Asymptomatic Middle East Respiratory Syndrome Coronavirus Infection in Rabbits

The ability of Middle East respiratory syndrome coronavirus (MERS-CoV) to infect small animal species may be restricted given the fact that mice, ferrets, and hamsters were shown to resist MERS-CoV infection. We inoculated rabbits with MERS-CoV. Although virus was detected in the lungs, neither significant histopathological changes nor clinical symptoms were observed. Infectious virus, however, was excreted from the upper respiratory tract, indicating a potential route of MERS-CoV transmission in some animal species.     

Protective Efficacy of Recombinant Modified Vaccinia Virus Ankara Delivering Middle East Respiratory Syndrome Coronavirus Spike Glycoprotein

Middle East respiratory syndrome coronavirus (MERS-CoV) causes severe respiratory disease in humans. We tested a recombinant modified vaccinia virus Ankara (MVA) vaccine expressing full-length MERS-CoV spike (S) glycoprotein by immunizing BALB/c mice with either intramuscular or subcutaneous regimens. In all cases, MVA-MERS-S induced MERS-CoV-specific CD8⁺ T cells and virus-neutralizing antibodies. Vaccinated mice were protected against MERS-CoV challenge infection after transduction with the human dipeptidyl peptidase 4 receptor. This MERS-CoV infection model demonstrates the safety and efficacy of the candidate vaccine.     

Pichinde virus-specific cell-associated suppression of primary footpad swelling in an inbred strain of Syrian hamsters

Pichinde virus causes a lethal disease after i.p. inoculation of adult MHA hamsters; other strains of Syrian hamsters are resistant to this lethal infection. During studies of cell-mediated immune responses to Pichinde virus, it was noted that MHA hamsters survived infection when the virus was given in the footpad. However, unlike the resistant LSH and LVG strains of hamsters, the MHA hamsters did not manifest a footpad swelling response. Failure of the MHA hamster to respond to a footpad inoculation of Pichinde virus was shown to be virus-specific and appeared to be mediated by a cell-associated suppressor mechanism.     

Crystal Structure of the Receptor-Binding Domain from Newly Emerged Middle East Respiratory Syndrome Coronavirus

The newly emerged Middle East respiratory syndrome coronavirus (MERS-CoV) has infected at least 77 people, with a fatality rate of more than 50%. Alarmingly, the virus demonstrates the capability of human-to-human transmission, raising the possibility of global spread and endangering world health and economy. Here we have identified the receptor-binding domain (RBD) from the MERS-CoV spike protein and determined its crystal structure. This study also presents a structural comparison of MERS-CoV RBD with other coronavirus RBDs, successfully positioning MERS-CoV on the landscape of coronavirus evolution and providing insights into receptor binding by MERS-CoV. Furthermore, we found that MERS-CoV RBD functions as an effective entry inhibitor of MERS-CoV. The identified MERS-CoV RBD may also serve as a potential candidate for MERS-CoV subunit vaccines. Overall, this study enhances our understanding of the evolution of coronavirus RBDs, provides insights into receptor recognition by MERS-CoV, and may help control the transmission of MERS-CoV in humans.     

Susceptibility of inbred Syrian golden hamsters (Mesocricetus auratus) to lethal disease by lymphocytic choriomeningitis virus

An acutely lethal LCMV disease model has been established in the Syrian golden hamster (Mesocricetus auratus) in which lethality and disease are dependent upon both the inbred hamster strain and the LCMV strain. Young adult inbred, male and female, hamsters were tested for lethal-disease susceptibility by lymphocytic choriomeningitis virus (LCMV) strains, WE or Armstrong (ARM). With WE inocula, PD4 and MHA inbred hamsters were highly susceptible to a wasting disease. LVG and LHC inbred hamsters were intermediate in susceptibility; some of these animals died of wasting illness, and others exhibited minimal disease and survived. CB and LSH hamsters were highly resistant to any disease by WE. Mean survival times of susceptible hamsters given lethal WE inocula approximated 2.5 weeks and were not dependent on virus dose. By 1.5 weeks after WE inoculation wasting disease signs were notable and consisted of lethargy, progressive body weight loss, and diarrhea. The LCMV strain, ARM, was avirulent for all hamster strains, causing neither death nor disease. Hamsters surviving WE or ARM inoculation appeared healthy, produced LCMV antibody, and acquired resistance to further lethal WE challenge. Despite hamster-lethality differences. WE and ARM appeared comparably immunogenic for all hamster strains, based on host antibody titers. A number of other differences between the LCMV strains were, however, noted which could be relevant to virus virulence and lethality for hamster hosts. These included guinea pig lethality, temperature sensitivity, and plaque morphology.     

Development of A MERS-CoV Replicon Cell Line for Antiviral Screening

Middle East respiratory syndrome coronavirus (MERS-CoV) is the causative agent of a severe respiratory disease with a high mortality of ~ 35%. The lack of approved treatments for MERS-CoV infection underscores the need for a user-friendly system for rapid drug screening. In this study, we constructed a MERS-CoV replicon containing the Renilla luciferase (Rluc) reporter gene and a stable luciferase replicon-carrying cell line. Using this cell line, we showed that MERS-CoV replication was inhibited by combined application of lopinavir and ritonavir, indicating that this cell line can be used to screen inhibitors of MERS-CoV replication. Importantly, the MERS-replicon cell line can be used for high-throughput screening of antiviral drugs without the need for live virus handling, providing an effective and safe tool for the discovery of antiviral drugs against MERS-CoV.     

Foodborne Transmission of Nipah Virus in Syrian Hamsters

Since 2001, outbreaks of Nipah virus have occurred almost every year in Bangladesh with high case-fatality rates. Epidemiological data suggest that in Bangladesh, Nipah virus is transmitted from the natural reservoir, fruit bats, to humans via consumption of date palm sap contaminated by bats, with subsequent human-to-human transmission. To experimentally investigate this epidemiological association between drinking of date palm sap and human cases of Nipah virus infection, we determined the viability of Nipah virus (strain Bangladesh/200401066) in artificial palm sap. At 22°C virus titers remained stable for at least 7 days, thus potentially allowing food-borne transmission. Next, we modeled food-borne Nipah virus infection by supplying Syrian hamsters with artificial palm sap containing Nipah virus. Drinking of 5×10⁸ TCID₅₀ of Nipah virus resulted in neurological disease in 5 out of 8 hamsters, indicating that food-borne transmission of Nipah virus can indeed occur. In comparison, intranasal (i.n.) inoculation with the same dose of Nipah virus resulted in lethal respiratory disease in all animals. In animals infected with Nipah virus via drinking, virus was detected in respiratory tissues rather than in the intestinal tract. Using fluorescently labeled Nipah virus particles, we showed that during drinking, a substantial amount of virus is deposited in the lungs, explaining the replication of Nipah virus in the respiratory tract of these hamsters. Besides the ability of Nipah virus to infect hamsters via the drinking route, Syrian hamsters infected via that route transmitted the virus through direct contact with naïve hamsters in 2 out of 24 transmission pairs. Although these findings do not directly prove that date palm sap contaminated with Nipah virus by bats is the origin of Nipah virus outbreaks in Bangladesh, they provide the first experimental support for this hypothesis. Understanding the Nipah virus transmission cycle is essential for preventing and mitigating future outbreaks.     

Severe Acute Respiratory Syndrome Coronavirus 2: Manifestations of Disease and Approaches to Treatment and Prevention in Humans

The coronavirus disease 2019 (COVID-19) pandemic was caused by a novel coronavirus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). This virus has challenged civilization and modern science in ways that few infectious diseases and natural disasters have previously, causing globally significant human morbidity and mortality and triggering economic downturns across financial markets that will be dealt with for generations. Despite this, the pandemic has also brought an opportunity for humanity to come together and participate in a shared scientific investigation. Clinically, SARS-CoV-2 is associated with lower mortality rates than other recently emerged coronaviruses, such as SARS-CoV and the Middle East respiratory syndrome coronavirus (MERS-CoV). However, SARS-CoV-2 exhibits efficient human-to-human spread, with transmission often occurring before symptom recognition; this feature averts containment strategies that had worked previously for SARS-CoV and MERS-CoV. Severe COVID-19 disease is characterized by dysregulated inflammatory responses associated with pulmonary congestion and intravascular coagulopathy leading to pneumonia, vascular insults, and multiorgan disease. Approaches to treatment have combined supportive care with antivirals, such as remdesivir, with immunomodulatory medications, including corticosteroids and cytokine-blocking antibody therapies; these treatments have advanced rapidly through clinical trials. Innovative approaches to vaccine development have facilitated rapid advances in design, testing, and distribution. Much remains to be learned about SARS-CoV-2 and COVID-19, and further biomedical research is necessary, including comparative medicine studies in animal models. This overview of COVID-19 in humans will highlight important aspects of disease, relevant pathophysiology, underlying immunology, and therapeutics that have been developed to date.

In December 2019, a cluster of cases of pneumonia without a clear etiology occurred in Wuhan, China. With remarkable speed and efficiency, the etiology of this illness was soon identified as a novel coronavirus; the complete viral genome was sequenced and published on January 10, 2020.¹⁸² These events introduced the world to coronavirus disease 2019 (COVID-19). The disease, now known to be caused by a novel coronavirus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has developed into the most significant pandemic of recent times. In less than a year since the virus was first recognized, multiple candidate vaccines were developed worldwide, and some of them rapidly progressed to clinical trials and widespread administration. As the pandemic continues, a number of sequence variants of the virus have emerged around the world. This continued viral evolution highlights the need for continued biomedical research to facilitate understanding of the pathogenesis of COVID-19, seeking innovative therapeutic and preventative strategies for the current and possibly future pandemics. This article will review aspects of SARS-CoV-2 infection of humans and COVID-19, focusing on important aspects of clinical disease, pathophysiology, immunology, and the development of therapeutic and preventative measures to provide context for discussion of the animal models used to study SARS-CoV-2 and COVID-19.     

A Lethal Disease Model for Hantavirus Pulmonary Syndrome in Immunosuppressed Syrian Hamsters Infected with Sin Nombre Virus

Sin Nombre virus (SNV) is a rodent-borne hantavirus that causes hantavirus pulmonary syndrome (HPS) predominantly in North America. SNV infection of immunocompetent hamsters results in an asymptomatic infection; the only lethal disease model for a pathogenic hantavirus is Andes virus (ANDV) infection of Syrian hamsters. Efforts to create a lethal SNV disease model in hamsters by repeatedly passaging virus through the hamster have demonstrated increased dissemination of the virus but no signs of disease. In this study, we demonstrate that immunosuppression of hamsters through the administration of a combination of dexamethasone and cyclophosphamide, followed by infection with SNV, results in a vascular leak syndrome that accurately mimics both HPS disease in humans and ANDV infection of hamsters. Immunosuppressed hamsters infected with SNV have a mean number of days to death of 13 and display clinical signs associated with HPS, including pulmonary edema. Viral antigen was widely detectable throughout the pulmonary endothelium. Histologic analysis of lung sections showed marked inflammation and edema within the alveolar septa of SNV-infected hamsters, results which are similar to what is exhibited by hamsters infected with ANDV. Importantly, SNV-specific neutralizing polyclonal antibody administered 5 days after SNV infection conferred significant protection against disease. This experiment not only demonstrated that the disease was caused by SNV, it also demonstrated the utility of this animal model for testing candidate medical countermeasures. This is the first report of lethal disease caused by SNV in an adult small-animal model.     

Coronaviruses: Important Emerging Human Pathogens

The identification of Middle East respiratory syndrome coronavirus (MERS-CoV) in 2012 reaffirmed the importance of understanding how coronaviruses emerge, infect, and cause disease. By comparing what is known about severe acute respiratory syndrome coronavirus (SARS-CoV) to what has recently been found for MERS-CoV, researchers are discovering similarities and differences that may be important for pathogenesis. Here we discuss what is known about each virus and what gaps remain in our understanding, especially concerning MERS-CoV.     

A novel human coronavirus: Middle East respiratory syndrome human coronavirus

In 2012,a novel coronavirus,initially named as human coronavirus EMC(HCoV-EMC) but recently renamed as Middle East respiratory syndrome human coronavirus(MERS-CoV),was identified in patients who suffered severe acute respiratory infection and subsequent renal failure that resulted in death.Ongoing epidemiological investigations together with retrospective studies have found 61 laboratory-confirmed cases of infection with this novel coronavirus,including 34 deaths to date.This novel coronavirus is culturable and two complete genome sequences are now available.Furthermore,molecular detection and indirect immunofluorescence assay have been developed.The present paper summarises the limited recent advances of this novel human coronavirus,including its discovery,genomic characterisation and detection.     

A global treatments for coronaviruses including COVID-19

In late December 2019 in Wuhan, China, several patients with viral pneumonia were identified as 2019 novel coronavirus (2019-nCoV). So far, there are no specific treatments for patients with coronavirus disease-19 (COVID-19), and the treatments available today are based on previous experience with similar viruses such as severe acute respiratory syndrome-related coronavirus (SARS-CoV), Middle East respiratory syndrome coronavirus (MERS-CoV), and Influenza virus. In this article, we have tried to reach a therapeutic window of drugs available to patients with COVID-19. Cathepsin L is required for entry of the 2019-nCoV virus into the cell as target teicoplanin inhibits virus replication. Angiotensin-converting-enzyme 2 (ACE2) in soluble form as a recombinant protein can prevent the spread of coronavirus by restricting binding and entry. In patients with COVID-19, hydroxychloroquine decreases the inflammatory response and cytokine storm, but overdose causes toxicity and mortality. Neuraminidase inhibitors such as oseltamivir, peramivir, and zanamivir are invalid for 2019-nCoV and are not recommended for treatment but protease inhibitors such as lopinavir/ritonavir (LPV/r) inhibit the progression of MERS-CoV disease and can be useful for patients of COVID-19 and, in combination with Arbidol, has a direct antiviral effect on early replication of SARS-CoV. Ribavirin reduces hemoglobin concentrations in respiratory patients, and remdesivir improves respiratory symptoms. Use of ribavirin in combination with LPV/r in patients with SARS-CoV reduces acute respiratory distress syndrome and mortality, which has a significant protective effect with the addition of corticosteroids. Favipiravir increases clinical recovery and reduces respiratory problems and has a stronger antiviral effect than LPV/r. currently, appropriate treatment for patients with COVID-19 is an ACE2 inhibitor and a clinical problem reducing agent such as favipiravir in addition to hydroxychloroquine and corticosteroids.     

Laboratory Investigation and Phylogenetic Analysis of an Imported Middle East Respiratory Syndrome Coronavirus Case in Greece

Rapid and reliable laboratory diagnosis of persons suspected of Middle East respiratory syndrome coronavirus (MERS-CoV) infection is important for timely implementation of infection control practices and disease management. In addition, monitoring molecular changes in the virus can help elucidate chains of transmission and identify mutations that might influence virus transmission efficiency. This was illustrated by a recent laboratory investigation we conducted on an imported MERS-CoV case in Greece. Two oropharyngeal swab specimens were collected on the 1^st and 2^nd day of patient hospitalization and tested using two real-time RT-PCR (rRT-PCR) assays targeting the UpE and Orf-1a regions of the MERS-CoV genome and RT-PCR and partial sequencing of RNA-dependent RNA polymerase and nucleocapsid genes. Serum specimens were also collected and serological test were performed. Results from the first swab sample were inconclusive while the second swab was strongly positive for MERS-CoV RNA by rRT-PCR and confirmed positive by RT-PCR and partial gene sequencing. Positive serologic test results further confirmed MERS-CoV infection. Full-length nucleocapsid and spike gene coding sequences were later obtained from the positive swab sample. Phylogenetic analysis revealed that the virus was closely related to recent human-derived MERS-CoV strains obtained in Jeddah and Makkah, Saudi Arabia, in April 2014 and dromedary camels in Saudi Arabia and Qatar. These findings were consistent with the patient’s history. We also identified a unique amino acid substitution in the spike receptor binding domain that may have implications for receptor binding efficiency. Our initial inconclusive rRT-PCR results highlight the importance of collecting multiple specimens from suspect MERS-CoV cases and particularly specimens from the lower respiratory tract.     

Middle East Respiratory Syndrome Coronavirus Spike Protein Delivered by Modified Vaccinia Virus Ankara Efficiently Induces Virus-Neutralizing Antibodies

Middle East respiratory syndrome coronavirus (MERS-CoV) has recently emerged as a causative agent of severe respiratory disease in humans. Here, we constructed recombinant modified vaccinia virus Ankara (MVA) expressing full-length MERS-CoV spike (S) protein (MVA-MERS-S). The genetic stability and growth characteristics of MVA-MERS-S make it a suitable candidate vaccine for clinical testing. Vaccinated mice produced high levels of serum antibodies neutralizing MERS-CoV. Thus, MVA-MERS-S may serve for further development of an emergency vaccine against MERS-CoV.     

Serological Evidence of MERS-CoV Antibodies in Dromedary Camels (Camelus dromedaries) in Laikipia County,Kenya

Middle East respiratory syndrome coronavirus (MERS-CoV) is a recently identified virus causing severe viral respiratory illness in people. Little is known about the reservoir in the Horn of Africa. In Kenya, where no human MERS cases have been reported, our survey of 335 dromedary camels, representing nine herds in Laikipia County, showed a high seroprevalence (46.9%) to MERS-CoV antibodies. Between herd differences were present (14.3%– 82.9%), but was not related to management type or herd isolation. Further research should focus on identifying similarity between MERS-CoV viral isolates in Kenya and clinical isolates from the Middle East and elsewhere.     

Dehydroepiandrosterone-induced reduction of Cryptosporidium parvum infections in aged Syrian golden hamsters.

Cryptosporidiosis, a parasitic disorder caused by Cryptosporidium parvum, is frequently a fulminating and life-threatening disease in immunocompromised hosts. The immune status of the host plays a critical role in determining the length and severity of the disease. Dehydroepiandrosterone (DHEA) is an immunomodulator that has been demonstrated to upregulate immune parameters. Ten aged (20-24 mo) Syrian golden hamsters were treated with DHEA for 7 days prior to intragastric inoculation with 1 x 10(6) C. parvum oocysts. An additional 10 aged hamsters were infected similarly but retained as untreated controls. The untreated hamsters presented with generalized infections as determined by oocyst shedding in the feces and parasite colonization of the small intestine. Hamsters treated with DHEA exhibited a significant reduction in cryptosporidial infection when compared to untreated hamsters. These results suggest that DHEA may be an effective prophylactic agent for cryptosporidiosis in immunocompromised patients.     

Middle East Respiratory Syndrome Coronavirus Infection Mediated by the Transmembrane Serine Protease TMPRSS2

The Middle East respiratory syndrome coronavirus (MERS-CoV) utilizes host proteases for virus entry into lung cells. In the current study, Vero cells constitutively expressing type II transmembrane serine protease (Vero-TMPRSS2 cells) showed larger syncytia at 18 h after infection with MERS-CoV than after infection with other coronaviruses. Furthermore, the susceptibility of Vero-TMPRSS2 cells to MERS-CoV was 100-fold higher than that of non-TMPRSS2-expressing parental Vero cells. The serine protease inhibitor camostat, which inhibits TMPRSS2 activity, completely blocked syncytium formation but only partially blocked virus entry into Vero-TMPRSS2 cells. Importantly, the coronavirus is thought to enter cells via two distinct pathways, one mediated by TMPRSS2 at the cell surface and the other mediated by cathepsin L in the endosome. Simultaneous treatment with inhibitors of cathepsin L and TMPRSS2 completely blocked virus entry into Vero-TMPRSS2 cells, indicating that MERS-CoV employs both the cell surface and the endosomal pathway to infect Vero-TMPRSS2 cells. In contrast, a single camostat treatment suppressed MERS-CoV entry into human bronchial submucosal gland-derived Calu-3 cells by 10-fold and virus growth by 270-fold, although treatment with both camostat and (23,25)-trans-epoxysuccinyl-l-leucylamindo-3-methylbutane ethyl ester, a cathepsin inhibitor, or treatment with leupeptin, an inhibitor of cysteine, serine, and threonine peptidases, was no more efficacious than treatment with camostat alone. Further, these inhibitors were not efficacious against MERS-CoV infection of MRC-5 and WI-38 cells, which were derived from lung, but these characters differed from those of mature pneumocytes. These results suggest that a single treatment with camostat is sufficient to block MERS-CoV entry into a well-differentiated lung-derived cell line.