A large-scale serological survey in pets from October 2020 through June 2021 in France shows significantly higher exposure to SARS-CoV-2 in cats (preprint)

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) can infect many animals, including pets such as dogs and cats. Many studies have documented infection in companion animals by bio-molecular and serological methods. However, only a few have compared seroprevalence in cats and dogs from the general population, and these studies were limited by small sample sizes and collections over short periods. Our goal was to obtain a more accurate evaluation of seroprevalence in companion animals in France and to determine whether cats and dogs differ in their exposure to SARS-CoV-2. For this purpose, we conducted an extensive SARS-CoV-2 serological survey of 2036 cats and 3577 dogs sampled by veterinarians during medical examinations in clinics throughout France. Sampling was carried out from October 2020 through June 2021, a period encompassing the second and third waves of SARS-CoV-2 infections in humans in the country. Using a microsphere immunoassay targeting receptor binding domain and trimeric spike protein, we found 7.1% seroprevalence in pets, in a subset of 308 seropositive samples, 26.3% had neutralizing antibodies. We found that cats were significantly more likely to test positive than dogs, with seropositivity rates of 9.3% and 5.9% in cats and dogs, respectively. Finally, data for both species showed that seroprevalence was lower in older animals and was not associated with the date of sampling or the sex of the animal. Our results show that cats are significantly more sensitive to SARS-CoV-2 than dogs, in line with experimental studies showing that cats are more susceptible than dogs. This study reinforces that pets are commonly infected or exposed to SARS-CoV-2, emphasizing the importance of a One-Health approach to the SARS-CoV-2 pandemic and raising the question of vaccination of companion animals in close contact with humans.

Immunogenicity and efficacy of heterologous ChadOx1/BNT162b2 vaccination (preprint)

Following severe adverse reactions in patients vaccinated with the AstraZeneca ChadOx1 (Chad) vaccine, European health authorities have recommended that patients under the age of 55 who received one dose of Chad vaccine receive a second dose of Pfizer BNT162b2 (BNT) vaccine as a booster. However, the effectiveness and the immunogenicity of this vaccination regimen have not been formally tested. Here, we show that the heterologous Chad/BNT combination confers better protection against SARS-CoV-2 infection than the homologous BNT/BNT combination in a population of health care workers. To understand the underlying mechanism, we monitored in a longitudinal way the anti-spike immunity conferred by each vaccinal combination. Both combinations induced strong anti-spike antibody responses after boost in all vaccinated individuals. However, sera from heterologous vaccinated individuals displayed a stronger neutralizing activity, regardless of the SARS-CoV-2 variant analyzed, and this was associated with more switched memory RBD-specific B cells with an activated phenotype and less IgA. The Chad vaccine induced a stronger T cell response than the BNT vaccine after the priming dose, and the reciprocal was true for the IgG response, which could explain the complementarity of both vaccines when used in an heterologous setting. This strongly protective vaccination regimen could be therefore particularly suitable for immunocompromised individuals.

New detection of SARS-CoV-2 in two cats height months after COVID-19 outbreak appearance in France (preprint)

Although there are several reports in the literature of SARS-CoV-2 infection in cats, few SARS-CoV-2 sequences from infected cats have been published. In this report, SARS-CoV-2 infection was evaluated in two cats by clinical observation, molecular biology (qPCR and NGS), and serology (Microsphere immunoassay and seroneutralization). Following the observation of symptomatic SARS-CoV-2-infection in two cats, infection status was confirmed by RT-qPCR and, in one cat, serological analysis for antibodies against N-protein and S-protein, as well as neutralizing antibodies. Comparative analysis of five SARS-CoV-2 sequence-fragments obtained from one of the cats showed that this infection was not with one of the three recently emerged variants of SARS-CoV-2. This study provides additional information on the clinical, molecular, and serological aspects of SARS-CoV-2 infection in cats.

Myocarditis in naturally infected pets with the British variant of COVID-19 (preprint)

Domestic pets can contract SARS-CoV-2 infection but, based on the limited information available to date, it is unknown whether the new British B.1.1.7 variant can more easily infect certain animal species or increase the possibility of human-to-animal transmission. In this study, we report the first cases of infection of domestic cats and dogs by the British B.1.1.7 variant of SARS-CoV-2 diagnosed at a specialist veterinary hospital in the South-East of England. Furthermore, we discovered that many owners and handlers of these pets had developed Covid-19 respiratory symptoms 3-6 weeks before their pets became ill and had also tested PCR positive for Covid-19. Interestingly, all these B.1.1.7 infected pets developed atypical clinical manifestations, including severe cardiac abnormalities secondary to myocarditis and a profound impairment of the general health status of the patient but without any primary respiratory signs. Together, our findings demonstrate for the first time the ability for companion animals to be infected by the B.1.1.7 variant of SARS-CoV-2 and raise questions regarding its pathogenicity in these animals. Moreover, given the enhanced infectivity and transmissibility of B.1.1.7 variant for humans, these findings also highlights more than ever the risk that companion animals may potentially play a significant role in SARS-CoV-2 outbreak dynamics than previously appreciated.

Impact of the SARS-COV-2 Outbreak on Epidemiology and Management of Major Trauma in France: Registry-Based Study. The COVITRAUMA Study (preprint)

Background: Evidence increases to suggest that the reallocation of health care resources during considerable the COVID-19 pandemic impacts considerably any health system. This study describes the epidemiology and the outcome of major trauma patients admitted to centers in France during the first wave of the COVID-19 outbreak. MethodsThis retrospective observational study included all consecutive trauma patients aged 15 years and older admitted into 15 centers participating to the TraumaBase ® registry in France during the first wave of the SARS-CoV-2 pandemic in France. ResultsOver a 4 years-study period, 5762 patients were admitted between the first week of February and mid-June. This cohort was split between patients admitted during the first 2020 pandemic wave in France (pandemic period, 1314 patients) and those admitted during the corresponding period in the three previous years (2017-2019, 4448 patients). Patient demographics changed substantially during the pandemic and more specifically during the lockdown period specially with a reduction in both absolute numbers admitted and the proportion of road traffic accidents (348 annually 2017-2019 [55.4 % of trauma admissions] vs 143 [36.8 %] in 2020 p<0.005). Mortality during the pandemic period and the difference between predicted and observed mortality was not different compared to the non-pandemic years. ConclusionsDuring this first wave of COVID-19 in France, management of trauma patients admitted to regional Traumacenters was not significantly altered, despite medical resources being reallocated and reorganized. Mortality as well as prehospital and in hospital care remained stable throughout the period of the first pandemic wave despite a massive increase in demand for acute care beds.

Protonation states in SARS-CoV-2 main protease mapped by neutron crystallography (preprint)

The main protease (3CL Mpro) from SARS-CoV-2, the etiological agent of COVID-19, is an essential enzyme for viral replication, possessing an unusual catalytic dyad composed of His41 and Cys145. A long-standing question in the field has been what the protonation states of the ionizable residues in the substrate-binding active site cavity are. Here, we present the room-temperature neutron structure of 3CL Mpro from SARS-CoV-2, which allows direct determination of hydrogen atom positions and, hence, protonation states. The catalytic site natively adopts a zwitterionic reactive state where His41 is doubly protonated and positively charged, and Cys145 is in the negatively charged thiolate state. The neutron structure also identified the protonation states of other amino acid residues, mapping electrical charges and intricate hydrogen bonding networks in the SARS-CoV-2 3CL Mpro active site cavity and dimer interface. This structure highlights the ability of neutron protein crystallography for experimentally determining protonation states at near-physiological temperature -- the critical information for structure-assisted and computational drug design.

High prevalence of SARS-CoV-2 antibodies in pets from COVID-19+ households (preprint)

In a survey of household cats and dogs of laboratory-confirmed COVID-19 patients, we found a high seroprevalence of SARS-CoV-2 antibodies, ranging from 21% to 53%, depending on the positivity criteria chosen. Seropositivity was significantly greater among pets from COVID-19+ households compared to those with owners of unknown status. Our results highlight the potential role of pets in the spread of the epidemic.

Bat and pangolin coronavirus spike glycoprotein structures provide insights into SARS-CoV-2 evolution (preprint)

In recognizing the host cellular receptor and mediating fusion of virus and cell membranes, the spike (S) glycoprotein of coronaviruses is the most critical viral protein for cross-species transmission and infection. Here we determined the cryo-EM structures of the spikes from bat (RaTG13) and pangolin (PCoV_GX) coronaviruses, which are closely related to SARS-CoV-2. All three receptor-binding domains (RBDs) of these two spike trimers are in the "down" conformation, indicating they are more prone to adopt this receptor-binding inactive state. However, we found that the PCoV_GX, but not the RaTG13, spike is comparable to the SARS-CoV-2 spike in binding the human ACE2 receptor and supporting pseudovirus cell entry. Through structure and sequence comparisons, we identified critical residues in the RBD that underlie the different activities of the RaTG13 and PCoV_GX/SARS-CoV-2 spikes and propose that N-linked glycans serve as conformational control elements of the RBD. These results collectively indicate that strong RBD-ACE2 binding and efficient RBD conformational sampling are required for the evolution of SARS-CoV-2 to gain highly efficient infection.

A longitudinal study of SARS-CoV-2 infected patients shows high correlation between neutralizing antibodies and COVID-19 severity (preprint)

Understanding the immune responses elicited by SARS-CoV-2 infection is critical in terms of protection from re-infection and, thus, for public health policy and for vaccine development against the COVID-19. Here, using either live SARS-CoV-2 particles or retroviruses pseudotyped with the SARS-CoV-2 S viral surface protein (Spike), we studied the neutralizing antibody (nAb) response in serum specimens from a cohort of 140 SARS-CoV-2 qPCR-confirmed patients, including patient with mild symptoms but also more severe form including those that require intensive care. We show that nAb titers were strongly correlated with disease severity and with anti-Spike IgG levels. Indeed, patients from intensive care units exhibited high nAb titers, whereas patients with milder disease symptoms displayed heterogenous nAb titers and asymptomatic or exclusive outpatient care patients had no or poor nAb levels. We found that the nAb activity in SARS-CoV-2-infected patients displayed a relatively rapid decline after recovery, as compared to individuals infected with alternative coronaviruses. We show the absence of cross-neutralization between endemic coronaviruses and SARS-CoV-2, indicating that previous infection by human coronaviruses may not generate protective nAb against SARS-CoV-2 infection. Finally, we found that the D614G mutation in the Spike protein, which has recently been identified as the major variant now found in Europe, does not allow neutralization escape. Altogether, our results contribute to the understanding of the immune correlate of SARS-CoV-2 induced disease and claim for a rapid evaluation of the role of the humoral response in the pathogenesis of SARS-CoV-2.

The SARS-CoV-2 Envelope and Membrane proteins modulate maturation and retention of the Spike protein, allowing optimal formation of VLPs in presence of Nucleoprotein (preprint)

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), a {beta}-coronavirus, is the causative agent of the COVID-19 pandemic. Like for other coronaviruses, its particles are composed of four structural proteins, namely Spike S, Envelope E, Membrane M and Nucleoprotein N proteins. The involvement of each of these proteins and their interplays during the assembly process of this new virus are poorly-defined and are likely {beta}-coronavirus-type different. Therefore, we sought to investigate how SARS-CoV-2 behaves for its assembly by expression assays of S, in combination with E, M and/or N. By combining biochemical and imaging assays, we showed that E and M regulate intracellular trafficking of S and hence its furin-mediated processing. Indeed, our imaging data revealed that S remains at ERGIC or Golgi compartments upon expression of E or M, like for SARS-CoV-2 infected cells. By studying a mutant of S, we showed that its cytoplasmic tail, and more specifically, its C-terminal retrieval motif, is required for the M-mediated retention in the ERGIC, whereas E induces S retention by modulating the cell secretory pathway. We also highlighted that E and M induce a specific maturation of S N-glycosylation, which is observed on particles and lysates from infected cells independently of its mechanisms of intracellular retention. Finally, we showed that both M, E and N are required for optimal production of virus-like-proteins. Altogether, our results indicated that E and M proteins influence the properties of S proteins to promote assembly of viral particles. Our results therefore highlight both similarities and dissimilarities in these events, as compared to other {beta}-coronaviruses. Author SummaryThe severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of the COVID-19 pandemic. Its viral particles are composed of four structural proteins, namely Spike S, Envelope E, Membrane M and Nucleoprotein N proteins, though their involvement in the virion assembly remain unknown for this particular coronavirus. Here we showed that presence of E and M influence the localization and maturation of S protein, in term of cleavage and N-glycosylation maturation. Indeed, E protein is able to slow down the cell secretory pathway whereas M-induced retention of S requires the retrieval motif in S C-terminus. We also highlighted that E and M might regulate the N glycosylation maturation of S independently of its intracellular retention mechanism. Finally, we showed that the four structural proteins are required for optimal formation of virus-like particles, highlighting the involvement of N, E and M in assembly of infectious particles. Altogether, our results highlight both similarities and dissimilarities in these events, as compared to other {beta}-coronaviruses.