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Middle East respiratory syndrome coronavirus (MERS-CoV) is the cause of a severe respiratory disease with a high case fatality rate in humans. Since its emergence in mid-2012, 2578 laboratory-confirmed cases in 27 countries have been reported by the World Health Organization, leading to 888 known deaths due to the disease and related complications. Dromedary camels are considered the major reservoir host for this virus leading to zoonotic infection in humans. Dromedary camels, llamas, and alpacas are susceptible to MERS-CoV, developing a mild-to-moderate upper respiratory tract infection characterized by epithelial hyperplasia as well as infiltration of neutrophils, lymphocytes, and some macrophages within epithelium, lamina propria, in association with abundant viral antigen. The very mild lesions in the lower respiratory tract of these camelids correlate with absence of overt illness following MERS-CoV infection. Unfortunately, there is no approved antiviral treatment or vaccine for MERS-CoV infection in humans. Thus, there is an urgent need to develop intervention strategies in camelids, such as vaccination, to minimize virus spillover to humans. Therefore, the development of camelid models of MERS-CoV infection is key not only to assess vaccine prototypes but also to understand the biologic mechanisms by which the infection can be naturally controlled in these reservoir species. This review summarizes information on virus-induced pathological changes, pathogenesis, viral epidemiology, and control strategies in camelids, as the intermediate hosts and primary source of MERS-CoV infection in humans. info:eu-repo/semantics/acceptedVersion

Investigadors de la Unitat Mixta IRTA-UAB en Salut Animal han desenvolupat i validat un tècnica de cultiu de teixit respiratori de llama que permet estudiar per què els camèlids superen la infecció causada pel MERS-CoV sense desenvolupar signes clínics. Aquesta metodologia permetrà testar productes terapèutics per als humans front a aquest coronavirus, reduint així l'ús d'animals amb finalitats experimentals. Investigadores de la Unidad Mixta IRTA-UAB en Salud Animal han desarrollado y validado un técnica de cultivo de tejido respiratorio de llama que permite estudiar por qué los camélidos superan la infección causada por el MERS-CoV sin desarrollar signos clínicos. La metodología permitirá testar productos terapéuticos para humanos frente a este coronavirus, reduciendo así el uso de animales con fines experimentales. Researchers at the IRTA-UAB Joint Research Unit in Animal Health have developed and validated a technique for the culture of llama respiratory tissue that permits to study why camelids overcome infection caused by MERS-CoV without developing clinical signs. The methodology will allow to test therapeutic products for humans against this coronavirus, thus reducing the use of animals for experimental purposes.

Additional file 2: MERS-CoV N protein detection by IHC in ATE.

Conventional piglets were inoculated with severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) through different routes, including intranasal, intratracheal, intramuscular and intravenous ones. Although piglets were not susceptible to SARS‐CoV‐2 and lacked lesions or viral RNA in tissues/swabs, seroconversion was observed in pigs inoculated parenterally (intramuscularly or intravenously).

Conventional piglets were inoculated with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) through different routes, including intranasal, intratracheal, intramuscular and intravenous ones. Although piglets were not susceptible to SARS-CoV-2 and lacked lesions or viral RNA in tissues/swabs, seroconversion was observed in pigs inoculated parenterally (intramuscularly or intravenously).

Trabajo presentado en el European Congress of Virology, celebrado en Rotterdam (Países Bajos), del 28 de abril al 1 de mayo de 2019, Introduction: With the ongoing outbreaks and 35% case fatality rate, Middle East respiratory syndrome coronavirus (MERS-CoV), continues to pose a global threat. However, licensed vaccines and therapeutics for MERS-CoV, including monoclonal antibodies, are not currently available. Camelids have unique heavy-chain-only antibodies with long finger-like complementary determining regions allowing accessibility to epitopes inaccessible to conventional antibodies. The variable domain of these antibodies (VHHs), known as nanobodies, have small size, high antigen affinity and high homology to human counterparts, making them potential candidates for clinical use. Aim: We aimed at generating MERS-CoV-specific VHHs targeting different epitopes within the MERS-CoV spike protein to prevent viral neutralization escape and provide efficient protection against viral infection. Methods: Using bone marrow from MERS-CoV vaccinated dromedary camels, we cloned and expressed the VHH repertoire in E. coli. From this library, we screened for and characterized MERS-CoV-specific VHHs targeting different domains on the vial spike protein. We selected potent VHHs and fused them to a human Fc, generating camel/human monoand bi-specific chimeric heavy-chain-only antibodies (cHCAbs). Results: We identified several potent MERS-CoV-specific VHHs targeting the viral spike receptor binding domain (RBD) and the sialic acid binding (S1A ) domain, thereby blocking virus entry and attachment, respectively. Those with the highest potencies were selected and fused to human Fc to extend their half-life in vivo. The cHCAbs directed against the RBD were capable of blocking MERS-CoV entry in vitro and in vivo. S1A -directed cHCAbs blocked virus attachment to sialic acids and reduced infection in vitro. Different combinations of cHCAbs and bi-specific cHCAbs were evaluated for their protective efficacy against different virus variants and escape mutants. Conclusion: The pathogenesis of a viral disease is the biological mechanism, or mechanisms, that lead to the diseased state. There are several possibilities for comparing the pathogenesis of viral diseases among host species: a viral disease, e.g. rabies, that naturally infects different host species; a viral disease in an experimentally infected laboratory animal species, e.g. avian influenza in ferrets, as a model for the disease in humans; an emerging viral disease, e.g. Marburg haemorrhagic fever, in its reservoir species and its new host species. We here focus on the latter possibility. Many emerging viral diseases in a given host species originate from another host species, which acts as a reservoir. While the clinical outcome of emerging virus infections ranges from subclinical to fatal, we usually focus on those virus infections that cause severe disease in the new host species. Currently, research concentrates on the pathogenesis of such a viral disease in its new host species or, if that is not possible, in a laboratory animal species acting as a model. However, research on the pathogenesis of emerging viral diseases in their reservoir host species is neglected. This is a pity, because there are several good reasons to compare the pathogenesis of emerging viral diseases between the new host species and the reservoir species. First, pathogenesis in the reservoir host species may predict several types of virus-host interaction in the new host species. Second, differences in the pathogenesis between reservoir host species and new host species may provide clues for the mechanism of increased severity of disease in the new host species. Third, pathogenesis in the reservoir host species may predict the future evolution of the emerging virus in the new host species. Therefore, we advocate an increased emphasis on understanding the pathogenesis of emerging viral diseases in their reservoir species.

The ongoing Middle East respiratory syndrome coronavirus (MERS-CoV) outbreaks pose a worldwide public health threat. Blocking MERS-CoV zoonotic transmission from dromedary camels, the animal reservoir, could potentially reduce the number of primary human cases. Here we report MERS-CoV transmission from experimentally infected llamas to naïve animals. Directly inoculated llamas shed virus for at least 6 days and could infect all in-contact naïve animals 4-5 days after exposure. With the aim to block virus transmission, we examined the efficacy of a recombinant spike S1-protein vaccine. In contrast to naïve animals, in-contact vaccinated llamas did not shed infectious virus upon exposure to directly inoculated llamas, consistent with the induction of strong virus neutralizing antibody responses. Our data provide further evidence that vaccination of the reservoir host may impede MERS-CoV zoonotic transmission to humans. info:eu-repo/semantics/publishedVersion

Dromedary camel heavy chain–only antibodies may provide novel intervention strategies against MERS coronavirus., Middle East respiratory syndrome coronavirus (MERS-CoV) continues to cause outbreaks in humans as a result of spillover events from dromedaries. In contrast to humans, MERS-CoV–exposed dromedaries develop only very mild infections and exceptionally potent virus-neutralizing antibody responses. These strong antibody responses may be caused by affinity maturation as a result of repeated exposure to the virus or by the fact that dromedaries—apart from conventional antibodies—have relatively unique, heavy chain–only antibodies (HCAbs). These HCAbs are devoid of light chains and have long complementarity-determining regions with unique epitope binding properties, allowing them to recognize and bind with high affinity to epitopes not recognized by conventional antibodies. Through direct cloning and expression of the variable heavy chains (VHHs) of HCAbs from the bone marrow of MERS-CoV–infected dromedaries, we identified several MERS-CoV–specific VHHs or nanobodies. In vitro, these VHHs efficiently blocked virus entry at picomolar concentrations. The selected VHHs bind with exceptionally high affinity to the receptor binding domain of the viral spike protein. Furthermore, camel/human chimeric HCAbs—composed of the camel VHH linked to a human Fc domain lacking the CH1 exon—had an extended half-life in the serum and protected mice against a lethal MERS-CoV challenge. HCAbs represent a promising alternative strategy to develop novel interventions not only for MERS-CoV but also for other emerging pathogens.