Integrating Veterinary Diagnostic Laboratories for Emergency Use Testing during Pandemics1.

The SARS-CoV-2 pandemic showed limitations in human outbreak testing. Veterinary diagnostic laboratories (VDLs) possess capabilities to bolster emergency test capacity. Surveys from 26 participating VDLs found human SARS-CoV-2 testing was mutually beneficial, including One Health benefits. VDLs indicated testing >3.8 million human samples during the pandemic, which included some challenges.

A self-amplifying mRNA SARS-CoV-2 vaccine candidate induces safe and robust protective immunity in preclinical models.

RNA vaccines have demonstrated efficacy against SARS-CoV-2 in humans, and the technology is being leveraged for rapid emergency response. In this report, we assessed immunogenicity and, for the first time, toxicity, biodistribution, and protective efficacy in preclinical models of a two-dose self-amplifying messenger RNA (SAM) vaccine, encoding a prefusion-stabilized spike antigen of SARS-CoV-2 Wuhan-Hu-1 strain and delivered by lipid nanoparticles (LNPs). In mice, one immunization with the SAM vaccine elicited a robust spike-specific antibody response, which was further boosted by a second immunization, and effectively neutralized the matched SARS-CoV-2 Wuhan strain as well as B.1.1.7 (Alpha), B.1.351 (Beta) and B.1.617.2 (Delta) variants. High frequencies of spike-specific germinal center B, Th0/Th1 CD4, and CD8 T cell responses were observed in mice. Local tolerance, potential systemic toxicity, and biodistribution of the vaccine were characterized in rats. In hamsters, the vaccine candidate was well-tolerated, markedly reduced viral load in the upper and lower airways, and protected animals against disease in a dose-dependent manner, with no evidence of disease enhancement following SARS-CoV-2 challenge. Therefore, the SARS-CoV-2 SAM (LNP) vaccine candidate has a favorable safety profile, elicits robust protective immune responses against multiple SARS-CoV-2 variants, and has been advanced to phase 1 clinical evaluation (NCT04758962).

Experimental infection of domestic dogs and cats with SARS-CoV-2: Pathogenesis, transmission, and response to reexposure in cats.

The pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has reached nearly every country in the world with extraordinary person-to-person transmission. The most likely original source of the virus was spillover from an animal reservoir and subsequent adaptation to humans sometime during the winter of 2019 in Wuhan Province, China. Because of its genetic similarity to SARS-CoV-1, it is probable that this novel virus has a similar host range and receptor specificity. Due to concern for human-pet transmission, we investigated the susceptibility of domestic cats and dogs to infection and potential for infected cats to transmit to naive cats. We report that cats are highly susceptible to infection, with a prolonged period of oral and nasal viral shedding that is not accompanied by clinical signs, and are capable of direct contact transmission to other cats. These studies confirm that cats are susceptible to productive SARS-CoV-2 infection, but are unlikely to develop clinical disease. Further, we document that cats developed a robust neutralizing antibody response that prevented reinfection following a second viral challenge. Conversely, we found that dogs do not shed virus following infection but do seroconvert and mount an antiviral neutralizing antibody response. There is currently no evidence that cats or dogs play a significant role in human infection; however, reverse zoonosis is possible if infected owners expose their domestic pets to the virus during acute infection. Resistance to reinfection holds promise that a vaccine strategy may protect cats and, by extension, humans.

Potential Use for Serosurveillance of Feral Swine to Map Risk for Anthrax Exposure, Texas, USA.

Anthrax is a disease of concern in many mammals, including humans. Management primarily consists of prevention through vaccination and tracking clinical-level observations because environmental isolation is laborious and bacterial distribution across large geographic areas difficult to confirm. Feral swine (Sus scrofa) are an invasive species with an extensive range in the southern United States that rarely succumbs to anthrax. We present evidence that feral swine might serve as biosentinels based on comparative seroprevalence in swine from historically defined anthrax-endemic and non-anthrax-endemic regions of Texas. Overall seropositivity was 43.7% (n = 478), and logistic regression revealed county endemicity status, age-class, sex, latitude, and longitude were informative for predicting antibody status. However, of these covariates, only latitude was statistically significant (ß = -0.153, p = 0.047). These results suggests anthrax exposure in swine, when paired with continuous location data, could serve as a proxy for bacterial presence in specific areas.

Inactivation of severe acute respiratory syndrome coronavirus 2 in plasma and platelet products using a riboflavin and ultraviolet light-based photochemical treatment.

BACKGROUND AND OBJECTIVE: Severe acute respiratory distress syndrome coronavirus-2 (SARS-CoV-2), the causative agent of coronavirus disease 2019 (COVID-19), is a member of the coronavirus family. Coronavirus infections in humans are typically associated with respiratory illnesses; however, viral RNA has been isolated in serum from infected patients. Coronaviruses have been identified as a potential low-risk threat to blood safety. The Mirasol Pathogen Reduction Technology (PRT) System utilizes riboflavin and ultraviolet (UV) light to render blood-borne pathogens noninfectious, while maintaining blood product quality. Here, we report on the efficacy of riboflavin and UV light against the pandemic virus SARS-CoV-2 when tested in both plasma and platelets units. MATERIALS AND METHODS: Stock SARS-CoV-2 was grown in Vero cells and inoculated into either plasma or platelet units. Those units were then treated with riboflavin and UV light. The infectious titres of SARS-CoV-2 were determined by plaque assay using Vero cells. A total of five (n = 5) plasma and three (n = 3) platelet products were evaluated in this study. RESULTS: In both experiments, the measured titre of SARS-CoV-2 was below the limit of detection following treatment with riboflavin and UV light. The mean log reductions in the viral titres were ≥3·40 and ≥4·53 for the plasma units and platelet units, respectively. CONCLUSION: Riboflavin and UV light effectively reduced the titre of SARS-CoV-2 in both plasma and platelet products to below the limit of detection in tissue culture. The data suggest that the process would be effective in reducing the theoretical risk of transfusion transmitted SARS-CoV-2.

UV-C Light Intervention as a Barrier against Airborne Transmission of SARS-CoV-2.

BACKGROUND: SARS-CoV-2 continues to impact human health globally, with airborne transmission being a significant mode of transmission. In addition to tools like vaccination and testing, countermeasures that reduce viral spread in indoor settings are critical. This study aims to assess the efficacy of UV-C light, utilizing the Violett sterilization device, as a countermeasure against airborne transmission of SARS-CoV-2 in the highly susceptible Golden Syrian hamster model. METHODS: Two cohorts of naïve hamsters were subjected to airborne transmission from experimentally infected hamsters; one cohort was exposed to air treated with UV-C sterilization, while the other cohort was exposed to untreated air. RESULTS: Treatment of air with UV-C light prevented the airborne transmission of SARS-CoV-2 from the experimentally exposed hamster to naïve hamsters. Notably, this protection was sustained over a multi-day exposure period during peak viral shedding by hamsters. CONCLUSIONS: These findings demonstrate the efficacy of the UV-C light to mitigate against airborne SARS-CoV-2 transmission. As variants continue to emerge, UV-C light holds promise as a tool for reducing infections in diverse indoor settings, ranging from healthcare facilities to households. This study reinforces the urgency of implementing innovative methods to reduce airborne disease transmission and safeguard public health against emerging biological threats.

An omicron-specific neutralizing antibody test predicts neutralizing activity against XBB 1.5.

Introduction: Understanding the immune status of an individual using neutralizing antibody testing is complicated by the continued evolution of the SARS-CoV-2 virus. Previous work showed that assays developed against the wildtype strain of SARS-CoV-2 were insufficient predictors of neutralization of omicron variants, thus we developed an omicron-specific flow cytometry-based neutralizing antibody test and performed experiments to assess how well it compared to an omicron-specific PRNT assay (gold standard) and whether it could predict neutralizing activity to more recent omicron subvariants such as XBB.1.5. Methods: Accuracy of a novel flow cytometry-based neutralizing antibody (FC-NAb) assay was determined by comparison with an omicron-specific PRNT assay. A series of samples were evaluated in both the omicron FC-NAb assay and a second test was designed to assess neutralization of XBB.1.5. Results: Good concordance between the omicron FC-NAb test and the omicron PRNT was demonstrated (AUC = 0.97, p <0.001; sensitivity = 94%, specificity = 100%, PPV = 100%, and NPV = 97%). A strong linear relationship between the omicron FC-NAb and neutralization of XBB1.5 was observed (r = 0.83, p<0.001). Additionally, the omicron FC-NAb test was a very strong predictor of positive XBB1.5 NAb activity (AUC = 0.96, p<0.001; sensitivity = 94%, specificity = 90%, positive predictive value = 90%, and negative predictive values = 94%). Discussion: Our data suggest that despite continued evolution of the SARS-CoV-2 spike protein, the omicron FC-NAb assay described here is a good predictor of XBB1.5 neutralizing activity, as evidenced by a strong correlation and good predictive performance characteristics.

SARS-CoV-2 surveillance in a veterinary health system provides insight into transmission risks.

OBJECTIVE: To investigate the prevalence and seropositivity of SARS-CoV-2 in companion and exotic animals in a veterinary healthcare system. SAMPLE: A total of 341 animals were sampled by a combination of oral and nasal swabs. Serum from whole blood was collected from a subset of animals (86 canines, 25 felines, and 6 exotic animals). METHODS: After informed owner consent, convenience samples from client-owned animals and the pets of students and staff members associated with Colorado State University's Veterinary Health System were collected between May 2021 and September 2022. Study samples were collected by trained veterinarians, Veterinary Health System staff, and veterinary students. RESULTS: SARS-CoV-2 RNA was detected by reverse transcription PCR in 1.6% (95% CI, 0.5% to 4.6%) of domestic canines and 1.1% (95% CI, 0.2% to 6.1%) of domestic felines. No RNA was detected in any of the exotic animal species tested (n = 66). Plaque reduction neutralization tests indicated that 12.8% (95% CI, 7.3% to 21.5%) of canines and 12.0% (95% CI, 4.2% to 30.0%) of felines had neutralizing antibodies against SARS-CoV-2. CLINICAL RELEVANCE: This study provides insight regarding SARS-CoV-2 spillover in domestic companion and exotic animals and contributes to our understanding of transmission risk in the veterinary setting.

Emerging Pathogen Threats in Transfusion Medicine: Improving Safety and Confidence with Pathogen Reduction Technologies.

Emerging infectious disease threats are becoming more frequent due to various social, political, and geographical pressures, including increased human-animal contact, global trade, transportation, and changing climate conditions. Since blood products for transfusion are derived from donated blood from the general population, emerging agents spread by blood contact or the transfusion of blood products are also a potential risk. Blood transfusions are essential in treating patients with anemia, blood loss, and other medical conditions. However, these lifesaving procedures can contribute to infectious disease transmission, particularly to vulnerable populations. New methods have been implemented on a global basis for the prevention of transfusion transmissions via plasma, platelets, and whole blood products. Implementing proactive pathogen reduction methods may reduce the likelihood of disease transmission via blood transfusions, even for newly emerging agents whose transmissibility and susceptibility are still being evaluated as they emerge. In this review, we consider the Mirasol PRT system for blood safety, which is based on a photochemical method involving riboflavin and UV light. We provide examples of how emerging threats, such as Ebola, SARS-CoV-2, hepatitis E, mpox and other agents, have been evaluated in real time regarding effectiveness of this method in reducing the likelihood of disease transmission via transfusions.

Pathogen reduction of monkeypox virus in plasma and whole blood using riboflavin and UV light.

BACKGROUND: Monkeypox virus has recently emerged from endemic foci in Africa and, since October 20, 2022, more than 73,000 human infections have been reported by the CDC from over 100 countries that historically have not reported monkeypox cases. The detection of virus in skin lesions, blood, semen, and saliva of infected patients with monkeypox infections raises the potential for disease transmission via routes that have not been previously documented, including by blood and plasma transfusions. Methods for protecting the blood supply against the threats of newly emerging disease agents exist and include Pathogen Reduction Technologies (PRT) which utilize photochemical treatment processes to inactivate pathogens in blood while preserving the integrity of plasma and cellular components. Such methods have been employed broadly for over 15 years, but effectiveness of these methods under routine use conditions against monkeypox virus has not been reported. STUDY DESIGN AND METHODS: Monkeypox virus (strain USA_2003) was used to inoculate plasma and whole blood units that were then treated with riboflavin and UV light (Mirasol Pathogen Reduction Technology System, Terumo BCT, Lakewood, CO). The infectious titers of monkeypox virus in the samples before and after riboflavin + UV treatment were determined by plaque assay on Vero cells. RESULTS: The levels of spiked virus present in whole blood and plasma samples exceeded 103 infectious particles per dose, corresponding to greater than 105 DNA copies per mL. Treatment of whole blood and plasma units under standard operating procedures for the Mirasol PRT System resulted in complete inactivation of infectivity to the limits of detection. This is equivalent to a reduction of ≥ 2.86 +/- 0.73 log10 pfu/mL of infectivity in whole blood and ≥ 3.47 +/-0.19 log10 pfu/mL of infectivity in plasma under standard operating conditions for those products. CONCLUSION: Based on this data and corresponding studies on infectivity in patients with monkeypox infections, use of Mirasol PRT would be expected to significantly reduce the risk of transfusion transmission of monkeypox.

Preliminary Evaluation of a Recombinant Rift Valley Fever Virus Glycoprotein Subunit Vaccine Providing Full Protection against Heterologous Virulent Challenge in Cattle.

Rift Valley fever virus (RVFV) is a mosquito-borne zoonotic pathogen that causes periodic outbreaks of abortion in ruminant species and hemorrhagic disease in humans in sub-Saharan Africa. These outbreaks have a significant impact on veterinary and public health. Its introduction to the Arabian Peninsula in 2003 raised concerns of further spread of this transboundary pathogen to non-endemic areas. These concerns are supported by the presence of competent vectors in many non-endemic countries. There is no licensed RVF vaccine available for humans and only a conditionally licensed veterinary vaccine available in the United States. Currently employed modified live attenuated virus vaccines in endemic countries lack the ability for differentiating infected from vaccinated animals (DIVA). Previously, the efficacy of a recombinant subunit vaccine based on the RVFV Gn and Gc glycoproteins, derived from the 1977 human RVFV isolate ZH548, was demonstrated in sheep. In the current study, cattle were vaccinated subcutaneously with the Gn only, or Gn and Gc combined, with either one or two doses of the vaccine and then subjected to heterologous virus challenge with the virulent Kenya-128B-15 RVFV strain, isolated from Aedes mosquitoes in 2006. The elicited immune responses by some vaccine formulations (one or two vaccinations) conferred complete protection from RVF within 35 days after the first vaccination. Vaccines given 35 days prior to RVFV challenge prevented viremia, fever and RVFV-associated histopathological lesions. This study indicates that a recombinant RVFV glycoprotein-based subunit vaccine platform is able to prevent and control RVFV infections in target animals.

A Whole Virion Vaccine for COVID-19 Produced via a Novel Inactivation Method and Preliminary Demonstration of Efficacy in an Animal Challenge Model.

The COVID-19 pandemic has generated intense interest in the rapid development and evaluation of vaccine candidates for this disease and other emerging diseases. Several novel methods for preparing vaccine candidates are currently undergoing clinical evaluation in response to the urgent need to prevent the spread of COVID-19. In many cases, these methods rely on new approaches for vaccine production and immune stimulation. We report on the use of a novel method (SolaVAX) for production of an inactivated vaccine candidate and the testing of that candidate in a hamster animal model for its ability to prevent infection upon challenge with SARS-CoV-2 virus. The studies employed in this work included an evaluation of the levels of neutralizing antibody produced post-vaccination, levels of specific antibody sub-types to RBD and spike protein that were generated, evaluation of viral shedding post-challenge, flow cytometric and single cell sequencing data on cellular fractions and histopathological evaluation of tissues post-challenge. The results from this preliminary evaluation provide insight into the immunological responses occurring as a result of vaccination with the proposed vaccine candidate and the impact that adjuvant formulations, specifically developed to promote Th1 type immune responses, have on vaccine efficacy and protection against infection following challenge with live SARS-CoV-2. This data may have utility in the development of effective vaccine candidates broadly. Furthermore, the results of this preliminary evaluation suggest that preparation of a whole virion vaccine for COVID-19 using this specific photochemical method may have potential utility in the preparation of one such vaccine candidate.

Pathogen reduction of SARS-CoV-2 virus in plasma and whole blood using riboflavin and UV light.

BACKGROUND: Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) has recently been identified as the causative agent for Coronavirus Disease 2019 (COVID-19). The ability of this agent to be transmitted by blood transfusion has not been documented, although viral RNA has been detected in serum. Exposure to treatment with riboflavin and ultraviolet light (R + UV) reduces blood-borne pathogens while maintaining blood product quality. Here, we report on the efficacy of R + UV in reducing SARS-CoV-2 infectivity when tested in human plasma and whole blood products. STUDY DESIGN AND METHODS: SARS-CoV-2 (isolate USA-WA1/2020) was used to inoculate plasma and whole blood units that then underwent treatment with riboflavin and UV light (Mirasol Pathogen Reduction Technology System, Terumo BCT, Lakewood, CO). The infectious titers of SARS-CoV-2 in the samples before and after R + UV treatment were determined by plaque assay on Vero E6 cells. Each plasma pool (n = 9) underwent R + UV treatment performed in triplicate using individual units of plasma and then repeated using individual whole blood donations (n = 3). RESULTS: Riboflavin and UV light reduced the infectious titer of SARS-CoV-2 below the limit of detection for plasma products at 60-100% of the recommended energy dose. At the UV light dose recommended by the manufacturer, the mean log reductions in the viral titers were ≥ 4.79 ± 0.15 Logs in plasma and 3.30 ± 0.26 in whole blood units. CONCLUSION: Riboflavin and UV light effectively reduced the titer of SARS-CoV-2 to the limit of detection in human plasma and by 3.30 ± 0.26 on average in whole blood. Two clades of SARS-CoV-2 have been described and questions remain about whether exposure to one strain confers strong immunity to the other. Pathogen-reduced blood products may be a safer option for critically ill patients with COVID-19, particularly those in high-risk categories.

A multiplex fluorescence microsphere immunoassay for increased understanding of Rift Valley fever immune responses in ruminants in Kenya.

Rift Valley fever virus (RVFV) is an important mosquito-borne pathogen with devastating impacts on agriculture and public health. With outbreaks being reported beyond the continent of Africa to the Middle East, there is great concern that RVFV will continue to spread to non-endemic areas such as the Americas and Europe. There is a need for safe and high throughput serological assays for rapid detection of RVFV during outbreaks and for surveillance. We evaluated a multiplexing fluorescence microsphere immunoassay (FMIA) for the detection of IgG and IgM antibodies in ruminant sera against the RVFV nucleocapsid Np, glycoprotein Gn, and non-structural protein NSs. Sheep and cattle sera from a region in Kenya with previous outbreaks were tested by FMIA and two commercially available competitive ELISAs (BDSL and IDvet). Our results revealed strong detection of RVFV antibodies against the Np, Gn and NSs antigen targets. Additionally, testing of samples with FMIA Np and Gn had 100% agreement with the IDvet ELISA. The targets developed in the FMIA assay provided a basis for a larger ruminant disease panel that can simultaneously screen several abortive and zoonotic pathogens.

Bactrian camels shed large quantities of Middle East respiratory syndrome coronavirus (MERS-CoV) after experimental infection.

In 2012, Middle East respiratory syndrome coronavirus (MERS-CoV) emerged. To date, more than 2300 cases have been reported, with an approximate case fatality rate of 35%. Epidemiological investigations identified dromedary camels as the source of MERS-CoV zoonotic transmission and evidence of MERS-CoV circulation has been observed throughout the original range of distribution. Other new-world camelids, alpacas and llamas, are also susceptible to MERS-CoV infection. Currently, it is unknown whether Bactrian camels are susceptible to infection. The distribution of Bactrian camels overlaps partly with that of the dromedary camel in west and central Asia. The receptor for MERS-CoV, DPP4, of the Bactrian camel was 98.3% identical to the dromedary camel DPP4, and 100% identical for the 14 residues which interact with the MERS-CoV spike receptor. Upon intranasal inoculation with 107 plaque-forming units of MERS-CoV, animals developed a transient, primarily upper respiratory tract infection. Clinical signs of the MERS-CoV infection were benign, but shedding of large quantities of MERS-CoV from the URT was observed. These data are similar to infections reported with dromedary camel infections and indicate that Bactrians are susceptible to MERS-CoV and given their overlapping range are at risk of introduction and establishment of MERS-CoV within the Bactrian camel populations.

Rift Valley Fever Viral RNA Detection by In Situ Hybridization in Formalin-Fixed, Paraffin-Embedded Tissues.

Sporadic outbreaks of Rift Valley fever virus (RVFV), a zoonotic, mosquito-borne Phlebovirus, cause abortion storms and death in sheep and cattle resulting in catastrophic economic impacts in endemic regions of Africa. More recently, with changes in competent vector distribution, growing international trade, and its potential use for bioterrorism, RVFV has become a transboundary animal disease of significant concern. New and sensitive techniques that determine RVFV presence, while lessening the potential for environmental contamination and human risk, through the use of inactivated, noninfectious samples such as formalin-fixed, paraffin-embedded (FFPE) tissues are needed. FFPE tissue in situ hybridization (ISH) enables the detection of nucleic acid sequences within the visual context of cellular and tissue morphology. Here, we present a chromogenic pan-RVFV ISH assay based on RNAscope® technology, which is able to detect multiple RVFV strains in FFPE tissues, enabling visual correlation of RVFV RNA presence with histopathologic lesions.

Investigating the Potential Role of North American Animals as Hosts for Zika Virus.

The recent emergence of the mosquito-borne Zika virus (ZIKV) in the Americas has become a global public health concern. We describe a series of experimental infections designed to investigate whether animals within certain taxonomic groups in North America have the potential to serve as ZIKV amplifying or maintenance hosts. Species investigated included armadillos, cottontail rabbits, goats, mink, chickens, pigeons, ground hogs, deer mice, cattle, raccoons, ducks, Syrian Golden hamsters, garter snakes, leopard frogs, house sparrows, and pigs. Infectious virus was isolated from blood only in frogs and armadillos; however, the magnitude of viremia was low. In addition, neutralizing antibodies were detected after infection in goats, rabbits, ducks, frogs, and pigs. This study indicates that the animals tested to date are unlikely to act as animal reservoirs for ZIKV, but that rabbits and pigs could potentially serve as sentinel species. Understanding the transmission cycle and maintenance of ZIKV in animals will help in developing effective surveillance programs and preventative measures for future outbreaks.

Successful conservative management of suspected Fournier's gangrene in cats: three cases.

CASE SERIES SUMMARY: Three cats with suspected Fournier's gangrene had an acute onset of clinical signs and bloodwork changes consistent with sepsis. All cases had similar progression of wounds that were managed without aggressive surgical debridement, which is the currently accepted treatment of choice. All cats survived and have maintained an excellent long-term quality of life. RELEVANCE AND NOVEL INFORMATION: Fournier's gangrene is a potentially fatal disease, with few cases reported in the veterinary literature. This retrospective case series describes the only known reports of survival from suspected Fournier's gangrene cats, none of which required aggressive surgical debridement.