SEV’s internalization. The neuraminidase effect was independent of HIV infection and insignificant for BEVs. Finally, these results were corroborated by enrichment of glycoproteins in SEVs versus BEVs. Taken together, these findings uncover fundamental tissue-specific differences in surface electrostatic properties of EVs and highlight the critical role of surface charge in EV/target cell interactions.

Background: Personal protective equipment (PPE) is a critical need during the coronavirus disease 2019 (COVID-19) pandemic. Alternative sources of surgical masks, including 3-dimensionally (3D) printed approaches that may be reused, are urgently needed to prevent PPE shortages. Few data exist identifying decontamination strategies to inactivate viral pathogens and retain 3D-printing material integrity.Objective: To test viral disinfection methods on 3D-printing materials.Methods: The viricidal activity of common disinfectants (10% bleach, quaternary ammonium sanitizer, 3% hydrogen peroxide, or 70% isopropanol and exposure to heat (50°C, and 70°C) were tested on four 3D-printed materials used in the healthcare setting, including a surgical mask design developed by the Veterans’ Health Administration. Inactivation was assessed for several clinically relevant RNA and DNA pathogenic viruses, including severe acute respiratory coronavirus virus 2 (SARS-CoV-2) and human immunodeficiency virus 1 (HIV-1).Results: SARS-CoV-2 and all viruses tested were completely inactivated by a single application of bleach, ammonium quaternary compounds, or hydrogen peroxide. Similarly, exposure to dry heat (70°C) for 30 minutes completely inactivated all viruses tested. In contrast, 70% isopropanol reduced viral titers significantly less well following a single application. Inactivation did not interfere with material integrity of the 3D-printed materials.Conclusions: Several standard decontamination approaches effectively disinfected 3D-printed materials. These approaches were effective in the inactivation SARS-CoV-2, its surrogates, and other clinically relevant viral pathogens. The decontamination of 3D-printed surgical mask materials may be useful during crisis situations in which surgical mask supplies are limited.

Background: Program death receptor 1 (PD-1) is a co-inhibitory receptor that is upregulated and contributes to T cell dysfunction (exhaustion) during chronic viral infections, including HIV and HCV. GB virus C (GBV-C) is a persistent human virus, and co-infection is associated with reduced immune activation and improved clinical outcomes in HIV- and Ebola-infected individuals. Methods: PD-1 levels were measured by flow cytometry on CD38+ T cells from 45 HIV-infected individuals, 20 of whom were co-infected with GBV-C. Jurkat cell lines that stably express GBV-C E2 protein and vector control were used to purify total cellular RNA before, and 24 h following, activation using anti-CD3/CD28 treatment. Gene expression was analyzed by RNA-seq and qRT-PCR. Results: HIV-infected individuals with GBV-C viremia had reduced PD-1 expression on activated CD4+ and CD8+ T cells compared to HIV-infected GBV-C negative individuals. GBV-C particles and GBV-C E2 protein each inhibited PD-1 expression on T cells in vitro. Consistent with this, GBV-C E2 reduced gene expression of PD-1, and its ligand PD-L1, in both resting and activated T cells. GBV-C E2 regulated transcription of the PD-1 signaling pathway and T cell activation associated genes, without downregulation of the interferon-stimulated and innate immunity-related genes needed to resolve viral infections. Conclusions: Our current understanding of chronic RNA virus infections is that upregulation of PD-1 with T cell exhaustion is critical for viral persistence. However, these data demonstrate that GBV-C infection reduced PD-1 expression on activated T cells during HIV infection, and that the GBV-C E2 protein inhibits PD-1 signaling in T cells. This may preserve T cell function and contribute to the lack of immune deficiency in people with chronic GBV-C infection. Understanding the mechanisms by which GBV-C E2 alters PD-1 signaling may aid in the development of novel immunomodulatory therapeutics to prevent T cell dysfunction (exhaustion) during chronic viral infections.

Background: We previously identified a yellow fever virus (YFV) genome-derived, short noncoding RNA (vsRNA) generated during infection that targets the 3’ untranslated region (UTR) of a Src-kinase regulatory phosphatase (PTPRE), reducing PTPRE translation. By contrast, a related flavivirus (Zika) did not regulate PTPRE. We assessed the role of PTPRE in YFV and Zika replication. Methods: Human PTPRE with a β -Globulin 3’UTR to remove the 3’UTR sequence targeted by the YFV vsRNA was stably expressed in Jurkat and Huh7D cells using a tetracycline-regulated promoter. YFV (17D strain) and Zika (PR strain) were used to infect the different cell lines, and viral replication was measured by viral RNA and infectivity. Attempts to knock out PTPRE by CRISPR were unsuccessful, suggesting that PTPRE may be required for cell survival. Results: The expression of PTPRE with nontargeted 3′UTR led to a 100-fold reduction in infectious virus released from Jurkat and Huh7D cells by infectivity and a 3–10-fold reduction in viral RNA levels. Infectivity was restored in cells grown in doxycycline. By contrast, nontargeted PTPRE expression had no effect on the release of infectious Zika virus. PTPRE expression did not alter YFV attachment or entry. Despite reduced infectivity of the supernatant virus produced by PTPRE-nontargeted cells, YFV envelope content in culture supernatants was similar to that released by cells not expressing nontargeted PTPRE. Electron microscopy demonstrated large, empty-appearing viral particles in YFV-infected cells expressing nontargeted PTPRE that were not present in cells not expressing nontargeted PTPRE. Conclusion s : PTPRE is a novel YFV restriction factor that appears to increase the proportion of defective YFV particles released into cell culture media, potentially by preventing YFV RNA encapsidation. PTPRE’s effects on viral replication are not conserved among flaviviruses, as Zika replication was not altered by PTPRE overexpression. Understanding how PTPRE inhibits YFV may provide insight into YFV assembly and release and potentially help to identify novel drug targets.

A total of 340 million sexually transmitted infections (STIs) are acquired each year. Antimicrobial agents that target multiple infectious pathogens are ideal candidates to reduce the number of newly acquired STIs. The antimicrobial and immunoregulatory properties of GML make it an excellent candidate to fit this critical need. Previous studies established the safety profile and antibacterial activity of GML against both Gram-positive and Gram-negative bacteria. GML protected against high-dose SIV infection and reduced inflammation, which can exacerbate disease, during infection. We found that GML inhibits HIV-1 and other human-pathogenic viruses (yellow fever virus, mumps virus, and Zika virus), broadening its antimicrobial range. Because GML targets diverse infectious pathogens, GML may be an effective agent against the broad range of sexually transmitted pathogens. Further, our data show that reutericyclin, a GML analog expressed by some lactobacillus species, also inhibits HIV-1 replication and thus may contribute to the protective effect of Lactobacillus in HIV-1 transmission.

Background: Extracellular vesicles (EVs) are cell-derived vesicles with diverse functions in intercellular communication including disease and infection, and EVs seem to influence HIV-1 pathogenesis. EVs isolated from HIV-1–uninfected semen (SE), but not blood (BE), contain factors that interfere with HIV-1 infection and replication in target cells. The reason for this dichotomy is unknown. Furthermore, the effect of HIV-1 infection and antiretroviral (ARV) drugs on the anti–HIV-1 effects of SE and BE is unknown. Here, we characterize EVs and EV-free plasma isolated from HIV-infected donor semen and blood and their effects on HIV infection.Methods: EVs and EV-free plasma were purified from autologous blood and semen of HIV-negative, HIV-infected antiretroviral therapy (ART)-naïve, and HIV-infected ART-treated participants. HIV infection was assessed in a TZM-bl cell reporter system. ARV concentrations were analyzed using liquid chromatography-mass spectrometry.Results: SE isolated from both HIV-negative and HIV-infected, ART-naïve donors inhibited HIV-1 infection, but BE and semen and blood EV-free plasma did not. By contrast, BE, SE, and EV-free plasma from HIV-infected, ART-treated donors inhibited HIV-1. Importantly, exosomes isolated from ART-treated donors contained concentrations of ARV drugs (ART-EVs) at biologically relevant inhibitory levels.Conclusions: The HIV-1–inhibitory phenotype of SE is independent of donor HIV-1 or ART status, and ARV drugs and their metabolites are SE- and BE-associated in vivo.

Semen exosomes (SE) inhibit HIV infection. However, the effect of SE on cell activation and inflammation remains unknown. We characterized the response of peripheral blood mononuclear cells (PBMCs) from HIV‐uninfected and antiretroviral therapy‐suppressed HIV‐infected (HIV+) subjects to SE. Quiescent PBMCs or T‐cell receptor (TCR)‐activated PBMCs from HIV− and HIV+ donors were stimulated with SE in the presence/absence of ex vivo HIV infection. In HIV‐infected PBMCs, SE did not reactivate HIV, did not induce lymphoblast development, nor increase CD69+/CD25+ numbers. Furthermore, SE inhibited de novo HIV infection without altering cell activation. SE also asynchronously downregulated HIV‐inducible IL1β, IL8, and TNFα and upregulated CXCL10. These data suggest that SE inhibits HIV infection and production of HIV‐induced proinflammatory cytokines while preserving lymphocyte activation.