Rift Valley fever (RVF) is an emerging zoonosis of major public health concern in Africa and Arabia. Previous outbreaks attributed camelids a significant role in the epidemiology of Rift Valley fever virus (RVFV), making them an important target species for vaccination. Using three alpacas as model-organisms for dromedary camels, the safety, immunogenicity and pathogenicity of the MP-12 vaccine were evaluated inthis study. To compare both acute and subacute effects, animals were euthanized at 3 and 31days post infection (dpi). Clinical monitoring, analysis of liver enzymes and hematological parameters demonstrated the tolerability of the vaccine, as no significant adverse effects were observed. Comprehensive analysis of serological parameters illustrated the immunogenicity of the vaccine, eliciting high neutralizing antibody titers and antibodies targeting different viral antigens. RVFV was detected in serum and liver of the alpaca euthanized 3dpi, whereas no viruswas detectable at 31dpi. Viral replication was confirmed by detection of various RVFV-antigens in hepatocytes by immunohistochemistry and the presence of mild multifocal necrotizing hepatitis. In conclusion, results indicate that MP-12 is a promising vaccine candidate but still has a residual pathogenicity, which requires further investigation.
Antibodies specific for Rift Valley fever virus (RVFV) can be detected by diverse methods, including ezyme-linked immunosortbent assay (ELISA) and virus neutralization test (VNT). The VNT is superior in sensitivity and specificity and is therefore considered the gold standard serological assay. Classical VNTs make use of virulent RVFV and therefore have to be performed in biosafety level 3 laboratories. Here, we report the development of a novel VNT that is based on an avirulent RVFV expressing the enhanced green fluorescent protein (eGFP), which can be performed safely outside level 3 biocontainment facilities. Evaluation with a broad panel of experimental sera and field sera demonstrated that this novel VNT is faster and more sensitive than the classical VNT.
Orthobunyaviruses are enveloped viruses that can cause human and animal diseases. A novel and major member is the Schmallenberg virus(SBV), the etiological agent of an emerging disease of ruminants that has been spreading all over Europe since 2011. The glycoproteins Gn and Gc of orthobunyaviruses mediate the viral entry, and specifically Gc is a major target for the humoral immune response. For example, the N terminal subdomain of the SBV glycoprotein Gc is targeted by neutralizing monoclonal antibodies that recognize conformational epitopes. Here, we determined the structural features of the N terminus of Gc, and analysed its interaction with monoclonal antibodies. We were able to demonstrate that one of two N-glycosylation sites is essential for secretion and interaction with a subset of Gc-specific monoclonal antibodies. Furthermore, four disulfide bonds (S-S) were identified and the deletion of the third S-S blocked reactivity with another subset of mAbs with virus-neutralizing and non-neutralizing activity. The mutagenesis of the N-glycosylation sites and the disulfide bonds strongly indicated the independent folding of two subdomains within the SBV Gc N terminus. Further, the epitopes recognized by a panel of mAbs could be grouped into two clusters, as revealed by fine mapping using chimeric proteins. Combining the disulfide bonding and epitope mapping allowed us to generate a structural model of the SBV Gc N-terminus. This novel information about the role and structure of the amino terminal region of SBV Gc is of general relevance for the design of antivirals and vaccines against this virus.
Cell surface aminopeptidase N (APN) is a membrane-bound ectoenzyme that hydrolyzes proteins and peptides and regulates numerous cell functions. APN participates in tumor cell expansion and motility, and is a target for cancer therapies. Small drugs that bind to the APN active site inhibit catalysis and suppress tumor growth. APN is also a major cell entry receptor for coronavirus, which binds to a region distant from the active site. Three crystal structures that we determined of human and pig APN ectodomains defined the dynamic conformation of the protein. These structures offered snapshots of closed, intermediate and open APN, which represent distinct functional states. Coronavirus envelope proteins specifically recognized the open APN form, prevented ectodomain progression to the closed form and substrate hydrolysis. In addition, drugs that bind the active site inhibited both coronavirus binding to cell surface APN and infection; the drugs probably hindered APN transition to the virus-specific open form. We conclude that allosteric inhibition of APN functions occurs by ligand suppression of ectodomain motions necessary for catalysis and virus cell entry, as validated by locking APN with disulfides. Blocking APN dynamics can thus be a valuable approach to development of drugs that target this ectoenzyme.
Emerging and re-emerging pathogens represent a substantial threat to public health, as demonstrated with numerous outbreaks over the past years, including the 2013-2016 outbreak of Ebola virus in western Africa. Coronaviruses are also a threat for humans, as evidenced in2002/2003 with infection by the severe acute respiratory syndrome coronavirus (SARS-CoV), which caused more than 8000 human infections with 10% fatality rate in 37 countries. Ten years later, a novel human coronavirus (Middle East respiratory syndrome coronavirus, MERS-CoV), associated with severe pneumonia, arose in the Kingdom of Saudi Arabia. Until December 2016, MERS has accounted for more than 1800 cases and 35% fatality rate. Finding an animal model of disease is key to develop vaccines or antivirals against such emerging pathogens and to understand its pathogenesis. Knowledge of the potential role of domestic livestock and other animal species inthe transmission of pathogens is of importance to understand the epidemiology of the disease. Little is known about MERS-CoV animal host range. In this paper, experimental data on potential hosts for MERS-CoV is reviewed. Advantages and limitations of different animal models are evaluated in relation to viral pathogenesis and transmission studies. Finally, the relevance of potential new target species is discussed.
Dromedary camels are the main reservoir of Middle East respiratory syndrome coronavirus (MERS-CoV), but other livestock species (i.e., alpacas, llamas, and pigs) are also susceptible to infection with MERS-CoV. Animal-to-animal transmission in alpacas was reported, but evidence for transmission in other species has not been proved. This study explored pig-to-pig MERS-CoV transmission experimentally. Virus was present in nasal swabs of infected animals, and limited amounts of viral RNA, but no infectious virus were detected in the direct contact pigs. No virus was detected in the indirect contact group. Furthermore, direct and indirect contact pigs did not develop specific antibodies against MERS-CoV. Therefore, the role of pigs as reservoir is probably negligible, although it deserves further confirmation.
Severe acute respiratory syndrome coronavirus (SARS-CoV) causes lethal disease in humans, which is characterized by exacerbated inflammatory response and extensive lung pathology. To address the relevance of small non-coding RNAs in SARS-CoV pathology, we deep sequenced RNAs from the lungs of infected mice and discovered three 18–22 nt small viral RNAs (svRNAs). The three svRNAs were derived from the nsp3 (svRNA-nsp3.1 and -nsp3.2) and N (svRNA-N) genomic regions of SARS-CoV. Biogenesis of CoV svRNAs was RNase III, cell type, and host species independent, but it was dependent on the extent of viral replication. Antagomir-mediated inhibition of svRNA-N significantly reduced in vivo lung pathology and pro-inflammatory cytokine expression. Taken together, these data indicate that svRNAs contribute to SARS-CoV pathogenesis and highlight the potential of svRNA-N antagomirs as antivirals.
Middle East respiratory syndrome coronavirus (MERS-CoV) is a cause of severe respiratory infection in humans, specifically the elderly and people with comorbidities. The re-emergence of lethal coronaviruses calls for international collaboration to produce coronavirus vaccines, which are still lacking to date. Ongoing efforts to develop MERS-CoV vaccines should consider the different target populations (dromedary camels and humans) and the correlates of protection. Extending on our current knowledge of MERS, vaccination of dromedary camels to induce mucosal immunity could be a promising approach to diminish MERS-CoV transmission to humans. In addition, it is equally important to develop vaccines for humans that induce broader reactivity against various coronaviruses to be prepared for a potential next CoV outbreak.
Dromedary camels from Africa and Arabia are an established source for zoonotic Middle East respiratory syndrome coronavirus (MERS-CoV) infection among humans. In Pakistan, we found specific neutralizing antibodies in samples from 39.5% of 565 dromedaries, documenting significant expansion of the enzootic range of MERS-CoV to Asia.
Middle East respiratory syndrome (MERS) cases continue to be reported, predominantly in Saudi Arabia and occasionally other countries. Although dromedaries are the main reservoir, other animal species might be susceptible to MERS coronavirus (MERS-CoV) infection and potentially serve as reservoirs. To determine whether other animals are potential reservoirs, we inoculated MERS-CoV into llamas, pigs, sheep, and horses and collected nasal and rectal swab samples at various times. The presence of MERS-CoV in the nose of pigs and llamas was confirmed by PCR, titration of infectious virus, immunohistochemistry, and in situ hybridization; seroconversion was detected in animals of both species. Conversely, in sheep and horses, virus-specific antibodies did not develop and no evidence of viral replication in the upper respiratory tract was found. These results prove the susceptibility of llamas and pigs to MERS-CoV infection. Thus, the possibility of MERS-CoV circulation in animals other than dromedaries, such as llamas and pigs, is not negligible.