Genetic diversity of hepatitis E virus (HEV) in imported and domestic camels in Saudi Arabia.

Camels gained attention since the discovery of MERS-CoV as intermediary hosts for potentially epidemic zoonotic viruses. DcHEV is a novel zoonotic pathogen associated with camel contact. This study aimed to genetically characterize DcHEV in domestic and imported camels in Saudi Arabia. DcHEV was detected by RT-PCR in serum samples, PCR-positive samples were subjected to sequencing and phylogenetic analyses. DcHEV was detected in 1.77% of samples with higher positivity in domestic DCs. All positive imported dromedaries were from Sudan with age declining prevalence. Domestic DcHEV sequences clustered with sequences from Kenya, Somalia, and UAE while imported sequences clustered with one DcHEV isolate from UAE and both sequences clustered away from isolates reported from Pakistan. Full-genome sequences showed 24 amino acid difference with reference sequences. Our results confirm the detection of DcHEV in domestic and imported DCs. Further investigations are needed in human and camel populations to identify DcHEV potential zoonosis threat.

Computational and In Vitro Experimental Investigations Reveal Anti-Viral Activity of Licorice and Glycyrrhizin against Severe Acute Respiratory Syndrome Coronavirus 2.

Without effective antivirals, the COVID-19 pandemic will likely continue to substantially affect public health. Medicinal plants and phytochemicals are attractive therapeutic options, particularly those targeting viral proteins essential for replication cycle. Herein, a total 179 phytochemicals of licorice (Glycyrrhiza glabra) were screened and scrutinized against the SARS-CoV-2 main protease (Mpro) with considerable binding affinities in the range of -9.831 to -2.710 kcal/mol. The top 10 compounds with the best docking scores, licuraside, glucoliquiritin apioside, 7,3'-Dihydroxy-5'-methoxyisoflavone, licuroside, kanzonol R, neoisoliquiritin, licochalcone-A, formononetin, isomucronulatol, and licoricone, were redocked using AutoDock Vina, yielding -8.7 to -7.3 kcal/mol binding energy against Glycyrrhizin (-8.0 kcal/mol) as a reference ligand. Four compounds, licuraside, glucoliquiritin apioside, 7,3'-Dihydroxy-5'-methoxyisoflavone, and licuroside, with glycyrrhizin (reference ligand) were considered for the 100 ns MD simulation and post-simulation analysis which support the stability of docked bioactive compounds with viral protein. In vitro studies demonstrated robust anti-SARS-CoV-2 activity of licorice and glycyrrhizin under different treatment protocols (simulations treatment with viral infection, post-infection treatment, and pre-treatment), suggesting multiple mechanisms for action. Although both compounds inhibited SARS-CoV-2 replication, the half-maximal inhibitory concentration (IC50) of glycyrrhizin was substantially lower than licorice. This study supports proceeding with in vivo experimentation and clinical trials and highlights licorice and glycyrrhizin as potential therapeutics for COVID-19.

Test-based de-isolation in COVID-19 immunocompromised patients: Cycle threshold value versus SARS-CoV-2 viral culture.

BACKGROUND: Immunocompromised patients with coronavirus disease 2019 (COVID-19) have prolonged infectious viral shedding for more than 20 days. A test-based approach is suggested for de-isolation of these patients. METHODS: The strategy was evaluated by comparing severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) viral load (cycle threshold (Ct) values) and viral culture at the time of hospital discharge in a series of 13 COVID-19 patients: six immunocompetent and seven immunocompromised (five solid organ transplant patients, one lymphoma patient, and one hepatocellular carcinoma patient). RESULTS: Three of the 13 (23%) patients had positive viral cultures: one patient with lymphoma (on day 16) and two immunocompetent patients (on day 7 and day 11). Eighty percent of the patients had negative viral cultures and had a mean Ct value of 20.5. None of the solid organ transplant recipients had positive viral cultures. CONCLUSIONS: The mean Ct value for negative viral cultures was 20.5 in this case series of immunocompromised patients. Unlike those with hematological malignancies, none of the solid organ transplant patients had positive viral cultures. Adopting the test-based approach for all immunocompromised patients may lead to prolonged quarantine. Large-scale studies in disease-specific populations are needed to determine whether a test-based approach versus a symptom-based approach or a combination is applicable for the de-isolation of various immunocompromised patients.

Amotosalen and ultraviolet A light treatment efficiently inactivates severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in human plasma.

BACKGROUND AND OBJECTIVES: During the ongoing pandemic of COVID-19, SARS-CoV-2 RNA was detected in plasma and platelet products from asymptomatic blood donors, raising concerns about potential risk of transfusion transmission, also in the context of the current therapeutic approach utilizing plasma from convalescent donors. The objective of this study was to assess the efficacy of amotosalen/UVA light treatment to inactivate SARS-CoV-2 in human plasma to reduce the risk of potential transmission through blood transfusion. METHODS: Pools of three whole-blood-derived human plasma units (630-650 ml) were inoculated with a clinical SARS-CoV-2 isolate. Spiked units were treated with amotosalen/UVA light (INTERCEPT Blood System™) to inactivate SARS-CoV-2. Infectious titres and genomic viral load were assessed by plaque assay and real-time quantitative PCR. Inactivated samples were subject to three successive passages on permissive tissue culture to exclude the presence of replication-competent viral particles. RESULTS: Inactivation of infectious viral particles in spiked plasma units below the limit of detection was achieved by amotosalen/UVA light treatment with a mean log reduction of >3·32 ± 0·2. Passaging of inactivated samples on permissive tissue showed no viral replication even after 9 days of incubation and three passages, confirming complete inactivation. The treatment also inhibited NAT detection by nucleic acid modification with a mean log reduction of 2·92 ± 0·87 PFU genomic equivalents. CONCLUSION: Amotosalen/UVA light treatment of SARS-CoV-2 spiked human plasma units efficiently and completely inactivated >3·32 ± 0·2 log of SARS-CoV-2 infectivity, showing that such treatment could minimize the risk of transfusion-related SARS-CoV-2 transmission.

Re-infection with a different SARS-CoV-2 clade and prolonged viral shedding in a hematopoietic stem cell transplantation patient.

Immunocompromised patients who have a severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection pose many clinical and public health challenges. We describe the case of a hematopoietic stem cell transplantation patient with lymphoma who had a protracted illness requiring three consecutive hospital admissions. Whole genome sequencing confirmed two different SARS-CoV-2 clades. Clinical management issues and the unanswered questions arising from this case are discussed.

A Reliable Indirect ELISA Protocol for Detection of Human Antibodies Directed to SARS-CoV-2 NP Protein.

A few months ago, the availability of a reliable and cost-effective testing capacity for COVID-19 was a concern for many countries. With the emergence and circulation of new SARS-CoV-2 variants, another layer of challenge can be added for COVID-19 testing at both molecular and serological levels. This is particularly important for the available tests principally designed to target the S gene/protein where multiple mutations have been reported. Herein, the SARS-CoV-2 NP recombinant protein was utilized to develop a simple and reliable COVID-19 NP human IgG ELISA. The optimized protocol was validated against a micro-neutralization (MN) assay, in-house S-based ELISA, and commercial chemiluminescence immunoassay (CLIA). The developed assay provides 100% sensitivity, 98.9% specificity, 98.9% agreement, and high overall accuracy with an area under curve equal to 0.9998 ± 0.0002 with a 95% confidence interval of 0.99 to 1.00. The optical density values of positive samples significantly correlated with their corresponding MN titers. The assay specifically detects IgG antibodies to the SARS-CoV-2 NP protein and does not cross-detect IgG to the viral S protein. Moreover, it does not cross-react with antibodies related to other coronaviruses (e.g., the Middle East respiratory syndrome coronavirus or human coronavirus HKU1). The availability of this reliable COVID-19 NP IgG ELISA protocol is highly valuable for its diagnostic and epidemiological applications.

Seroprevalence of neutralizing antibodies to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) among healthcare workers in Makkah, Saudi Arabia.

OBJECTIVE: The new coronavirus disease 2019 (COVID-19) is a major health problem worldwide. The surveillance of seropositive individuals serves as an indicator to the extent of infection spread and provides an estimation of herd immunity status among population. Reports from different countries investigated this issue among healthcare workers (HCWs) who are "at risk" and "sources of risk" for COVID-19. This study aims to investigate the seroprevalence of COVID-19 among HCWs in one of the COVID-19 referral centers in Makkah, Saudi Arabia using three different serological methods. METHODS: In-house developed enzyme-linked immunoassay (ELISA), commercially available electro-chemiluminescence immunoassay (ECLIA), and microneutralization (MN) assay were utilized to determine the seroprevalence rate among the study population. 204 HCWs participated in the study. Both physicians and nurses working in the COVID-19 and non COVID-19 areas were included. Twelve out of 204 were confirmed cases of COVID-19 with variable disease severity. Samples from recovered HCWs were collected four weeks post diagnosis. RESULTS: The overall seroprevalence rate was 6.3% (13 out of 204) using the in-house ELISA and MN assay and it was 5.8% (12 out of 204) using the commercial ECLIA. Among HCWs undiagnosed with COVID-19, the seroprevalence was 2% (4 out 192). Notably, neutralizing antibodies were not detected in 3 (25%) out 12 confirmed cases of COVID-19. CONCLUSIONS: Our study, similar to the recent national multi-center study, showed a low seroprevalence of SARS-Cov-2 antibodies among HCWs. Concordance of results between the commercial electro-chemiluminescence immunoassay (ECLIA), in-house ELISA and MN assay was observed. The in-house ELISA is a promising tool for the serological diagnosis of SARS-CoV-2 infection. However, seroprevalence studies may underestimate the extent of COVID-19 infection as some cases with mild disease did not have detectable antibody responses.

Lack of Antibodies to SARS-CoV-2 among Blood Donors during COVID-19 Lockdown: A Study from Saudi Arabia.

In response to the coronavirus disease 2019 (COVID-19), Saudi Arabia have imposed timely restrictions to minimize the infection spread, lower the risk for vulnerable groups, and reduce the pressure on healthcare services. The effectiveness of these measures has not been assessed comprehensively and, thereby, remains uncertain. Besides monitoring the number of COVID-19 cases diagnosed by molecular assays, the seroprevalence can serve as an indicator for the incidence rate among the general population. This study aimed to evaluate seroprevalence status of all healthy blood donors who attended one of the main largest hospital located in the western region of Saudi Arabia from 1 January to 31 May 2020. The study period covered two months prior to reporting the first COVID-19 case in the country on 2 March 2020. Importantly, it covered the period when "lock-down type" measures have been enforced. Samples were subjected to in-house enzyme-linked immunosorbent assay (ELISA), chemiluminescence immunoassay (CLIA), and microneutralization (MN). The sero statuses of all samples were confirmed negative, demonstrating the lack of antibodies to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) among blood donors during COVID-19 lockdown period. This study supports the hypothesis that COVID-19 restrictions have potential for limiting the extent of the infection.

Development and Optimization of In-house ELISA for Detection of Human IgG Antibody to SARS-CoV-2 Full Length Spike Protein.

The ongoing coronavirus disease 19 (COVID-19) pandemic, caused by the novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), poses a threat to human health. Despite this, many affected countries are now in the process of gradual lifting of COVID-19 restrictions that were initially implemented in response to the pandemic. The success of the so-called "exit strategy" requires continued surveillance of virus circulation in the community and evaluation of the prevalence of protective immunity among population. Serology tests are valuable tools for these purposes. Herein, SARS-CoV-2 full-length spike (S) recombinant protein was utilized to develop and optimize an indirect enzyme-linked immunoassay (ELISA) that enables a reliable detection of virus-specific IgG antibody in human sera. Importantly, the performance of this assay was evaluated utilizing micro-neutralization (MN) assay as a reference test. Our developed ELISA offers 100% sensitivity, 98.4% specificity, 98.8% agreement, and high overall accuracy. Moreover, the optical density (OD) values of positive samples significantly correlated with their MN titers. The assay specifically detects human IgG antibodies directed against SARS-CoV-2, but not those to Middle East respiratory syndrome coronavirus (MERS-CoV) or human coronavirus HKU1 (HCoV-HKU1). The availability of this in-house ELISA protocol would be valuable for various diagnostic and epidemiological applications.

Cross-sectional prevalence study of MERS-CoV in local and imported dromedary camels in Saudi Arabia, 2016-2018.

The Middle East Respiratory Syndrome-Coronavirus (MERS-CoV) is an endemic virus in dromedaries. Annually, Saudi Arabia imports thousands of camels from the Horn of Africa, yet the epidemiology of MERS-CoV in these animals is largely unknown. Here, MERS-CoV prevalence was compared in imported African camels and their local counterparts. A total of 1399 paired sera and nasal swabs were collected from camels between 2016 and 2018. Imported animals from Sudan (n = 829) and Djibouti (n = 328) were sampled on incoming ships at Jeddah Islamic seaport before unloading, and local camels were sampled from Jeddah (n = 242). Samples were screened for neutralizing antibodies (nAbs) and MERS-CoV viral RNA. The overall seroprevalence was 92.7% and RNA detection rate was 17.2%. Imported camels had higher seroprevalence compared to resident herds (93.8% vs 87.6%, p <0.01) in contrast to RNA detection (13.3% vs 35.5%, p <0.0001). Seroprevalence significantly increased with age (p<0.0001) and viral RNA detection rate was ~2-folds higher in camels <2-year-old compared to older animals. RNA detection was higher in males verses females (24.3% vs 12.6%, p<0.0001) but seroprevalence was similar. Concurrent positivity for viral RNA and nAbs was found in >87% of the RNA positive animals, increased with age and was sex-dependent. Importantly, reduced viral RNA load was positively correlated with nAb titers. Our data confirm the widespread of MERS-CoV in imported and domestic camels in Saudi Arabia and highlight the need for continuous active surveillance and better prevention measures. Further studies are also warranted to understand camels correlates of protection for proper vaccine development.