Early Circulation of SARS-CoV-2, Congo, 2020.

To determine when severe acute respiratory syndrome coronavirus 2 arrived in Congo, we retrospectively antibody tested 937 blood samples collected during September 2019-February 2020. Seropositivity significantly increased from 1% in December 2019 to 5.3% in February 2020, before the first officially reported case in March 2020, suggesting unexpected early virus circulation.

NS2 proteases from hepatitis C virus and related hepaciviruses share composite active sites and previously unrecognized intrinsic proteolytic activities.

Over the recent years, several homologues with varying degrees of genetic relatedness to hepatitis C virus (HCV) have been identified in a wide range of mammalian species. HCV infectious life cycle relies on a first critical proteolytic event of its single polyprotein, which is carried out by nonstructural protein 2 (NS2) and allows replicase assembly and genome replication. In this study, we characterized and evaluated the conservation of the proteolytic mode of action and regulatory mechanisms of NS2 across HCV and animal hepaciviruses. We first demonstrated that NS2 from equine, bat, rodent, New and Old World primate hepaciviruses also are cysteine proteases. Using tagged viral protein precursors and catalytic triad mutants, NS2 of equine NPHV and simian GBV-B, which are the most closely and distantly related viruses to HCV, respectively, were shown to function, like HCV NS2 as dimeric proteases with two composite active sites. Consistent with the reported essential role for NS3 N-terminal domain (NS3N) as HCV NS2 protease cofactor via NS3N key hydrophobic surface patch, we showed by gain/loss of function mutagenesis studies that some heterologous hepacivirus NS3N may act as cofactors for HCV NS2 provided that HCV-like hydrophobic residues are conserved. Unprecedently, however, we also observed efficient intrinsic proteolytic activity of NS2 protease in the absence of NS3 moiety in the context of C-terminal tag fusions via flexible linkers both in transiently transfected cells for all hepaciviruses studied and in the context of HCV dicistronic full-length genomes. These findings suggest that NS3N acts as a regulatory rather than essential cofactor for hepacivirus NS2 protease. Overall, unique features of NS2 including enzymatic function as dimers with two composite active sites and additional NS3-independent proteolytic activity are conserved across hepaciviruses regardless of their genetic distances, highlighting their functional significance in hepacivirus life cycle.

Evidence for circulation of Rift Valley fever virus in wildlife and domestic animals in a forest environment in Gabon, Central Africa.

Rift Valley fever (RVF) is a mosquito-borne viral zoonosis caused by the Rift Valley fever virus (RVFV) that can infect domestic and wild animals. Although the RVFV transmission cycle has been well documented across Africa in savanna ecosystems, little is known about its transmission in tropical rainforest settings, particularly in Central Africa. We therefore conducted a survey in northeastern Gabon to assess RVFV circulation among wild and domestic animals. Among 163 wildlife samples tested using RVFV-specific RT-qPCR, four ruminants belonging to subfamily Cephalophinae were detected positive. The phylogenetic analysis revealed that the four RVFV sequences clustered together with a virus isolated in Namibia within the well-structured Egyptian clade. A cross-sectional survey conducted on sheep, goats and dogs living in villages within the same area determined the IgG RVFV-specific antibody prevalence using cELISA. Out of the 306 small ruminants tested (214 goats, 92 sheep), an overall antibody prevalence of 15.4% (95% CI [11.5-19.9]) was observed with a higher rate in goats than in sheep (20.1% versus 3.3%). RVFV-specific antibodies were detected in a single dog out of the 26 tested. Neither age, sex of domestic animals nor season was found to be significant risk factors of RVFV occurrence. Our findings highlight sylvatic circulation of RVFV for the first time in Gabon. These results stress the need to develop adequate surveillance plan measures to better control the public health threat of RVFV.

Molecular Identification of Enteric Viruses in Domestic Animals in Northeastern Gabon, Central Africa.

Astroviruses (AstVs), enteroviruses (EVs), and caliciviruses (CaVs) infect several vertebrate taxa. Transmitted through the fecal-oral route, these enteric viruses are highly resistant and can survive in the environment, thereby increasing their zoonotic potential. Here, we screened for AstVs, EVs, and CaVs to investigate the role of domestic animals in the emergence of zoonoses, because they are situated at the human/wildlife interface, particularly in rural forested areas in Central Africa. Rectal swabs were obtained from 123 goats, 41 sheep, and 76 dogs in 10 villages located in northeastern Gabon. Extracted RNA reverse-transcribed into cDNA was used to detect AstVs, EVs, and CaVs by amplification of the RNA-dependent RNA polymerase (RdRp), or capsid protein (VP1) gene using PCR. A total of 23 samples tested positive, including 17 goats for AstVs, 2 goats, 2 sheep, 1 dog for EVs, and 1 dog for CaVs. Phylogenetic analyses revealed that AstV RdRp sequences clustered with sheep-, goat-, or bovine-related AstVs. In addition, one goat and two sheep VP1 sequences clustered with caprine/ovine-related Evs within the Enterovirus G species, and the CaV was a canine vesivirus. However, human-pathogenic Evs, EV-B80 and EV-C99, were detected in goats and dogs, raising questions on the maintenance of viruses able to infect humans.

Absence of Coronavirus RNA in Faecal Samples from Wild Primates in Gabon, Central Africa.

Coronaviruses (CoVs, Coronaviridae) are a diverse group of viruses that infect mammals, birds, and fish. Seven CoVs infect humans, among which Severe Acute Respiratory Syndrome CoVs-1 and -2 and Middle East respiratory syndrome CoVs have shown how they can impact global health and the economy. Their spillover from bats-the natural reservoir-to humans has required intermediary hosts. Prevention requires that active surveillance be conducted on animals. Today, there is no data concerning the genetic diversity of CoVs naturally circulating in wild primates. This study aimed to screen wild great apes and mandrills in Gabon for CoVs. A total of 229 faecal samples of great apes and mandrills collected from 2009 to 2012 in forests and national parks were used for the detection of CoVs by nested PCR using primers targeting a conserved region of the RNA-dependent RNA polymerase. While all samples were negative, this lack of detection could be related to sample size, the transient nature of the infection, or because faecal samples are not suitable for detecting CoVs in primates. A longitudinal study should be performed and other non-invasive methods used to collect respiratory samples to better evaluate the circulation of CoVs in these primates.

Whole-Genome Characterization of SARS-CoV-2 Reveals Simultaneous Circulation of Three Variants and a Putative Recombination (20B/20H) in Pets, Brazzaville, Republic of the Congo.

Following the emergence of SARS-CoV-2, cases of pets infected with variants circulating among humans were reported. In order to evaluate the occurrence of SARS-CoV-2 circulation among pets in the Republic of the Congo, we conducted a ten-month study of dogs and cats living in COVID-19-positive households in Brazzaville and neighboring localities. Real-time PCR and the Luminex platform were used to detect SARS-CoV-2 RNA and antibodies to SARS-CoV-2 RBD and S proteins, respectively. Our results show for the first time the simultaneous circulation of several variants of SARS-CoV-2, including viruses from clades 20A and 20H and a putative recombinant variant between viruses from clades 20B and 20H. We found a high seroprevalence of 38.6%, with 14% of tested pets positive for SARS-CoV-2 RNA. Thirty-four percent of infected pets developed mild clinical signs, including respiratory and digestive signs, and shed the virus for about one day to two weeks. These results highlight the potential risk of SARS-CoV-2 interspecies transmission and the benefits of a "One Health" approach that includes SARS-CoV-2 diagnosis and surveillance of viral diversity in pets. This approach aims to prevent transmission to surrounding wildlife as well as spillback to humans.

First Evidence of Natural SARS-CoV-2 Infection in Domestic Rabbits.

We tested 144 pet rabbits sampled in France between November 2020 and June 2021 for antibodies to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) by microsphere immunoassay. We reported the first evidence of a natural SARS-CoV-2 infection in rabbits with a low observed seroprevalence between 0.7% and 1.4%.

Detection of SARS-CoV-2 in two cats during the second wave of the COVID-19 pandemic in France.

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 study, 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.

Infection with SARS-CoV-2 variant B.1.1.7 detected in a group of dogs and cats with suspected myocarditis.

BACKGROUND: Domestic pets can contract severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection; however, it is unknown whether the UK B.1.1.7 variant can more easily infect certain animal species or increase the possibility of human-to-animal transmission. METHODS: This is a descriptive case series reporting SARS-CoV-2 B.1.1.7 variant infections in a group of dogs and cats with suspected myocarditis. RESULTS: The study describes the infection of domestic cats and dogs by the B.1.1.7 variant. Two cats and one dog were positive to SARS-CoV-2 PCR on rectal swab, and two cats and one dog were found to have SARS-CoV-2 antibodies 2-6 weeks after they developed signs of cardiac disease. Many owners of these pets had developed respiratory symptoms 3-6 weeks before their pets became ill and had also tested positive for COVID-19. Interestingly, all these pets were referred for acute onset of cardiac disease, including severe myocardial disorders of suspected inflammatory origin but without primary respiratory signs. CONCLUSIONS: These findings demonstrate, for the first time, the ability for pets to be infected by the B.1.1.7 variant and question its possible pathogenicity in these animals.

Report of One-Year Prospective Surveillance of SARS-CoV-2 in Dogs and Cats in France with Various Exposure Risks: Confirmation of a Low Prevalence of Shedding, Detection and Complete Sequencing of an Alpha Variant in a Cat.

Despite the probable zoonotic origin of SARS-CoV-2, only limited research efforts have been made to understand the role of companion animals in SARS-CoV-2 epidemiology. According to recent serological prevalence studies, human-to-companion animal transmission is quite frequent, which led us to consider that the risk of SARS-CoV-2 transmission from animal to human, albeit negligible in the present context, may have been underestimated. In this study, we provide the results of a prospective survey that was conducted to evaluate the SARS-CoV-2 isolation rate by qRT-PCR in dogs and cats with different exposure risks and clinical statuses. From April 2020 to April 2021, we analyzed 367 samples and investigated the presence of SARS-CoV-2 RNA using qRT-PCR. Only four animals tested positive, all of them being cats. Three cats were asymptomatic and one presented a coryza-like syndrome. We describe in detail the infection in two cats and the associated clinical characteristics. Importantly, we obtained SARS-CoV-2 genomes from one infected animal and characterized them as Alpha variants. This represents the first identification of the SARS-CoV-2 Alpha variant in an infected animal in France.

High prevalence of SARS-CoV-2 antibodies in pets from COVID-19+ households.

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.

Detection of Ebola Virus Antibodies in Fecal Samples of Great Apes in Gabon.

Based on a large study conducted on wild great ape fecal samples collected in regions of Gabon where previous human outbreaks of Ebola virus disease have occurred between 1994 and 2002, we provide evidence for prevalence of Zaire ebolavirus (EBOV)-specific antibodies of 3.9% (immunoglobulin G (IgG)) and 3.5% (immunoglobulin M (IgM)) in chimpanzees and 8.8% (IgG) and 2.4% (IgM) in gorillas. Importantly, we observed a high local prevalence (31.2%) of anti-EBOV IgG antibodies in gorilla samples. This high local rate of positivity among wild great apes raises the question of a spatially and temporally localized increase in EBOV exposure risk and the role that can be played by these animals as sentinels of the virus's spread or reemergence in a given area.

Re-emergence of chikungunya in the Republic of the Congo in 2019 associated with a possible vector-host switch.

In January 2019, an outbreak of chikungunya virus fever was reported in a rural region near Pointe-Noire, Republic of the Congo. Molecular and phylogenetic analysis of this new CHIKV strain demonstrated the presence of the A226V substitution and a surprisingly close relation with Aedes aegypti-associated Central Africa chikungunya strains. These results, combined with the preponderance of Aedes albopictus in the outbreak area, suggest a recent vector-host switch facilitated by the emergence and spread of the A226V mutation from a related CHIKV strain previously circulating in Aedes aegypti. The proximity of this outbreak to the large city of Pointe-Noire alerts us to a possibly devastating future outbreak in the absence of measures limiting the proliferation of Ae. albopictus mosquitoes.

Immature particles and capsid-free viral RNA produced by Yellow fever virus-infected cells stimulate plasmacytoid dendritic cells to secrete interferons.

Plasmacytoid dendritic cells (pDCs) are specialized in the production of interferons (IFNs) in response to viral infections. The Flaviviridae family comprises enveloped RNA viruses such as Hepatitis C virus (HCV) and Dengue virus (DENV). Cell-free flaviviridae virions poorly stimulate pDCs to produce IFN. By contrast, cells infected with HCV and DENV potently stimulate pDCs via short-range delivery of viral RNAs, which are either packaged within immature virions or secreted exosomes. We report that cells infected with Yellow fever virus (YFV), the prototypical flavivirus, stimulated pDCs to produce IFNs in a TLR7- and cell contact- dependent manner. Such stimulation was unaffected by the presence of YFV neutralizing antibodies. As reported for DENV, cells producing immature YFV particles were more potent at stimulating pDCs than cells releasing mature virions. Additionally, cells replicating a release-deficient YFV mutant or a YFV subgenomic RNA lacking structural protein-coding sequences participated in pDC stimulation. Thus, viral RNAs produced by YFV-infected cells reach pDCs via at least two mechanisms: within immature particles and as capsid-free RNAs. Our work highlights the ability of pDCs to respond to a variety of viral RNA-laden carriers generated from infected cells.

Divergent assembly mechanisms of the manganese/iron cofactors in R2lox and R2c proteins.

A manganese/iron cofactor which performs multi-electron oxidative chemistry is found in two classes of ferritin-like proteins, the small subunit (R2) of class Ic ribonucleotide reductase (R2c) and the R2-like ligand-binding oxidase (R2lox). It is unclear how a heterodimeric Mn/Fe metallocofactor is assembled in these two related proteins as opposed to a homodimeric Fe/Fe cofactor, especially considering the structural similarity and proximity of the two metal-binding sites in both protein scaffolds and the similar first coordination sphere ligand preferences of MnII and FeII. Using EPR and Mössbauer spectroscopies as well as X-ray anomalous dispersion, we examined metal loading and cofactor activation of both proteins in vitro (in solution). We find divergent cofactor assembly mechanisms for the two systems. In both cases, excess MnII promotes heterobimetallic cofactor assembly. In the absence of FeII, R2c cooperatively binds MnII at both metal sites, whereas R2lox does not readily bind MnII at either site. Heterometallic cofactor assembly is favored at substoichiometric FeII concentrations in R2lox. FeII and MnII likely bind to the protein in a stepwise fashion, with FeII binding to site 2 initiating cofactor assembly. In R2c, however, heterometallic assembly is presumably achieved by the displacement of MnII by FeII at site 2. The divergent metal loading mechanisms are correlated with the putative in vivo functions of R2c and R2lox, and most likely with the intracellular MnII/FeII concentrations in the host organisms from which they were isolated.