SARS-CoV-2 Rapidly Infects Peripheral Sensory and Autonomic Neurons, Contributing to Central Nervous System Neuroinvasion before Viremia.

Neurological symptoms associated with COVID-19, acute and long term, suggest SARS-CoV-2 affects both the peripheral and central nervous systems (PNS/CNS). Although studies have shown olfactory and hematogenous invasion into the CNS, coinciding with neuroinflammation, little attention has been paid to susceptibility of the PNS to infection or to its contribution to CNS invasion. Here we show that sensory and autonomic neurons in the PNS are susceptible to productive infection with SARS-CoV-2 and outline physiological and molecular mechanisms mediating neuroinvasion. Our infection of K18-hACE2 mice, wild-type mice, and golden Syrian hamsters, as well as primary peripheral sensory and autonomic neuronal cultures, show viral RNA, proteins, and infectious virus in PNS neurons, satellite glial cells, and functionally connected CNS tissues. Additionally, we demonstrate, in vitro, that neuropilin-1 facilitates SARS-CoV-2 neuronal entry. SARS-CoV-2 rapidly invades the PNS prior to viremia, establishes a productive infection in peripheral neurons, and results in sensory symptoms often reported by COVID-19 patients.

Herpes Simplex Virus 1 (HSV-1) Infected Cell Protein 0 (ICP0) Targets of Ubiquitination during Productive Infection of Primary Adult Sensory Neurons.

Herpes simplex virus 1 (HSV-1) enters sensory neurons with the potential for productive or latent infection. For either outcome, HSV-1 must curtail the intrinsic immune response, regulate viral gene expression, and remove host proteins that could restrict viral processes. Infected cell protein 0 (ICP0), a virus-encoded E3 ubiquitin ligase, supports these processes by mediating the transfer of ubiquitin to target proteins to change their location, alter their function, or induce their degradation. To identify ubiquitination targets of ICP0 during productive infection in sensory neurons, we immunoprecipitated ubiquitinated proteins from primary adult sensory neurons infected with HSV-1 KOS (wild-type), HSV-1 n212 (expressing truncated, defective ICP0), and uninfected controls using anti-ubiquitin antibody FK2 (recognizing K29, K48, K63 and monoubiquitinated proteins), followed by LC-MS/MS and comparative analyses. We identified 40 unique proteins ubiquitinated by ICP0 and 17 ubiquitinated by both ICP0 and host mechanisms, of which High Mobility Group Protein I/Y (HMG I/Y) and TAR DNA Binding Protein 43 (TDP43) were selected for further analysis. We show that ICP0 ubiquitinates HMG I/Y and TDP43, altering protein expression at specific time points during productive HSV-1 infection, demonstrating that ICP0 manipulates the sensory neuronal environment in a time-dependent manner to regulate infection outcome in neurons.

Amyloid-ß and p-Tau Anti-Threat Response to Herpes Simplex Virus 1 Infection in Primary Adult Murine Hippocampal Neurons.

Alzheimer's Disease (AD) is the sixth leading cause of death in the United States. Recent studies have established a potential link between herpes simplex virus 1 (HSV-1) infection and the development of AD. HSV-1 DNA has been detected in AD amyloid plaques in human brains, and treatment with the antiviral acyclovir (ACV) was reported to block the accumulation of the AD-associated proteins beta-amyloid (Aß) and hyper-phosphorylated tau (p-tau) in Vero and glioblastoma cells. Our goal was to determine whether the accumulation of AD-related proteins is attributable to acute and/or latent HSV-1 infection in mature hippocampal neurons, a region of the brain severely impacted by AD. Primary adult murine hippocampal neuronal cultures infected with HSV-1, with or without antivirals, were assessed for Aß and p-tau expression over 7 days postinfection. P-tau expression was transiently elevated in HSV-1-infected neurons, as well as in the presence of antivirals alone. Infected neurons, as well as uninfected neurons treated with antivirals, had a greater accumulation of Aß42 than uninfected untreated neurons. Furthermore, Aß42 colocalized with HSV-1 latency-associated transcript (LAT) expression. These studies suggest that p-tau potentially acts as an acute response to any perceived danger-associated molecular pattern (DAMP) in primary adult hippocampal neurons, while Aß aggregation is a long-term response to persistent threats, including HSV-1 infection.IMPORTANCE Growing evidence supports a link between HSV-1 infection and Alzheimer's disease (AD). Although AD is clearly a complex multifactorial disorder, an infectious disease etiology provides alternative therapy opportunities for this devastating disease. Understanding the impact that HSV-1 has on mature neurons and the proteins most strongly associated with AD pathology may identify specific mechanisms that could be manipulated to prevent progression of neurodegeneration and dementia.

Herpes Simplex Virus 2 in Autonomic Ganglia: Evidence for Spontaneous Reactivation.

Herpes simplex virus 2 (HSV-2) can be transmitted in the presence or absence of lesions, allowing efficient spread among the general population. Recurrent HSV genital lesions are thought to arise from reactivated latent virus in sensory cell bodies of the dorsal root ganglia (DRG). However, HSV-2 has also been found latent in autonomic ganglia. Spontaneous reactivation or a low level of chronic infection could theoretically also occur in these peripheral nervous tissues, contributing to the presence of infectious virus in the periphery and to viral transmission. Use of a recently described, optimized virus with a monomeric mNeonGreen protein fused to viral capsid protein 26 (VP26) permitted detection of reactivating virus in explanted ganglia and cryosections of DRG and the sacral sympathetic ganglia (SSG) from latently infected guinea pigs. Immediate early, early, and late gene expression were quantified by droplet digital reverse transcription-PCR (ddRT-PCR), providing further evidence of viral reactivation not only in the expected DRG but also in the sympathetic SSG. These findings indicate that viral reactivation from autonomic ganglia is a feature of latent viral infection and that these reactivations likely contribute to viral pathogenesis.IMPORTANCE HSV-2 is a ubiquitous important human pathogen that causes recurrent infections for the life of its host. We hypothesized that the autonomic ganglia have important roles in viral reactivation, and this study sought to determine whether this is correct in the clinically relevant guinea pig vaginal infection model. Our findings indicate that sympathetic ganglia are sources of reactivating virus, helping explain how the virus causes lifelong recurrent disease.

Passivated-electrode insulator-based dielectrophoretic separation of heterogeneous cell mixtures.

Rapid and accurate purification of various heterogeneous mixtures is a critical step for a multitude of molecular, chemical, and biological applications. Dielectrophoresis has shown to be a promising technique for particle separation due to its exploitation of the intrinsic electrical properties, simple fabrication, and low cost. Here, we present a geometrically novel dielectrophoretic channel design which utilizes an array of localized electric fields to separate a variety of unique particle mixtures into distinct populations. This label-free device incorporates multiple winding rows with several nonuniform structures on to sidewalls to produce high electric field gradients, enabling high locally generated dielectrophoretic forces. A balance between dielectrophoretic forces and Stokes' drag is used to effectively isolate each particle population. Mixtures of polystyrene beads (500 nm and 2 µm), breast cancer cells spiked in whole blood, and for the first time, neuron and satellite glial cells were used to study the separation capabilities of the design. We found that our device was able to rapidly separate unique particle populations with over 90% separation yields for each investigated mixture. The unique architecture of the device uses passivated-electrode insulator-based dielectrophoresis in an innovative microfluidic device to separate a variety of heterogeneous mixture without particle saturation in the channel.

Stress Hormones Epinephrine and Corticosterone Selectively Modulate Herpes Simplex Virus 1 (HSV-1) and HSV-2 Productive Infections in Adult Sympathetic, but Not Sensory, Neurons.

Herpes simplex viruses 1 and 2 (HSV-1 and HSV-2) infect and establish latency in peripheral neurons, from which they can reactivate to cause recurrent disease throughout the life of the host. Stress is associated with the exacerbation of clinical symptoms and the induction of recurrences in humans and animal models. The viruses preferentially replicate and establish latency in different subtypes of sensory neurons, as well as in neurons of the autonomic nervous system that are highly responsive to stress hormones. To determine if stress-related hormones modulate productive HSV-1 and HSV-2 infections within sensory and autonomic neurons, we analyzed viral DNA and the production of viral progeny after treatment of primary adult murine neuronal cultures with the stress hormones epinephrine and corticosterone. Both sensory trigeminal ganglion (TG) and sympathetic superior cervical ganglion (SCG) neurons expressed adrenergic receptors (activated by epinephrine) and the glucocorticoid receptor (activated by corticosterone). Productive HSV infection colocalized with these receptors in SCG but not in TG neurons. In productively infected neuronal cultures, epinephrine treatment significantly increased the levels of HSV-1 DNA replication and production of viral progeny in SCG neurons, but no significant differences were found in TG neurons. In contrast, corticosterone significantly decreased the levels of HSV-2 DNA replication and production of viral progeny in SCG neurons but not in TG neurons. Thus, the stress-related hormones epinephrine and corticosterone selectively modulate acute HSV-1 and HSV-2 infections in autonomic, but not sensory, neurons.IMPORTANCE Stress exacerbates acute disease symptoms resulting from HSV-1 and HSV-2 infections and is associated with the appearance of recurrent skin lesions in millions of people. Although stress hormones are thought to impact HSV-1 and HSV-2 through immune system suppression, sensory and autonomic neurons that become infected by HSV-1 and HSV-2 express stress hormone receptors and are responsive to hormone fluctuations. Our results show that autonomic neurons are more responsive to epinephrine and corticosterone than are sensory neurons, demonstrating that the autonomic nervous system plays a substantial role in HSV pathogenesis. Furthermore, these results suggest that stress responses have the potential to differentially impact HSV-1 and HSV-2 so as to produce divergent outcomes of infection.

Melanopsin expression in the cornea.

A unique class of intrinsically photosensitive retinal ganglion cells in mammalian retinae has been recently discovered and characterized. These neurons can generate visual signals in the absence of inputs from rods and cones, the conventional photoreceptors in the visual system. These light sensitive ganglion cells (mRGCs) express the non-rod, non-cone photopigment melanopsin and play well documented roles in modulating pupil responses to light, photoentrainment of circadian rhythms, mood, sleep and other adaptive light functions. While most research efforts in mammals have focused on mRGCs in retina, recent studies reveal that melanopsin is expressed in non-retinal tissues. For example, light-evoked melanopsin activation in extra retinal tissue regulates pupil constriction in the iris and vasodilation in the vasculature of the heart and tail. As another example of nonretinal melanopsin expression we report here the previously unrecognized localization of this photopigment in nerve fibers within the cornea. Surprisingly, we were unable to detect light responses in the melanopsin-expressing corneal fibers in spite of our histological evidence based on genetically driven markers and antibody staining. We tested further for melanopsin localization in cell bodies of the trigeminal ganglia (TG), the principal nuclei of the peripheral nervous system that project sensory fibers to the cornea, and found expression of melanopsin mRNA in a subset of TG neurons. However, neither electrophysiological recordings nor calcium imaging revealed any light responsiveness in the melanopsin positive TG neurons. Given that we found no light-evoked activation of melanopsin-expressing fibers in cornea or in cell bodies in the TG, we propose that melanopsin protein might serve other sensory functions in the cornea. One justification for this idea is that melanopsin expressed in Drosophila photoreceptors can serve as a temperature sensor.

Herpes Simplex Virus 1 Reactivates from Autonomic Ciliary Ganglia Independently from Sensory Trigeminal Ganglia To Cause Recurrent Ocular Disease.

Herpes simplex virus 1 (HSV-1) and HSV-2 establish latency in sensory and autonomic neurons after ocular or genital infection, but their recurrence patterns differ. HSV-1 reactivates from latency to cause recurrent orofacial disease, and while HSV-1 also causes genital lesions, HSV-2 recurs more efficiently in the genital region and rarely causes ocular disease. The mechanisms regulating these anatomical preferences are unclear. To determine whether differences in latent infection and reactivation in autonomic ganglia contribute to differences in HSV-1 and HSV-2 anatomical preferences for recurrent disease, we compared HSV-1 and HSV-2 clinical disease, acute and latent viral loads, and viral gene expression in sensory trigeminal and autonomic superior cervical and ciliary ganglia in a guinea pig ocular infection model. HSV-2 produced more severe acute disease, correlating with higher viral DNA loads in sensory and autonomic ganglia, as well as higher levels of thymidine kinase expression, a marker of productive infection, in autonomic ganglia. HSV-1 reactivated in ciliary ganglia, independently from trigeminal ganglia, to cause more frequent recurrent symptoms, while HSV-2 replicated simultaneously in autonomic and sensory ganglia to cause more persistent disease. While both HSV-1 and HSV-2 expressed the latency-associated transcript (LAT) in the trigeminal and superior cervical ganglia, only HSV-1 expressed LAT in ciliary ganglia, suggesting that HSV-2 is not reactivation competent or does not fully establish latency in ciliary ganglia. Thus, differences in replication and viral gene expression in autonomic ganglia may contribute to differences in HSV-1 and HSV-2 acute and recurrent clinical disease.

Risk factors and sources of foodborne hepatitis E virus infection in the United States.

Hepatitis E virus (HEV) is an important human pathogen with pigs and other species serving as natural animal reservoirs. Ample evidence documents sporadic cases of hepatitis E acquired via consumption of undercooked meat. Chronic hepatitis E cases in immunosuppressed individuals are mostly caused by zoonotic HEV of swine origin. We report here the identification of genotype 3 HEV from non-liver commercial pork from local grocery stores in southwest Virginia, and association of HEV seropositivity to the consumption of undercooked meat in healthy young adults at a university in the United States. These results raise concerns about foodborne HEV transmission in the United States. J. Med. Virol. 88:1641-1645, 2016. © 2016 Wiley Periodicals, Inc.

Different mechanisms regulate productive herpes simplex virus 1 (HSV-1) and HSV-2 infections in adult trigeminal neurons.

Herpes simplex virus 1 (HSV-1) and HSV-2 establish latency in different neuronal subtypes (A5+ and KH10+) in murine trigeminal ganglia, results which correlate with restricted productive infection in these neurons in vitro. HSV-2 latency-associated transcript (LAT) contains a cis-acting regulatory element near the transcription start site that promotes productive infection in A5+ neurons and a second element in exon 1 that inhibits productive infection in KH10+ neurons. HSV-1 contains no such regulatory sequences, demonstrating different mechanisms for regulating productive HSV infection in neurons.

Oral Sampling of Little Brown Bat (Myotis lucifugus) Maternity Colonies for SARS-CoV-2 in the Northeast and Mid-Atlantic, USA.

The potential introduction of SARS-CoV-2, the virus responsible for the COVID-19 pandemic, into North American bat populations is of interest to wildlife managers due to recent disease-mediated declines of several species. Populations of little brown bats (Myotis lucifugus) have collapsed due to white-nose syndrome (WNS), a disease caused by the introduction and spread of the fungal pathogen Pseudogymnoascus destructans (Pd). Throughout much of the United States and southern Canada, large colonies of the species routinely established diurnal roosts in anthropogenic structures, thereby creating the potential for direct human contact and cross-species disease transmission. Given recent declines and the potential for further disease impacts, we collected oral swabs from eight little brown bat maternity colonies to assess the presence and prevalence of SARS-CoV-2 by RT-qPCR analysis. Little brown bat colonies in Maryland (n = 1), New Hampshire (n = 1), New Jersey (n = 2), New York (n = 1), Rhode Island (n = 2), and Virginia (n = 1) were taken during May-August, 2022. From 235 assayed individuals, no bat tested positive for SARS-CoV-2. Our results indicate that little brown bats may not contract SARS-CoV-2 or that the virus persists at undetectable levels in populations of the Mid-Atlantic and Northeast during summer months. Nonetheless, continued monitoring and future work addressing other seasons may still be warranted to conclusively determine infection status.

Spread of herpes simplex virus to the spinal cord is independent of spread to dorsal root ganglia.

Levels of herpes simplex virus 1 (HSV-1) and HSV-2 DNA in dorsal root ganglia (DRG) and spinal cord (SC) were quantified after inoculation of guinea pig genitals and footpads. In genital infection, viral DNA reached SC and DRG simultaneously (at 2 to 3 days after inoculation) but was more abundant in SC than in DRG. After inoculation of footpads, which lack parasympathetic innervation, the viruses spread more efficiently to DRG than to SC. These results show important differences between genital and footpad infections, including independence of spread to DRG and SC, and imply that autonomic neurons may play an important role in the pathogenesis of viral latency after genital inoculation.

A5-positive primary sensory neurons are nonpermissive for productive infection with herpes simplex virus 1 in vitro.

Herpes simplex viruses 1 and 2 (HSV-1 and HSV-2) establish latency and express the latency-associated transcript (LAT) preferentially in different murine sensory neuron populations, with most HSV-1 LAT expression in A5(+) neurons and most HSV-2 LAT expression in KH10(+) neurons. To study the mechanisms regulating the establishment of HSV latency in specific subtypes of neurons, cultured dissociated adult murine trigeminal ganglion (TG) neurons were assessed for relative permissiveness for productive infection. In contrast to that for neonatal TG, the relative distribution of A5(+) and KH10(+) neurons in cultured adult TG was similar to that seen in vivo. Productive infection with HSV was restricted, and only 45% of cultured neurons could be productively infected with either HSV-1 or HSV-2. A5(+) neurons supported productive infection with HSV-2 but were selectively nonpermissive for productive infection with HSV-1, a phenomenon that was not due to restricted viral entry or DNA uncoating, since HSV-1 expressing ß-galactosidase under the control of the neurofilament promoter was detected in ∼90% of cultured neurons, with no preference for any neuronal subtype. Infection with HSV-1 reporter viruses expressing enhanced green fluorescent protein (EGFP) from immediate early (IE), early, and late gene promoters indicated that the block to productive infection occurred before IE gene expression. Trichostatin A treatment of quiescently infected neurons induced productive infection preferentially from non-A5(+) neurons, demonstrating that the nonpermissive neuronal subtype is also nonpermissive for reactivation. Thus, HSV-1 is capable of entering the majority of sensory neurons in vitro; productive infection occurs within a subset of these neurons; and this differential distribution of productive infection is determined at or before the expression of the viral IE genes.

Herpes simplex virus 2 microRNA miR-H6 is a novel latency-associated transcript-associated microRNA, but reduction of its expression does not influence the establishment of viral latency or the recurrence phenotype.

The herpes simplex virus 2 (HSV-2) viral microRNA (miRNA) designated miR-H6 is located upstream of the latency-associated transcript (LAT) promoter region on the strand opposite the LAT. Deletion of the LAT promoter and part of LAT exon 1 abolished HSV-2 miR-H6 expression in acutely and latently infected guinea pig dorsal root ganglia (DRG), suggesting that this region is needed both for the expression of LAT-encoded miRNAs and for miR-H6 expression in vivo. Relative to cells infected with a viral rescuant, miR-H6 expression was significantly reduced in cells infected with a mutant HSV-2 virus, NotPolyA, with an insertion of a simian virus (SV40) polyadenylation signal sequence between the LAT promoter and miR-H6 sequences. In addition, expression of miR-H6, but not LAT or viral DNA, was significantly reduced in both mouse trigeminal ganglia (TG) and guinea pig DRG latently infected with the NotPolyA mutant. Guinea pigs infected with NotPolyA experienced reduced neurological complications of acute infection relative to those infected with the rescuant, but the recurrence phenotype of the NotPolyA mutant was similar to those of its rescuant and wild-type HSV-2, indicating that reduction of miR-H6 expression is not by itself able to alter the establishment of latency for the wild-type virus or the recurrence phenotype. Furthermore, the mutation in NotPolyA did not affect the propensity of wild-type HSV-2 to establish latency in neurons positive for subtype marker KH10. In contrast to published reports regarding its HSV-1 homolog, HSV-2 miR-H6 did not affect ICP4 expression in transfected or infected cells. We hypothesize that viral miRNAs associated with LAT expression are likely to work collectively, contributing to the phenotype attributed to the LAT.

SARS-CoV-2 Survival in Common Non-Alcoholic and Alcoholic Beverages.

SARS-CoV-2, the causative agent of COVID-19, is known to be transmitted by respiratory droplets and aerosols. Since the virus is shed at high concentrations in respiratory secretions and saliva, SARS-CoV-2 would also be expected to be transmitted through activities that involve the transfer of saliva from one individual to another, such as kissing or sharing beverages. To assess the survival of infectious SARS-CoV-2 in common beverages, we quantified infectious virus by plaque assays one hour after inoculation into 18 non-alcoholic and 16 alcoholic beverages, plus saliva, and also 7 days later for 5 of these beverages. SARS-CoV-2 remains infectious with minimal reductions in several common beverages, including milk and beer. However, cocoa, coffee, tea, fruit juices, and wine contain antiviral compounds that inactivate SARS-CoV-2. Although hard liquors containing 40% alcohol immediately inactivate SARS-CoV-2, mixing with non-alcoholic beverages reduces the antiviral effects. In summary, SARS-CoV-2 can be recovered from commonly consumed beverages in a beverage type and time-dependent manner. Although aerosol or droplet transmission remains the most likely mode of transmission, our findings combined with others suggest that beverages contaminated with SARS-CoV-2 during handling, serving, or through sharing of drinks should be considered as a potential vehicle for virus transmission.

SARS-CoV-2 Remains Infectious on Refrigerated Deli Food, Meats, and Fresh Produce for up to 21 Days.

SARS-CoV-2, the virus that causes COVID-19, has been detected on foods and food packaging and the virus can infect oral cavity and intestinal cells, suggesting that infection could potentially occur following ingestion of virus-contaminated foods. To determine the relative risk of infection from different types of foods, we assessed survival of SARS-CoV-2 on refrigerated ready-to-eat deli items, fresh produce, and meats (including seafood). Deli items and meats with high protein, fat, and moisture maintained infectivity of SARS-CoV-2 for up to 21 days. However, processed meat, such as salami, and some fresh produce exhibited antiviral effects. SARS-CoV-2 also remained infectious in ground beef cooked rare or medium, but not well-done. Although infectious SARS-CoV-2 was inactivated on the foods over time, viral RNA was not degraded in similar trends, regardless of food type; thus, PCR-based assays for detection of pathogens on foods only indicate the presence of viral RNA, but do not correlate with presence or quantity of infectious virus. The survival and high recovery of SARS-CoV-2 on certain foods support the possibility that food contaminated with SARS-CoV-2 could potentially be a source of infection, highlighting the importance of proper food handling and cooking to inactivate any contaminating virus prior to consumption.

Stress Hormones Epinephrine and Corticosterone Selectively Reactivate HSV-1 and HSV-2 in Sympathetic and Sensory Neurons.

Herpes simplex viruses 1 and 2 (HSV-1 and HSV-2) establish latency in sensory and autonomic neurons, from which they can reactivate to cause recurrent disease throughout the life of the host. Stress is strongly associated with HSV recurrences in humans and animal models. However, the mechanisms through which stress hormones act on the latent virus to cause reactivation are unknown. We show that the stress hormones epinephrine (EPI) and corticosterone (CORT) induce HSV-1 reactivation selectively in sympathetic neurons, but not sensory or parasympathetic neurons. Activation of multiple adrenergic receptors is necessary for EPI-induced HSV-1 reactivation, while CORT requires the glucocorticoid receptor. In contrast, CORT, but not EPI, induces HSV-2 reactivation in both sensory and sympathetic neurons through either glucocorticoid or mineralocorticoid receptors. Reactivation is dependent on different transcription factors for EPI and CORT, and coincides with rapid changes in viral gene expression, although genes differ for HSV-1 and HSV-2, and temporal kinetics differ for EPI and CORT. Thus, stress-induced reactivation mechanisms are neuron-specific, stimulus-specific and virus-specific. These findings have implications for differences in HSV-1 and HSV-2 recurrent disease patterns and frequencies, as well as development of targeted, more effective antivirals that may act on different responses in different types of neurons.

An acutely and latently expressed herpes simplex virus 2 viral microRNA inhibits expression of ICP34.5, a viral neurovirulence factor.

Latency-associated transcript (LAT) sequences regulate herpes simplex virus (HSV) latency and reactivation from sensory neurons. We found a HSV-2 LAT-related microRNA (miRNA) designated miR-I in transfected and infected cells in vitro and in acutely and latently infected ganglia of guinea pigs in vivo. miR-I is also expressed in human sacral dorsal root ganglia latently infected with HSV-2. miR-I is expressed under the LAT promoter in vivo in infected sensory ganglia. We also predicted and identified a HSV-1 LAT exon-2 viral miRNA in a location similar to miR-I, implying a conserved mechanism in these closely related viruses. In transfected and infected cells, miR-I reduces expression of ICP34.5, a key viral neurovirulence factor. We hypothesize that miR-I may modulate the outcome of viral infection in the peripheral nervous system by functioning as a molecular switch for ICP34.5 expression.

Survival of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) and Herpes Simplex Virus 1 (HSV-1) on Foods Stored at Refrigerated Temperature.

Outbreaks of coronavirus infectious disease 2019 (COVID-19) in meat processing plants and media reports of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) detection on foods have raised concerns of a public health risk from contaminated foods. We used herpes simplex virus 1, a non-Biosafety Level 3 (non-BSL3) enveloped virus, as a surrogate to develop and validate methods before assessing the survival of infectious SARS-CoV-2 on foods. Several food types, including chicken, seafood, and produce, were held at 4 °C and assessed for infectious virus survival (herpes simplex virus 1 (HSV-1) and SARS-CoV-2) at 0 h, 1 h, and 24 h post-inoculation (hpi) by plaque assay. At all three time points, recovery of SARS-CoV-2 was similar from chicken, salmon, shrimp, and spinach, ranging from 3.4 to 4.3 log PFU/mL. However, initial (0 h) virus recovery from apples and mushrooms was significantly lower than that from poultry and seafood, and infectious virus decreased over time, with recovery from mushrooms becoming undetectable by 24 hpi. Comparing infectious virus titers with viral genome copies confirmed that PCR-based tests only indicate presence of viral nucleic acid, which does not necessarily correlate with the quantity of infectious virus. The survival and high recovery of SARS-CoV-2 on certain foods highlight the importance of safe food handling practices in mitigating any public health concerns related to potentially contaminated foods.

Assessment of Two Novel Live-Attenuated Vaccine Candidates for Herpes Simplex Virus 2 (HSV-2) in Guinea Pigs.

Treatment to ameliorate the symptoms of infection with herpes simplex virus 2 (HSV-2) and to suppress reactivation has been available for decades. However, a safe and effective preventative or therapeutic vaccine has eluded development. Two novel live-attenuated HSV-2 vaccine candidates (RVx201 and RVx202) have been tested preclinically for safety. Hartley guinea pigs were inoculated vaginally (n = 3) or intradermally (n = 16) with either vaccine candidate (2 × 107 PFU) and observed for disease for 28 days. All animals survived to study end without developing HSV-2-associated disease. Neither vaccine candidate established latency in dorsal root or sacral sympathetic ganglia, as determined by viral DNA quantification, LAT expression, or explant reactivation. Infectious virus was shed in vaginal secretions for three days following vaginal inoculation with RVx202, but not RVx201, although active or latent HSV-2 was not detected at study end. In contrast, guinea pigs inoculated with wild-type HSV-2 MS (2 × 105 PFU) vaginally (n = 5) or intradermally (n = 16) developed acute disease, neurological signs, shed virus in vaginal secretions, experienced periodic recurrences throughout the study period, and had latent HSV-2 in their dorsal root and sacral sympathetic ganglia at study end. Both vaccine candidates generated neutralizing antibody. Taken together, these findings suggest that these novel vaccine candidates are safe in guinea pigs and should be tested for efficacy as preventative and/or therapeutic anti-HSV-2 vaccines.