Alyson Ann Kelvin

Mpox (formerly monkeypox) is caused by monkeypox virus (MPXV) and has prompted two recent global health emergencies. Clade IIb MPXV, a recently recognized subclade, has been associated with oral and genital lesions and transmission among men who have sex with men (MSM); however, mechanisms of genital pathogenesis and sexual transmission are not understood. We investigated several routes of MPXV Clade IIb virus infection (intranasal, oral, anal, and intraperitoneal) and found high and prolonged viral titres in the testes after IP inoculation still detectable at 21 days. The testes had significant changes to tissue architecture including loss of spermatogenesis, disorganization of spermatozoa, loss of Leydig cells, and breakdown of the seminiferous tubule membranes. Viral antigen positive cells were present in the interstitial spaces between the seminiferous tubules with macrophage infiltration also evident. This work provides insights into potential of sexual transmission for MPXV viruses as well as mechanisms of Mpox disease which may significantly impact long-term fertility or sex organ health of infected males. Author Summary: Mpox (formerly monkeypox) is a painful disease similar to smallpox caused by the monkeypox virus (MPXV) which is an emerging/re-emerging virus. Recently the WHO has declared two global health emergencies due to human-to-human transmission of MPXV. Clade IIb MPXV is a newly recognized subclade that has been associated with genital lesions and sexual transmission/contact; however, how the virus causes disease in sex-organs or how it is passed from person-to-person is not understood. We investigated several routes of MPXV Clade IIb inoculation and found IP inoculation in male mice led to high and prolonged viral titres in testes. Additionally, the testes were significantly affected having loss of sperm and cellular organization. Evidence of inflammatory cell infiltration, specifically, macrophages, are suspected to be the cause of testes specific disease. This work provides insights into potential of sexual transmission for MPXV viruses as well as mechanisms of Mpox disease.

The mammary gland is an essential organ for milk production, providing essential immune and nutritional support to offspring and supplying dairy products for human consumption. In both humans and animals, the lactating mammary gland is susceptible to bacterial and viral infections, which can lead to mastitis and, in some cases, vertical transmission to offspring, with potential adverse effects on infant health. However, until recently, the role of respiratory viruses in mammary gland infection has been relatively understudied, particularly their ability to infect mammary epithelial cells and transmit through lactation. The recent emergence of highly pathogenic avian influenza H5N1 clade 2.3.4.4b in dairy cattle has demonstrated the virus’s capacity to replicate in the mammary gland, cause mastitis, and produce high viral loads in milk. This raises significant concerns about the potential for zoonotic transmission to humans and other animals in contact with infected dairy cows and unpasteurized milk. In this mini-review, we highlight key studies that demonstrate the replication of influenza and other viruses in the mammary gland, summarize recent findings from experimental and natural H5N1 clade 2.3.4.4b infections in dairy cows and small animal models, and discuss the broader One Health implications of the current H5N1 outbreak. We emphasize the urgent need for an interdisciplinary collaboration across sectors to mitigate the risks posed by influenza viruses with pandemic potential.

Despite all the progress in treating SARS-CoV-2, escape mutants to current therapies remain a constant concern. Promising alternative treatments for current and future coronaviruses are those that limit escape mutants by inhibiting multiple pathogenic targets, analogous to the current strategies for treating HCV and HIV. With increasing popularity and ease of manufacturing of RNA technologies for vaccines and drugs, therapeutic microRNAs represent a promising option. In the present work, miR-24-3p was identified to inhibit SARS-CoV-2 entry, replication, and production; furthermore, this inhibition was retained against common mutations improving SARS-CoV-2 fitness. To determine the mechanism of action, bioinformatic tools were employed, identifying numerous potential effectors promoting infection targeted by miR-24-3p. Of these targets, several key host proteins for priming and facilitating SARS-CoV-2 entry were identified: furin, NRP1, NRP2, and SREBP2. With further experimental analysis, we show that miR-24-3p directly downregulates these viral entry factors to impede infection when producing virions and when infecting the target cell. Furthermore, we compare the findings with coronavirus, HCoV-229E, which relies on different factors strengthening the miR-24-3p mechanism. Taken together, the following work suggests that miR-24-3p could be an avenue to treat current coronaviruses and those likely to emerge.

The spread of H5N1 clade 2.3.4.4b in dairy herds raises concerns about zoonotic transmission due to its high viral load in milk, a key contact point between livestock and humans. H5N1 clade 2.3.4.4b exhibits tropism for the mammary gland, with milk from infected animals containing high levels of infectious virus, posing potential risks to offspring via breastfeeding. Using a lactating ferret model, we demonstrate that mammary gland infection with bovine H5N1 transmits the virus to suckling kits, resulting in neonatal mortality. Viral RNA levels increased in milk and remained high in mammary tissue, with infected kits exhibiting elevated viral RNA in the oral and nasal cavities and feces. Additionally, we detected the H5N1 receptor, α2,3 sialic acid, in ferret and human mammary tissue. These data demonstrate that H5N1 clade 2.3.4.4b infection in lactating dams leads to mastitis-related disease and transmits to suckling pups, resulting in mortality among neonates.

Post-acute sequelae of COVID-19 (PASC) or the continuation of COVID-19 (Coronavirus disease 2019) symptoms past 12 weeks may affect as many as 30% of people recovering from a SARS-CoV-2 (severe acute respiratory coronavirus 2) infection. The mechanisms regulating the development of PASC are currently not known; however, hypotheses include virus reservoirs, pre-existing conditions, microblood clots, immune dysregulation, as well as poor antibody responses. Importantly, virus neutralizing antibodies are essential for COVID-19 recovery and protection from reinfection but there is currently limited information on these immune regulators and associated cytokines in PASC patients. Understanding the key drivers of general and specific symptoms associated with Long COVID and the presence of virus neutralizing antibodies in PASC will aid in the development of therapeutics, diagnostics, and vaccines which currently do not exist. We designed a cross-sectional study to investigate systemic antibody and cytokine responses during COVID-19 recovery and PASC. In total, 195 participants were recruited in one of four groups: (1) Those who never had COVID-19 (No COVID); (2) Those in acute COVID-19 recovery (Acute Recovery) (4–12 weeks post infection); (3) Those who recovered from COVID-19 (Recovered) (+ 12 weeks from infection); and (4) those who had PASC (PASC) (+ 12 weeks from infection). Participants completed a questionnaire on health history, sex, gender, demographics, experiences with COVID-19 acute and COVID-19 recovery/continuing symptoms. Serum samples collected were evaluated for antibody binding to viral proteins, virus neutralizing antibody titers, and serum cytokine levels using Ella SimplePlex Immunoassay™ panels. We found participants with PASC reported more pre-existing conditions (e.g. such as hypertension, asthma, and obesity), and PASC symptoms (e.g. fatigue, brain fog, headaches, and shortness of breath) following COVID-19 than COVID-19 Recovered individuals. Importantly, we found PASC individuals to have significantly decreased levels of neutralizing antibodies toward both SARS-CoV-2 and the Omicron BA.1 variant. Sex analysis indicated that female PASC study participants had sustained antibody levels as well as levels of the inflammatory cytokines GM-CSF and ANG-2 over time following COVID-19. Our study reports people experiencing PASC had lower levels of virus neutralizing antibodies; however, the results are limited by the collection time post-COVID-19 and post-vaccination. Moreover, we found females experiencing PASC had sustained levels of GM-CSF and ANG-2. With lower levels of virus neutralizing antibodies, this data suggests that PASC individuals not only have had a suboptimal antibody response during acute SARS-CoV-2 infection but may also have increased susceptibility to subsequent infections which may exacerbate or prolong current PASC illnesses. We also provide evidence suggesting GM-CSF and ANG-2 to play a role in the sex-bias of PASC. Taken together, our findings maybe important for understanding immune molecular drivers of PASC and PASC subgroups.

Aleutian disease (AD), a severe immune-complex disease, brings tremendous financial losses to the mink industry. Resilience is the ability of an animal to minimize the influences caused by disturbances and maintain its performance under pathogen exposure. Many AD-positive farms have selected AD-resilient mink based on AD tests and/or AD-resilience indicator traits, such as growth, pelt quality, and reproduction. Selection based on these indicator traits may have changed the patterns of genetic variation and potentially represent a genomic signature that can be used to identify genes subjected to selection. Therefore, the main objective of this study was to identify the selection signatures related to immune response (IRE) and resilience to AD. . A total of 1,411 mink in five color types (black, demi, mahogany, pastel, and stardust) from the Canadian Centre for Fur Animal Research (Truro, Nova Scotia, Canada), which is an AD-positive facility, were genotyped using the Axiom Affymetrix Mink 70K single nucleotide polymorphism (SNP) panel. For the IRE trait, 264 mink were grouped into pairwise groups based on the combined results of counter-immunoelectrophoresis (CIEP) and enzyme-linked immunosorbent assay (ELISA) tests. Individuals in black (n = 29), demi (n = 131), mahogany (n = 76), and pastel (36) color types were also grouped based on combined results of CIEP and ELISA tests, respectively. For resilience traits, two methods were used to group CIEP-positive individuals into pairwise groups: 1) based on their general resilience performance (GRP, n = 30), which was measured by considering individual performance for feed conversion ratio, Kleiber ratio, and pelt quality, and 2) based on female reproductive performance (FRP, CIEP-positive dams only, n = 36), which was measured by the number of kits alive 24h after birth. The pairwise fixation index, nucleotide diversity, and cross-population extended haplotype homozygosity were used to detect selection signatures. Only SNPs detected by at least two methods were considered selection signatures candidates, including 619, 569, and 526 SNPs for IRE, GRP, and FRP traits, respectively. The genes annotated from the candidate SNPs were associated with previously reported traits influenced by AD, including immune system process, growth, reproduction, and pigmentation. Notably, two olfactory-related gene ontology (GO) terms were significant (q < 0.05) for all studied traits, suggesting AD might cause loss of smell in infected mink. Additionally, the differences in genes and GO terms detected across different color types for IRE indicated that mink of different color types may differ in immune response to AD. The mitogen-activated protein kinase (MAPK) signalling pathway was significant (q < 0.05) in Kyoto Encyclopedia of Genes and Genomes pathway analyses for FRP, indicating AD infection may disorder the MAPK signalling pathway, in turn influencing female reproductive performance. Our study has enhanced the understanding of genomic architecture underlying resilience of mink to AD and sheds light on the underlying biological mechanisms involved.

Post-acute sequelae of COVID-19 (PASC) or the continuation of COVID-19 (Coronavirus disease 2019) symptoms past 12 weeks may affect as many as 30% of people recovering from a SARS-CoV-2 (severe acute respiratory coronavirus 2) infection. The mechanisms regulating the development of PASC are currently not known; however, hypotheses including poor antibody responses have been suggested. Due to the importance of virus neutralizing antibodies during COVID-19 recovery and protection from reinfection, we designed a cross-sectional study to investigate systemic antibody and cytokine responses during COVID-19 recovery and PASC. In total, 195 participants were recruited in one of five groups: 1.) those who had PASC (PASC); 2.) those who recovered from COVID-19 (Recovered); 3.) those in acute recovery (Acute Recovery); 4.) those experiencing acute COVID-19 (Acute COVID-19); and 5.) those who never had COVID-19 (No COVID). Participants completed a questionnaire detailing their demographics, as well as COVID-19 experiences. Serum samples were evaluated for virus binding and neutralizing antibodies as well as serum cytokine levels. We found that participants with PASC reported more pre-existing conditions (such as hypertension), and PASC symptoms (ie., shortness of breath) following COVID-19 than Recovered individuals. PASC individuals also had significantly decreased levels of neutralizing antibodies toward both SARS-CoV-2 and the Omicron BA.1 variant. Sex analysis indicated that female PASC study participants had sustained antibody levels as well as inflammatory cytokines (GM-CSF) over time following COVID-19 while males had decreasing concentrations. Our study reports for the first time that people experiencing PASC have lower levels of virus neutralizing antibodies and females experiencing PASC have sustained levels of antibodies and inflammatory markers. With lower levels of virus neutralizing antibodies, this data suggests that PASC individuals not only have had a suboptimal antibody response during acute SARS-CoV-2 infection but may also have increased susceptibility to subsequent infections which may exacerbate or prolong current PASC illnesses. The work may be applied directly to developing PASC diagnostic screening tools, treatments, as well as public health policies.

SARS-CoV-2 variants and seasonal coronaviruses continue to cause disease and coronaviruses in the animal reservoir pose a constant spillover threat. Importantly, understanding of how previous infection may influence future exposures, especially in the context of seasonal coronaviruses and SARS-CoV-2 variants, is still limited. Here we adopted a step-wise experimental approach to examine the primary immune response and subsequent immune recall toward antigenically distinct coronaviruses using male Syrian hamsters. Hamsters were initially inoculated with seasonal coronaviruses (HCoV-NL63, HCoV-229E, or HCoV-OC43), or SARS-CoV-2 pango B lineage virus, then challenged with SARS-CoV-2 pango B lineage virus, or SARS-CoV-2 variants Beta or Omicron. Although infection with seasonal coronaviruses offered little protection against SARS-CoV-2 challenge, HCoV-NL63-infected animals had an increase of the previously elicited HCoV-NL63-specific neutralizing antibodies during challenge with SARS-CoV-2. On the other hand, primary infection with HCoV-OC43 induced distinct T cell gene signatures. Gene expression profiling indicated interferon responses and germinal center reactions to be induced during more similar primary infection-challenge combinations while signatures of increased inflammation as well as suppression of the antiviral response were observed following antigenically distant viral challenges. This work characterizes and analyzes seasonal coronaviruses effect on SARS-CoV-2 secondary infection and the findings are important for pan-coronavirus vaccine design.

With the emergence of the novel betacoronavirus Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), there has been an urgent need for the development of fast-acting antivirals, particularly in dealing with different variants of concern (VOC). SARS-CoV-2, like other RNA viruses, depends on host cell machinery to propagate and misregulate metabolic pathways to its advantage. Herein, we discovered that the immunometabolic microRNA-185 (miR-185) restricts SARS-CoV-2 propagation by affecting its entry and infectivity. The antiviral effects of miR-185 were studied in SARS-CoV-2 Spike protein pseudotyped virus, surrogate virus (HCoV-229E), as well as live SARS-CoV-2 virus in Huh7, A549, and Calu-3 cells. In each model, we consistently observed microRNA-induced reduction in lipid metabolism pathways-associated genes including SREBP2, SQLE, PPARG, AGPAT3, and SCARB1. Interestingly, we also observed changes in angiotensin-converting enzyme 2 (ACE2) levels, the entry receptor for SARS-CoV-2. Taken together, these data show that miR-185 significantly restricts host metabolic and other pathways that appear to be essential to SAR-CoV-2 replication and propagation. Overall, this study highlights an important link between non-coding RNAs, immunometabolic pathways, and viral infection. miR-185 mimics alone or in combination with other antiviral therapeutics represent possible future fast-acting antiviral strategies that are likely to be broadly antiviral against multiple variants as well as different virus types of potential pandemics.