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OBJECTIVE: Lopinavir/ritonavir has been used for the treatment of Severe Acute Respiratory Syndrome (SARS) and Middle East Respiratory Syndrome (MERS) coronavirus infections. It has been suggested that, based on this experience, this drug should also be studied in SARS-CoV2 infection. MATERIALS AND METHODS: We performed a systematic review of the literature regarding the use of lopinavir/ritonavir for the treatment of these three infections. We systematically searched the PubMed database from inception to April 30th, 2020, to identify in-vitro and animal studies and any reports of human use of lopinavir/ritonavir for the treatment of SARS, MERS and COVID-19. We also searched the Clinicatrial.gov to identify ongoing trials. RESULTS: Five in-vitro studies evaluated the effect of lopinavir/ritonavir in SARS. Three additional in-vitro studies reported the EC50 of the antiviral activity of lopinavir/ritonavir in MERS. We identified no in vitro studies evaluating the effect of lopinavir/ritonavir on the novel coronavirus. Two retrospective matched-cohort studies reported the use of lopinavir/ritonavir in combination with ribavirin for SARS patients. Three case reports and one retrospective study described the use of lopinavir/ritonavir in MERS. Twenty-two papers describe the use of lopinavir/ritonavir in adult patients with COVID-19. CONCLUSIONS: The existing literature does not suffice for assessing whether Lopinavir/ ritonavir has any benefit in SARS, MERS or COVID-19.

Due to their need for living cells, viruses have developed adaptive evolutionary strategies to survive and perpetuate in reservoir hosts that play a crucial role in the ecology of emerging pathogens. Pathogenic and potentially pandemic betacoronaviruses arose in humans in 2002 (SARS-CoV, disappeared in July 2003), 2012 (MERS-CoV, still circulating in Middle East areas), and 2019 (SARS-CoV-2, causing the current global pandemic). As universally recognized, bats host ancestors of the above-mentioned zoonotic viruses. However, hedgehogs have been recently identified in Europe and Asia as possible reservoirs of MERS-CoV-like strains classified as Erinaceus coronavirus (EriCoV). To elucidate the evolution and genetics of EriCoVs, NGS (next generation sequencing) and Sanger sequencing were used to examine fecal samples collected in Northern Italy in 2018/2019 from 12 hedgehogs previously found EriCoV-positive by RT-PCR. By sequence analysis, eight complete EriCoV genomes, obtained by NGS, showed a high phylogenetic correlation with EriCoV strains previously reported in Eurasia. Interestingly, eight viral strains presented an additional ORF encoding for the CD200 ortholog located between the genes encoding for the Spike and the ORF3a proteins. The CD200 ortholog sequences were closely similar to the host CD200 protein but varying among EriCoVs. The result, confirmed by Sanger sequencing, demonstrates for the first time that CoVs can acquire host genes potentially involved in the immune-modulatory cascade and possibly enabling the virus to escape the host defence.

The emergence of severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2), the etiological agent of the 2019 coronavirus disease (COVID-19), has erupted into a global pandemic that has led to tens of millions of infections and hundreds of thousands of deaths worldwide. The development of therapeutics to treat infection or as prophylactics to halt viral transmission and spread is urgently needed. SARS-CoV-2 relies on structural rearrangements within a spike (S) glycoprotein to mediate fusion of the viral and host cell membranes. Here, we describe the development of a lipopeptide that is derived from the C-terminal heptad repeat (HRC) domain of SARS-CoV-2 S that potently inhibits infection by SARS-CoV-2. The lipopeptide inhibits cell-cell fusion mediated by SARS-CoV-2 S and blocks infection by live SARS-CoV-2 in Vero E6 cell monolayers more effectively than previously described lipopeptides. The SARS-CoV-2 lipopeptide exhibits broad-spectrum activity by inhibiting cell-cell fusion mediated by SARS-CoV-1 and Middle East respiratory syndrome coronavirus (MERS-CoV) and blocking infection by live MERS-CoV in cell monolayers. We also show that the SARS-CoV-2 HRC-derived lipopeptide potently blocks the spread of SARS-CoV-2 in human airway epithelial (HAE) cultures, an ex vivo model designed to mimic respiratory viral propagation in humans. While viral spread of SARS-CoV-2 infection was widespread in untreated airways, those treated with SARS-CoV-2 HRC lipopeptide showed no detectable evidence of viral spread. These data provide a framework for the development of peptide therapeutics for the treatment of or prophylaxis against SARS-CoV-2 as well as other coronaviruses. IMPORTANCE SARS-CoV-2, the causative agent of COVID-19, continues to spread globally, placing strain on health care systems and resulting in rapidly increasing numbers of cases and mortalities. Despite the growing need for medical intervention, no FDA-approved vaccines are yet available, and treatment has been limited to supportive therapy for the alleviation of symptoms. Entry inhibitors could fill the important role of preventing initial infection and preventing spread. Here, we describe the design, synthesis, and evaluation of a lipopeptide that is derived from the HRC domain of the SARS-CoV-2 S glycoprotein that potently inhibits fusion mediated by SARS-CoV-2 S glycoprotein and blocks infection by live SARS-CoV-2 in both cell monolayers (in vitro) and human airway tissues (ex vivo). Our results highlight the SARS-CoV-2 HRC-derived lipopeptide as a promising therapeutic candidate for SARS-CoV-2 infections.

Middle East respiratory syndrome coronavirus (MERS-CoV) originated in bats and spread to humans via zoonotic transmission from camels. We analyzed the evolution of the spike (S) gene in betacoronaviruses (betaCoVs) isolated from different mammals, in bat coronavirus populations, as well as in MERS-CoV strains from the current outbreak. Results indicated several positively selected sites located in the region comprising the two heptad repeats (HR1 and HR2) and their linker. Two sites (R652 and V1060) were positively selected in the betaCoVs phylogeny and correspond to mutations associated with expanded host range in other coronaviruses. During the most recent evolution of MERS-CoV, adaptive mutations in the HR1 (Q/R/H1020) arose in camels or in a previous host and spread to humans. We determined that different residues at position 1020 establish distinct inter- and intra-helical interactions and affect the stability of the six-helix bundle formed by the HRs. A similar effect on stability was observed for a nearby mutation (T1015N) that increases MERS-CoV infection efficiency in vitro. Data herein indicate that the heptad repeat region was a major target of adaptive evolution in MERS-CoV-related viruses; these results are relevant for the design of fusion inhibitor peptides with antiviral function.