Discovery of imidazo[1,2-b]pyridazine-containing TAK1 kinase inhibitors with excellent activities against multiple myeloma.

Current treatment options for patients with multiple myeloma (MM) include proteasome inhibitors, anti-CD38 antibodies, and immunomodulatory agents. However, if patients have continued disease progression after administration of these treatments, there are limited options. There is a need for effective targeted therapies of MM. Recent studies have shown that the transforming growth factor-ß activated kinase (TAK1) is upregulated and overexpressed in MM. We have discovered that 6-substituted morpholine or piperazine imidazo[1,2-b]pyridazines, with an appropriate aryl substituent at position-3, inhibit TAK1 at nanomolar concentrations. The lead compound, 26, inhibits the enzymatic activity of TAK1 with an IC50 of 55 nM. Under similar conditions, the known TAK1 inhibitor, takinib, inhibits the kinase with an IC50 of 187 nM. Compound 26 and analogs thereof inhibit the growth of multiple myeloma cell lines MPC-11 and H929 with GI50 values as low as 30 nM. These compounds have the potential to be translated into anti-MM therapeutics.

Emodin improves glucose metabolism and ovarian function in PCOS mice via the HMGB1/TLR4/NF-κB molecular pathway.

In brief: Obese PCOS mice display metabolic and endocrine disorders that manifest as abnormal metabolism of glucose and dysfunctions in the reproductive system. This study demonstrates that emodin alleviates most of these conditions possibly via the HMGB1/TLR4/NF-kB pathway. Abstract: PCOS is a reproductive disorder with an unclear etiology. It affects 5-10% of women worldwide and is largely associated with impaired glucose metabolism and obesity. HMGB1 is a nuclear protein associated with impaired glucose metabolism and PCOS. We sought to investigate the potential therapeutic effects of emodin on glucose metabolism and ovarian functions in PCOS mice via the HMGB1 molecular pathway. A high-fat diet (HFD) and dehydroepiandrosterone (DHEA)- induced PCOS mouse model comprising four experimental groups was established: control, PCOS, PCOS plus emodin, and PCOS plus vehicle groups. Emodin administration attenuated obesity, elevated fasting glucose levels, impaired glucose tolerance, and insulin resistance, and improved the polycystic ovarian morphology of PCOS mice. Additionally, it lowered elevated serum HMGB1, LH, and testosterone levels in PCOS mice. Elevated ovarian protein and mRNA levels of HMGB1 and TLR4 in PCOS mice were also lowered following emodin treatment. Furthermore, emodin lowered high NF-ĸB/65 protein levels in the ovaries of PCOS mice. Immunohistochemical staining of the ovaries revealed strong HMGB1, TLR4, and AR expressions in PCOS mice, which were lowered by emodin treatment. Moreover, emodin significantly increased GLUT4, IRS2, and INSR levels that were lowered by PCOS. Overall, our study showed that emodin alleviated the impaired glucose metabolism and improved ovarian function in PCOS mice, possibly via the HMGB1/TLR4/NF-ĸB signaling pathway. Thus, emodin could be considered a potential therapeutic agent in the management of PCOS.

STING antagonists, synthesized via Povarov-Doebner type multicomponent reaction.

The cGAS-STING axis plays an important role in protecting higher organisms against invading pathogens or cancer by promoting the production of cytokines and interferons. However, persistent or uncontrolled activation of this pathway could lead to inflamed environments, which is detrimental to the host in the long run. Persistent activation of STING is known to be the cause of STING-associated vasculopathy with onset in infancy (SAVI) and activated STING is believed to play important roles in worsening various diseased states, such as traumatic brain injury, diabetic kidney disease and colitis. Thus, antagonists of STING could play important roles in managing various inflammatory diseases. Herein, we report the discovery of small molecule STING inhibitors, HSD1077 and analogs, which are facilely synthesized via a Povarov-Doebner type three-component reaction involving an amine, ketone, and aldehyde. Structure-activity relationship, SAR, studies indicate that both the 3H-pyrazolo[4,3-f]quinoline and pyrazole moieties in HSD1077 are critical for STING binding. At concentrations as low as 20 nM, HSD1077 suppressed type-1 interferon expression in both murine RAW macrophages and human THP-1 monocytes upon treatment with 100 µM 2'-3' cGAMP. Compounds containing the 3H-pyrazolo[4,3-f]quinoline moiety have the potential to be translated into anti-inflammatory compounds via STING inhibition.

Re-sensitization of Multidrug-Resistant and Colistin-Resistant Gram-Negative Bacteria to Colistin by Povarov/Doebner-Derived Compounds.

Colistin, typically viewed as the antibiotic of last resort to treat infections caused by multidrug-resistant (MDR) Gram-negative bacteria, had fallen out of favor due to toxicity issues. The recent increase in clinical usage of colistin has resulted in colistin-resistant isolates becoming more common. To counter this threat, we have investigated previously reported compounds, HSD07 and HSD17, and developed 13 compounds with more desirable drug-like properties for colistin sensitization against 16 colistin-resistant bacterial strains, three of which harbor the plasmid-borne mobile colistin resistance (mcr-1). Lead compound HSD1624, which has a lower LogDpH7.4 (2.46) compared to HSD07 (>5.58), reduces the minimum inhibitory concentration (MIC) of colistin against Pseudomonas aeruginosa strain TRPA161 to 0.03 µg/mL from 1024 µg/mL (34,000-fold reduction). Checkerboard assays revealed that HSD1624 and analogues are also synergistic with colistin against colistin-resistant strains of Escherichia coli, Acinetobacter baumannii, and Klebsiella pneumoniae. Preliminary mechanism of action studies indicate that HSD1624 exerts its action differently depending on the bacterial species. Time-kill studies suggested that HSD1624 in combination with 0.5 µg/mL colistin was bactericidal to extended-spectrum beta-lactamase (ESBL)-producing E. coli, as well as to E. coli harboring mcr-1, while against P. aeruginosa TRPA161, the combination was bacteriostatic. Mechanistically, HSD1624 increased membrane permeability in K. pneumoniae harboring a plasmid containing the mcr-1 gene but did not increase radical oxygen species (ROS), while a combination of 15 µM HSD1624 and 0.5 µg/mL colistin significantly increased ROS in P. aeruginosa TRPA161. HSD1624 was not toxic to mammalian red blood cells (up to 226 µM).

Role of CPXM1 in Impaired Glucose Metabolism and Ovarian Dysfunction in Polycystic Ovary Syndrome.

Polycystic ovary syndrome (PCOS), a common female endocrinopathy associated with both reproductive and metabolic disorders, has an unclear etiology and unsatisfactory management methods. Carboxypeptidase X, M14 family member 1 (CPXM1) is a protein involved in follicular atresia, insulin production, and adipose tissue production, though its role in PCOS is not fully understood. We used a 60% high-fat diet (HFD) plus dehydroepiandrosterone (DHEA)-induced PCOS mouse model to determine the role of CPXM1 in abnormal glucose metabolism and ovarian dysfunction in PCOS. We found that serum CPXM1 concentrations were higher in PCOS mice and positively correlated with increased levels of serum testosterone and insulin. In both ovarian and adipose tissues of PCOS mice, CPXM1 mRNA and protein levels were significantly increased but GLUT4 levels were significantly decreased. Immunohistochemistry (IHC) staining of the ovary showed increased CPXM1 expression in PCOS. In addition, the protein expression of phosphorylated protein kinase B (p-Akt) was also significantly decreased in PCOS mice. Furthermore, mRNA levels of inflammatory markers such as TNF-α, IL-6, IFN-α, and IFN-γ were increased in ovarian and adipose tissues of PCOS mice. However, IRS-1, IRS-2, and INSR levels were significantly decreased. Our results indicated for the first time that abnormally high expression of CPXM1, increased adiposity, impaired glucose tolerance, and chronic low-grade inflammation may act together in a vicious cycle in the pathophysiology of PCOS. Our research suggests the possibility of CPXM1 as a potential therapeutic target for the treatment of PCOS.

Fetal exposure of Aristolochic Acid I undermines ovarian reserve by disturbing primordial folliculogenesis.

The primordial follicle pool established in early life determines the ovarian reserve in the female reproductive lifespan. Premature exhaustion of primordial follicles contributes to primary ovarian insufficiency (POI), that is dependent by the initial size of the primordial follicle pool and by the rate of its activation and depletion. AAI, a powerful nephrotoxin with carcinogenic potential, is present in the Aristolochiaceae species, which can release AAI into soil as a persistent pollutant. In order to assess the potential risk of Aristolochic Acid I (AAI) exposure on mammalian oogenesis, we uncovered its adverse effect on primordial folliculogenesis in the neonatal mouse ovary and its effect on female fertility in adulthood. Pregnant mice were orally administrated with doses of AAI without hepatic or renal toxicity during late-gestation. Ovaries from offspring of administered female displayed gross aberrations during primordial folliculogenesis. Also, unenclosed oocytes in germ-cell cysts showed increased DNA damage. Furthermore, several key factors, including NANOS3, SOX9, KLF4, that govern early gonad's differentiation were abnormally expressed in the exposed ovary, while the follicle formation was partially restored by knockdown of Nanos3 or sox9. In adulthood, these aberrations evolved into a significant reduction in offspring number and impaired ovarian reserve. Together, our results show that AAI influences primordial folliculogenesis and, importantly, affected female fertility. This study shows that administration of drugs herbs or consumption of vegetables that contain AAs during pregnancy may adversely influence the fertility of offspring.

Surveillance and laboratory collaboration in response to an outbreak of Vibrio parahaemolyticus, Plesiomonas shigelloides, and Aeromonas hydrophila in Sekondi-Takoradi, Ghana: a case series.

BACKGROUND: The detection of epidemic-prone pathogens is important in strengthening global health security. Effective public health laboratories are critical for reliable, accurate, and timely testing results in outbreak situations. Ghana received funding as one of the high-risk non-Ebola affected countries to build and strengthen public health infrastructure to meet International Health Regulation core capacities. A key objective was to build laboratory capacities to detect epidemic-prone diseases. CASE PRESENTATION: In June 2018, a local hospital received eight patients who presented with acute diarrhea. A sample referral system for Ghana has not been established, but the Sekondi Zonal Public Health Laboratory staff and mentors collaborated with Disease Surveillance Officers (DSOs) to collect, package, and transport stool specimens from the outbreak hospital to the Public Health Laboratory for laboratory testing. The patients included seven females and one male, of Fante ethnicity from the Fijai township of Sekondi-Takoradi Municipality. The median age of the patients was 20 years (interquartile range: 20-29 years). Vibrio parahaemolyticus was identified within 48 hours from four patients, Plesiomonas shigelloides from one patient, and Aeromonas hydrophila from another patient. There was no bacteria growth from the samples from the two other patients. All patients were successfully treated and discharged. CONCLUSION: This is the first time these isolates have been identified at the Sekondi Zonal Public Health Laboratory, demonstrating how rapid response, specimen transportation, laboratory resourcing, and public health coordination are important in building capacity towards achieving health security. This capacity building was part of the United States Centers for Disease Control and Prevention engagement of international and local partners to support public health laboratories with supplies, diagnostic equipment, reagents, and logistics.

The role of fascin in carcinogenesis and embryo implantation.

The cytoskeleton, with its actin bundling proteins, plays crucial roles in a host of cellular function, such as cancer metastasis, antigen presentation and trophoblast migration and invasion, as a result of cytoskeletal remodeling. A key player in cytoskeletal remodeling is fascin. Upregulation of fascin induces the transition of epithelial phenotypes to mesenchymal phenotypes through complex interaction with transcription factors. Fascin expression also regulates mitochondrial F-actin to promote oxidative phosphorylation (OXPHOS) in some cancer cells. Trophoblast cells, on the other hand, exhibit similar physiological functions, involving the upregulation of genes crucial for its migration and invasion. Owing to the similar tumor-like characteristics among cancer and trophoblats, we review recent studies on fascin in relation to cancer and trophoblast cell biology; and based on existing evidence, link fascin to the establishment of the maternal-fetal interface.

Iodothyronine deiodinase 2 (DiO2) regulates trophoblast cell line cycle, invasion and apoptosis; and its downregulation is associated with early recurrent miscarriage.

INTRODUCTION: Trophoblast development is a crucial event in placentation and pregnancy complications but its underlying mechanisms remain unclear. Thus, we aimed at investigating the role of DiO2 in trophoblast cell line decisions and assessing its placental villous expression in early recurrent miscarriage (ERM) patients. METHODS: The placental villous expression of DiO2 was determined with immunofluorescence. Cell proliferation was measured with the CCK8 kit while cell-cycle and apoptosis were studied with flow-cytometry. Cell migration and invasion were measured with wound-healing and transwell assays, respectively. Gene expression was then assessed with RT-qPCR and western blotting. RESULTS: DiO2 is expressed in the CTB, PCT, DCT and STB of the placenta. Its overexpression arrested trophoblast cell line proliferation at the G1 phase of the cell-cycle by downregulating cyclin-D1 and PCNA, while promoting apoptosis via increased caspase-3 activity and inhibition of the AKT and ERK1/2 signaling pathways. Also, it augmented trophoblast cell line migration and invasion via the upregulation of N-cadherin, vimentin, fascin-1, twist-1 and other epithelial-mesenchymal transition genes. DiO2 knockdown elicited the opposite effects. Surprisingly, each of these effects of DiO2 manipulation was not mediated by thyroid hormone metabolism. Assessment of the ERM placental villi revealed a downregulation of DiO2, N-cadherin, vimentin, fascin-1 and twist-1. The expression of E-cadherin remained unchanged in these placentae. DISCUSSION: During placentation, DiO2 may inhibit trophoblast proliferation while facilitating their differentiation into an invasive phenotype; and that its downregulation may contribute to the shallow trophoblast invasion that precedes ERM. Hence, DiO2 is a potential therapeutic target against ERM.

Sero-molecular epidemiology of hepatitis E virus in pigs and human contacts in Ghana.

BACKGROUND: Hepatitis E virus (HEV) is among the leading causes of viral hepatitis in most developing countries. Zoonotic acquisition of HEV genotype 3 from swine has come into focus more recently. Available studies on HEV in Ghana and other countries in the region do not provide enough information towards understanding the epidemiology of HEV in human and animal populations. Towards this end, we conducted a comparative cross-sectional study to determine the seroprevalence and risk factors associated with HEV exposure, both in swine and humans working on pig farms in typical local settings. The presence of viral RNA in human and swine samples was also evaluated, along with classification of viral sequences from HEV-positive samples. METHODS: Structured questionnaires soliciting information on pigs reared, as well as socio-demographic information including age, sex and educational background of humans was collected. A total of 10 ml and 5 ml of whole blood was collected from pigs and human participants respectively. ELISA and real-time RT-PCR were performed on the sera for the qualitative detection of IgG antibodies to hepatitis E virus and viral RNA, respectively. RESULTS: Five hundred and forty-four (544) human participants including 264 swine contacts and 280 swine non-contacts were enrolled in the study. Although the proportion of HEV IgG antibodies was higher in contact groups (114; 54.3%) than non-contact groups (96; 45.7%), a multivariate analysis did not show any significant difference. No HEV RNA was detected in human samples. Similarly, 720 pigs were sampled from 18 farms located in five regions in Ghana. Twenty-three (23) of the pigs (3.2, 95%CI = 2.0-4.8) were positive for HEV RNA by real-time RT-PCR testing. Sequences obtained from HEV-positive samples were found to share high sequence identities with each other and clustered with other genotype 3 viruses indicating the existence of circulating zoonotic genotype 3 viruses on farms. Although we did not find evidence of pig to human transmission of HEV genotype 3, the presence of this genotype in pigs shows the potential for possible zoonotic transmission in African farm settings and buttresses the importance of active surveillance for the infection among at risk populations.

Downregulation of fascin in the first trimester placental villi is associated with early recurrent miscarriage.

Inadequate trophoblast proliferation, shallow invasion and exaggerated rate of trophoblast apoptosis are implicated in early recurrent miscarriage (ERM). However, the mechanistic bases of this association have not been fully established. We aimed at investigating the involvement of fascin, an actin-bundling protein, in trophoblast activities and ERM. We found that fascin was downregulated in the cytotrophoblasts (CTBs) and distal cytotrophoblasts (DCTs) of ERM placentae. Knockdown of fascin altered cellular and nucleolar morphology, and inhibited the proliferation but increased apoptosis of trophoblastic HTR8/SVneo cells. Furthermore, fascin knockdown decreased the expression of transcription factors such as Snail1/2, Twist and Zeb1/2, mesenchymal molecules such as Vimentin and N-cadherin, and the protein expression of phosphorylated extracellular signal-regulated kinase 1/2 (ERK1/2) and phosphorylates signal transducer and activator of transcript 3 (STAT3). Exposure of HTR-8/SVneo cells to hypoxia reoxygenation (H/R) decreased fascin expression to affect the cells' invasion. Our results indicate for the first time that the downregulation of fascin is involved in the pathogenesis of early recurrent miscarriage; and hence a potential therapeutic target against the disease.

Genomic and epidemiological characteristics of SARS-CoV-2 in Africa.

Since late 2019, the coronavirus disease 2019 (COVID-19) outbreak, caused by SARS-CoV-2, has rapidly evolved to become a global pandemic. Each country was affected but with a varying number of infected cases and mortality rates. Africa was hit late by the pandemic but the number of cases rose sharply. In this study, we investigated 224 SARS-CoV-2 genome sequences from the Global Initiative on Sharing Avian Influenza Data (GISAID) in the early part of the outbreak, of which 69 were from Africa. We analyzed a total of 550 mutations by comparing them with the reference SARS-CoV-2 sequence from Wuhan. We classified the mutations observed based on country and region, and afterwards analyzed common and unique mutations on the African continent as a whole. Correlation analyses showed that the duo variants ORF1ab/RdRp 4715L and S protein 614G variants, which are strongly linked to fatality rate, were not significantly and positively correlated with fatality rates (r = -0.03757, P = 0.5331 and r = -0.2876, P = 0.6389, respectively), although increased number of cases correlated with number of deaths (r = 0.997, P = 0.0002). Furthermore, most cases in Africa were mainly imported from American and European countries, except one isolate with no mutation and was similar to the original isolate from Wuhan. Moreover, unique mutations specific to countries were identified in the early phase of the outbreak but these mutations were not regional-specific. There were common mutations in all isolates across the continent as well as similar isolate-specific mutations in different regions. Our findings suggest that mutation is rapid in SARS-CoV-2 in Africa and although these mutations spread across the continent, the duo variants could not possibly be the sole cause of COVID-19 deaths in Africa in the early phase of the outbreak.

Molecular-based cross-species evaluation of bovine coronavirus infection in cattle, sheep and goats in Ghana.

BACKGROUND: Apart from the huge worldwide economic losses often occasioned by bovine coronavirus (BCoV) to the livestock industry, particularly with respect to cattle rearing, continuous surveillance of the virus in cattle and small ruminants is essential in monitoring variations in the virus that could enhance host switching. In this study, we collected rectal swabs from a total of 1,498 cattle, sheep and goats. BCoV detection was based on reverse transcriptase polymerase chain reaction. Sanger sequencing of the partial RNA-dependent RNA polymerase (RdRp) region for postive samples were done and nucleotide sequences were compared with homologous sequences from the GenBank. RESULTS: The study reports a BCoV prevalence of 0.3%, consisting of 4 positive cases; 3 goats and 1 cattle. Less than 10% of all the animals sampled showed clinical signs such as diarrhea and respiratory distress except for high temperature which occurred in > 1000 of the animals. However, none of the 4 BCoV positive animals manifested any clinical signs of the infection at the time of sample collection. Bayesian majority-rule cladogram comparing partial and full length BCoV RdRp genes obtained in the study to data from the GenBank revealed that the sequences obtained from this study formed one large monophyletic group with those from different species and countries. The goat sequences were similar to each other and clustered within the same clade. No major variations were thus observed between our isolates and those from elsewhere. CONCLUSIONS: Given that Ghana predominantly practices the extensive and semi-intensive systems of animal rearing, our study highlights the potential for spillover of BCoV to small ruminants in settings with mixed husbandry and limited separation between species.

Activin and inhibin signaling: From regulation of physiology to involvement in the pathology of the female reproductive system.

Activins and inhibins - comprising activin A, B, AB, C and E, and inhibin A and B isoforms - belong to the transforming growth factor beta (TGFß) superfamily. They regulate several biological processes, including cellular proliferation, differentiation and invasiveness, to enhance the formation and functioning of many human tissues and organs. In this review, we have discussed the role of activin and inhibin signaling in the physiological and female-specific pathological events that occur in the female reproductive system. The up-to-date evidence indicates that these cytokines regulate germ cell development, follicular development, ovulation, uterine receptivity, decidualization and placentation through the activation of several signaling pathways; and that their dysregulated expression is involved in the pathogenesis and pathophysiology of the numerous diseases, including pregnancy complications, that disturb reproduction. Hence, some of the isoforms have been suggested as potential biomarkers and therapeutic targets for the management of some of these diseases. Tackling the research directions highlighted in this review will enhance a detailed comprehension and the clinical utility of these cytokines.

Sero-prevalence, cross-species infection and serological determinants of prevalence of Bovine Coronavirus in Cattle, Sheep and Goats in Ghana.

Cattle, goats and sheep are dominant livestock species in sub-Saharan Africa, with sometimes limited information on the prevalence of major infectious diseases. Restrictions due to notifiable epizootics complicate the exchange of samples in surveillance studies and suggest that laboratory capacities should be established domestically. Bovine Coronavirus (BCoV) causes mainly enteric disease in cattle. Spillover to small ruminants is possible. Here we established BCoV serology based on a recombinant immunofluorescence assay for cattle, goats and sheep, and studied the seroprevalence of BCoV in these species in four different locations in the Greater Accra, Volta, Upper East, and Northern provinces of Ghana. The whole sampling and testing was organized and conducted by a veterinary school in Kumasi, Ashanti Region of Ghana. Among sampled sheep (n = 102), goats (n = 66), and cattle (n = 1495), the seroprevalence rates were 25.8 %, 43.1 % and 55.8 %. For cattle, seroprevalence was significantly higher on larger farms (82.2 % vs 17.8 %, comparing farms with >50 or <50 animals; p = 0.027). Highest prevalence was seen in the Northern province with dry climate, but no significant trend following the north-south gradient of sampling sites was detected. Our study identifies a considerable seroprevalence for BCoV in Ghana and provides further support for the spillover of BCoV to small ruminants in settings with mixed husbandry and limited separation between species.

The role of adiponectin in placentation and preeclampsia.

Preeclampsia is not fully understood; and few biomarkers, therapeutic targets, and therapeutic agents for its management have been identified. Original investigative findings suggest that abnormal placentation triggers preeclampsia and leads to hypertension, proteinuria, endothelial dysfunction, and inflammation, which are characteristics of the disease. Because of the regulatory roles that it plays in several metabolic processes, adiponectin has become a cytokine of interest in metabolic medicine. In this review, we have discussed the role of adiponectin in trophoblast proliferation, trophoblast differentiation, trophoblast invasion of the decidua, and decidual angiogenesis, which are the major phases of placentation. Also, we have highlighted the physiological profile of adiponectin in the course of normal pregnancy. Moreover, we have discussed the involvement of adiponectin in hypertension, endothelial dysfunction, inflammation, and proteinuria. Furthermore, we have summarized the reported relationship between the maternal serum adiponectin level and preeclampsia. The available evidence indicates that adiponectin level physiologically falls as pregnancy advances, regulates placentation, and exhibits protective effects against the symptoms of preeclampsia and that while hyperadiponectinemia is evident in normal-weight preeclamptic women, hypoadiponectinemia is evident in overweight and obese preeclamptic women. Therefore, the clinical use of adiponectin as a biomarker, therapeutic target, or therapeutic agent against the disease looks promising and should be considered.

The transforming growth factor ß superfamily as possible biomarkers of preeclampsia: a comprehensive review.

The etiology of preeclampsia - an abnormal placentation-mediated disease - is not fully understood; and there are very few biomarkers with which to predict and diagnose it. Early prediction and diagnosis of this pathology can lead to a significant improvement in maternal and perinatal outcomes. Since members of the transforming growth factor ß superfamily influence placentation, and are released from the placenta into the maternal circulatory system, several studies have investigated the involvement of these cytokines in preeclampsia and the possibility of using their serum levels as biomarkers of the disease. In this review, we have summarized the reported relationships between the levels of this superfamily of cytokines and preeclampsia. The available information indicates that altered levels of some of these cytokines are involved in the pathogenesis and pathophysiology of preeclampsia, suggesting their likelihood of serving as predictive and diagnostic biomarkers of the disease.

Potential Intermediate Hosts for Coronavirus Transmission: No Evidence of Clade 2c Coronaviruses in Domestic Livestock from Ghana.

The emergence of Middle East Respiratory Syndrome Coronavirus (MERS-CoV), nearly a decade ago with worldwide distribution, was believed to be of zoonotic origin from bats with dromedary camels as intermediate hosts. There is a likelihood of other domestic livestock serving as intermediate hosts for this virus. The presence of coronaviruses, closely related to MERS-CoV in Ghanaian bats, presented the opportunity to test the hypothesis of transmissibility of this virus through domestic livestock species. The possible interactions between livestock and bats in 31 household farms were accessed by observation and interviews with farmers. Rectal swabs and serum from cattle, sheep, goats, donkeys, and swine from commercial and household farms were tested for MERS-CoV and a Nycteris sp. bat coronavirus, previously detected in Ghana. A pan-PCR assay to detect clade 2c viruses and recombinant immunofluorescence assay to detect anti-spike IgG antibodies against the target viruses were used. Likely contact between livestock and bats was determined for 13 farms (41.9%) that reported confining their livestock and also observing bats in their homes. Livestock were left unconfined on eight farms (25.8%) that also observed bats roosting in trees close to their homes. No viral RNA or antibodies against the two coronaviruses were detected in any of the livestock species tested. Cattle, sheep, goats, donkeys, and swine are not likely hosts of clade 2c coronaviruses.

Development of a Whole-Virus ELISA for Serological Evaluation of Domestic Livestock as Possible Hosts of Human Coronavirus NL63.

Known human coronaviruses are believed to have originated in animals and made use of intermediate hosts for transmission to humans. The intermediate hosts of most of the human coronaviruses are known, but not for HCoV-NL63. This study aims to assess the possible role of some major domestic livestock species as intermediate hosts of HCoV-NL63. We developed a testing algorithm for high throughput screening of livestock sera with ELISA and confirmation with recombinant immunofluorescence assay testing for antibodies against HCoV-NL63 in livestock. Optimization of the ELISA showed a capability of the assay to significantly distinguish HCoV-NL63 from HCoV-229E (U = 27.50, p < 0.001) and HCoV-OC43 (U = 55.50, p < 0.001) in coronavirus-characterized sera. Evaluation of the assay with collected human samples showed no significant difference in mean optical density values of immunofluorescence-classified HCoV-NL63-positive and HCoV-NL63-negative samples (F (1, 215) = 0.437, p = 0.509). All the top 5% (n = 8) most reactive human samples tested by ELISA were HCoV-NL63 positive by immunofluorescence testing. In comparison, only a proportion (84%, n = 42) of the top 25% were positive by immunofluorescence testing, indicating an increased probability of the highly ELISA reactive samples testing positive by the immunofluorescence assay. None of the top 5% most ELISA reactive livestock samples were positive for HCoV-NL63-related viruses by immunofluorescence confirmation. Ghanaian domestic livestock are not likely intermediate hosts of HCoV-NL63-related coronaviruses.