SARS-CoV-2 Spike Protein Induces Hemagglutination: Implications for COVID-19 Morbidities and Therapeutics and for Vaccine Adverse Effects.

Experimental findings for SARS-CoV-2 related to the glycan biochemistry of coronaviruses indicate that attachments from spike protein to glycoconjugates on the surfaces of red blood cells (RBCs), other blood cells and endothelial cells are key to the infectivity and morbidity of COVID-19. To provide further insight into these glycan attachments and their potential clinical relevance, the classic hemagglutination (HA) assay was applied using spike protein from the Wuhan, Alpha, Delta and Omicron B.1.1.529 lineages of SARS-CoV-2 mixed with human RBCs. The electrostatic potential of the central region of spike protein from these four lineages was studied through molecular modeling simulations. Inhibition of spike protein-induced HA was tested using the macrocyclic lactone ivermectin (IVM), which is indicated to bind strongly to SARS-CoV-2 spike protein glycan sites. The results of these experiments were, first, that spike protein from these four lineages of SARS-CoV-2 induced HA. Omicron induced HA at a significantly lower threshold concentration of spike protein than the three prior lineages and was much more electropositive on its central spike protein region. IVM blocked HA when added to RBCs prior to spike protein and reversed HA when added afterward. These results validate and extend prior findings on the role of glycan bindings of viral spike protein in COVID-19. They furthermore suggest therapeutic options using competitive glycan-binding agents such as IVM and may help elucidate rare serious adverse effects (AEs) associated with COVID-19 mRNA vaccines, which use spike protein as the generated antigen.

Control of CDH1/E-Cadherin Gene Expression and Release of a Soluble Form of E-Cadherin in SARS-CoV-2 Infected Caco-2 Intestinal Cells: Physiopathological Consequences for the Intestinal Forms of COVID-19.

COVID-19 is the biggest pandemic the world has seen this century. Alongside the respiratory damage observed in patients with severe forms of the disease, gastrointestinal symptoms have been frequently reported. These symptoms (e.g., diarrhoea), sometimes precede the development of respiratory tract illnesses, as if the digestive tract was a major target during early SARS-CoV-2 dissemination. We hypothesize that in patients carrying intestinal SARS-CoV-2, the virus may trigger epithelial barrier damage through the disruption of E-cadherin (E-cad) adherens junctions, thereby contributing to the overall gastrointestinal symptoms of COVID-19. Here, we use an intestinal Caco-2 cell line of human origin which expresses the viral receptor/co-receptor as well as the membrane anchored cell surface adhesion protein E-cad to investigate the expression of E-cad after exposure to SARS-CoV-2. We found that the expression of CDH1/E-cad mRNA was significantly lower in cells infected with SARS-CoV-2 at 24 hours post-infection, compared to virus-free Caco-2 cells. The viral receptor ACE2 mRNA expression was specifically down-regulated in SARS-CoV-2-infected Caco-2 cells, while it remained stable in HCoV-OC43-infected Caco-2 cells, a virus which uses HLA class I instead of ACE2 to enter cells. It is worth noting that SARS-CoV-2 induces lower transcription of TMPRSS2 (involved in viral entry) and higher expression of B0AT1 mRNA (that encodes a protein known to co-express with ACE2 on intestinal cells). At 48 hours post-exposure to the virus, we also detected a small but significant increase of soluble E-cad protein (sE-cad) in the culture supernatant of SARS-CoV-2-infected Caco-2 cells. The increase of sE-cad release was also found in the intestinal HT29 cell line when infected by SARS-CoV-2. Beside the dysregulation of E-cad, SARS-CoV-2 infection of Caco-2 cells also leads to the dysregulation of other cell adhesion proteins (occludin, JAMA-A, zonulin, connexin-43 and PECAM-1). Taken together, these results shed light on the fact that infection of Caco-2 cells with SARS-CoV-2 affects tight-, adherens-, and gap-junctions. Moreover, intestinal tissues damage was associated to the intranasal SARS-CoV-2 infection in human ACE2 transgenic mice.

Drug repurposing against SARS-CoV-1, SARS-CoV-2 and MERS-CoV.

Since the beginning of the COVID-19 pandemic, large in silico screening studies and numerous in vitro studies have assessed the antiviral activity of various drugs on SARS-CoV-2. In the context of health emergency, drug repurposing represents the most relevant strategy because of the reduced time for approval by international medicines agencies, the low cost of development and the well-known toxicity profile of such drugs. Herein, we aim to review drugs with in vitro antiviral activity against SARS-CoV-2, combined with molecular docking data and results from preliminary clinical studies. Finally, when considering all these previous findings, as well as the possibility of oral administration, 11 molecules consisting of nelfinavir, favipiravir, azithromycin, clofoctol, clofazimine, ivermectin, nitazoxanide, amodiaquine, heparin, chloroquine and hydroxychloroquine, show an interesting antiviral activity that could be exploited as possible drug candidates for COVID-19 treatment.

Isolation of 4000 SARS-CoV-2 shows that contagiousness is associated with viral load, not vaccine or symptomatic status.

Culture inoculation of 6722 nasopharyngeal samples since February 2020 allowed isolation of 3637 SARS-CoV-2 and confirmed that isolation rate is correlated to viral load, regardless symptomatology or vaccination status. Moreover, the delta variant is associated with higher viral loads and therefore higher rates of viral isolation, explaining its greater contagiousness.

Monitoring the Circulation of SARS-CoV-2 Variants by Genomic Analysis of Wastewater in Marseille, South-East France.

The monitoring of SARS-CoV-2 RNA in sewage has been proposed as a simple and unbiased means of assessing epidemic evolution and the efficiency of the COVID-19 control measures. The past year has been marked by the emergence of variants that have led to a succession of epidemic waves. It thus appears that monitoring the presence of SARS-CoV-2 in wastewater alone is insufficient, and it may be important in the future to also monitor the evolution of these variants. We used a real-time RT-PCR screening test for variants in the wastewater of our city to assess the effectiveness of direct SARS-CoV-2 sequencing from the same wastewater. We compared the genome sequencing results obtained over the large RS network and the smaller B7 network with the different distributions of the variants observed by RT-PCR screening. The prevalence of the "UK variant" in the RS and B7 networks was estimated to be 70% and 8% using RT-PCR screening compared to 95% and 64% using genome sequencing, respectively. The latter values were close to the epidemiology observed in patients of the corresponding area, which were 91% and 58%, respectively. Genome sequencing in sewage identified SARS-CoV-2 of lineage B.1.525 in B7 at 27% (37% in patients), whereas it was completely missed by RT-PCR. We thus determined that direct sequencing makes it possible to observe, in wastewater, a distribution of the variants comparable to that revealed by genomic monitoring in patients and that this method is more accurate than RT-PCR. It also shows that, rather than a single large sample, it would be preferable to analyse several targeted samples if we want to more appropriately assess the geographical distribution of the different variants. In conclusion, this work supports the wider surveillance of SARS-CoV-2 variants in wastewater by genome sequencing and targeting small areas on the condition of having a sequencing capacity and, when this is not the case, to developing more precise screening tests based on the multiplexed detection of the mutations of interest.

Coculture at the crossroads of the new microbiology techniques for the isolation of strict intracellular bacteria.

Coculture played a major role in clinical microbiology by elucidating strict intracellular bacteria era. Since some of these bacteria are human pathogens, in-depth studies at the strain level are necessary to better understand their pathologies and treatment. Over the last decades, culture-independent tools have taken over the diagnostic procedure at the expense of coculture. These tools, although proven to be rapid and efficient, cannot overcome the need to culture the bacteria, as strain isolation remains a key factor to understanding its epidemiology, virulence and antibiotic susceptibility testing. Moreover, strain availability allows the development of molecular and serological tools, and remains crucial for taxonomy. This review revisits the current status of culture, its advantages, drawbacks and future needs.

Enteroviruses from Humans and Great Apes in the Republic of Congo: Recombination within Enterovirus C Serotypes.

Enteroviruses (EVs) are viruses of the family Picornaviridae that cause mild to severe infections in humans and in several animal species, including non-human primates (NHPs). We conducted a survey and characterization of enteroviruses circulating between humans and great apes in the Congo. Fecal samples (N = 24) of gorillas and chimpanzees living close to or distant from humans in three Congolese parks were collected, as well as from healthy humans (N = 38) living around and within these parks. Enteroviruses were detected in 29.4% of gorilla and 13.15% of human feces, including wild and human-habituated gorillas, local humans and eco-guards. Two identical strains were isolated from two humans coming from two remote regions. Their genomes were similar and all genes showed their close similarity to coxsackieviruses, except for the 3C, 3D and 5'-UTR regions, where they were most similar to poliovirus 1 and 2, suggesting recombination. Recombination events were found between these strains, poliovirus 1 and 2 and EV-C99. It is possible that the same EV-C species circulated in both humans and apes in different regions in the Congo, which must be confirmed in other investigations. In addition, other studies are needed to further investigate the circulation and genetic diversity of enteroviruses in the great ape population, to draw a definitive conclusion on the different species and types of enteroviruses circulating in the Republic of Congo.

A Phylogenomic Study of Acanthamoeba polyphaga Draft Genome Sequences Suggests Genetic Exchanges With Giant Viruses.

Acanthamoeba are ubiquitous phagocytes predominant in soil and water which can ingest many microbes. Giant viruses of amoebae are listed among the Acanthamoeba-resisting microorganisms. Their sympatric lifestyle within amoebae is suspected to promote lateral nucleotide sequence transfers. Some Acanthamoeba species have shown differences in their susceptibility to giant viruses. Until recently, only the genome of a single Acanthamoeba castellanii Neff was available. We analyzed the draft genome sequences of Acanthamoeba polyphaga through several approaches, including comparative genomics, phylogeny, and sequence networks, with the aim of detecting putative nucleotide sequence exchanges with giant viruses. We identified a putative sequence trafficking between this Acanthamoeba species and giant viruses, with 366 genes best matching with viral genes. Among viruses, Pandoraviruses provided the greatest number of best hits with 117 (32%) for A. polyphaga. Then, genes from mimiviruses, Mollivirus sibericum, marseilleviruses, and Pithovirus sibericum were best hits in 67 (18%), 35 (9%), 24 (7%), and 2 (0.5%) cases, respectively. Phylogenetic reconstructions showed in a few cases that the most parsimonious evolutionary scenarios were a transfer of gene sequences from giant viruses to A. polyphaga. Nevertheless, in most cases, phylogenies were inconclusive regarding the sense of the sequence flow. The number and nature of putative nucleotide sequence transfers between A. polyphaga, and A. castellanii ATCC 50370 on the one hand, and pandoraviruses, mimiviruses and marseilleviruses on the other hand were analyzed. The results showed a lower number of differences within the same giant viral family compared to between different giant virus families. The evolution of 10 scaffolds that were identified among the 14 Acanthamoeba sp. draft genome sequences and that harbored ≥ 3 genes best matching with viruses showed a conservation of these scaffolds and their 46 viral genes in A. polyphaga, A. castellanii ATCC 50370 and A. pearcei. In contrast, the number of conserved genes decreased for other Acanthamoeba species, and none of these 46 genes were present in three of them. Overall, this work opens up several potential avenues for future studies on the interactions between Acanthamoeba species and giant viruses.

Genome Characterization of the First Mimiviruses of Lineage C Isolated in Brazil.

The family Mimiviridae, comprised by giant DNA viruses, has been increasingly studied since the isolation of the Acanthamoeba polyphaga mimivirus (APMV), in 2003. In this work, we describe the genome analysis of two new mimiviruses, each isolated from a distinct Brazilian environment. Furthermore, for the first time, we are reporting the genomic characterization of mimiviruses of group C in Brazil (Br-mimiC), where a predominance of mimiviruses from group A has been previously reported. The genomes of the Br-mimiC isolates Mimivirus gilmour (MVGM) and Mimivirus golden (MVGD) are composed of double-stranded DNA molecules of ∼1.2 Mb, each encoding more than 1,100 open reading frames. Genome functional annotations highlighted the presence of mimivirus group C hallmark genes, such as the set of seven aminoacyl-tRNA synthetases. However, the set of tRNA encoded by the Br-mimiC was distinct from those of other group C mimiviruses. Differences could also be observed in a genome synteny analysis, which demonstrated the presence of inversions and loci translocations at both extremities of Br-mimiC genomes. Both phylogenetic and phyletic analyses corroborate previous results, undoubtedly grouping the new Brazilian isolates into mimivirus group C. Finally, an updated pan-genome analysis of genus Mimivirus was performed including all new genomes available until the present moment. This last analysis showed a slight increase in the number of clusters of orthologous groups of proteins among mimiviruses of group A, with a larger increase after addition of sequences from mimiviruses of groups B and C, as well as a plateau tendency after the inclusion of the last four mimiviruses of group C, including the Br-mimiC isolates. Future prospective studies will help us to understand the genetic diversity among mimiviruses.

Identification of giant Mimivirus protein functions using RNA interference.

Genomic analysis of giant viruses, such as Mimivirus, has revealed that more than half of the putative genes have no known functions (ORFans). We knocked down Mimivirus genes using short interfering RNA as a proof of concept to determine the functions of giant virus ORFans. As fibers are easy to observe, we targeted a gene encoding a protein absent in a Mimivirus mutant devoid of fibers as well as three genes encoding products identified in a protein concentrate of fibers, including one ORFan and one gene of unknown function. We found that knocking down these four genes was associated with depletion or modification of the fibers. Our strategy of silencing ORFan genes in giant viruses opens a way to identify its complete gene repertoire and may clarify the role of these genes, differentiating between junk DNA and truly used genes. Using this strategy, we were able to annotate four proteins in Mimivirus and 30 homologous proteins in other giant viruses. In addition, we were able to annotate >500 proteins from cellular organisms and 100 from metagenomic databases.

Pan-Genome Analysis of Brazilian Lineage A Amoebal Mimiviruses.

Since the recent discovery of Samba virus, the first representative of the family Mimiviridae from Brazil, prospecting for mimiviruses has been conducted in different environmental conditions in Brazil. Recently, we isolated using Acanthamoeba sp. three new mimiviruses, all of lineage A of amoebal mimiviruses: Kroon virus from urban lake water; Amazonia virus from the Brazilian Amazon river; and Oyster virus from farmed oysters. The aims of this work were to sequence and analyze the genome of these new Brazilian mimiviruses (mimi-BR) and update the analysis of the Samba virus genome. The genomes of Samba virus, Amazonia virus and Oyster virus were 97%-99% similar, whereas Kroon virus had a low similarity (90%-91%) with other mimi-BR. A total of 3877 proteins encoded by mimi-BR were grouped into 974 orthologous clusters. In addition, we identified three new ORFans in the Kroon virus genome. Additional work is needed to expand our knowledge of the diversity of mimiviruses from Brazil, including if and why among amoebal mimiviruses those of lineage A predominate in the Brazilian environment.

Current laboratory diagnosis of Q fever.

Q fever is a worldwide zoonosis caused by the strictly intracellular bacterium Coxiella burnetii. Among symptomatic patients (one-half of patients remain asymptomatic), acute Q fever most frequently manifests as a self-limited febrile illness, pneumonia, or hepatitis. Endocarditis is the predominant form of chronic Q fever. All the classical techniques of bacteriology may be used for diagnosis of C burnetii infection. Nonetheless, because of the risk of contamination, isolation must be performed in biosafety level 3 laboratories. Moreover, to date no diagnostic tests for detection by polymerase chain reaction or specific antibodies for immunochemistry are available commercially. Hence, Q fever is diagnosed in most cases by serology. The most reliable technique appears to be micro-immunofluorescence, which exhibits both good sensitivity and specificity. A wider use of this serology in cases of blood culture-negative endocarditis, atypical pneumonia, unexplained fever, and hepatitis should lead to an increase of diagnosed cases.

Protein candidates for the serodiagnosis of rickettsioses.

The laboratory diagnosis of rickettsioses is based on serology (reference method), cell culture and/or molecular tools. However, the main drawback of serology is its incapacity to provide identification of Rickettsiae at the level of species. The aim of this study was to propose the versatile protein markers able to discriminate the patients with murine typhus from those with Mediterranean spotted fever. We have cloned and expressed 20 proteins of Rickettsia prowazekii and Rickettsia rickettsii, respectively, using the GATEWAY approach. These recombinant proteins were screened by ELISA with sera of infected patients with Rickettsia typhi and Rickettsia conorii, respectively. We identified several potential markers which allowed infection due to R. typhi to be discriminated from those due to R. conorii. However, the values of test-operating parameters were not sufficient for its 'routine' clinical use. Our diagnostic test requires further optimization for be applied as a point-of-care strategy in the management of patients with suspected cases of rickettsiosis.

Usefulness of rpoB gene sequencing for identification of Afipia and Bosea species, including a strategy for choosing discriminative partial sequences.

Bacteria belonging to the genera Afipia and Bosea are amoeba-resisting bacteria that have been recently reported to colonize hospital water supplies and are suspected of being responsible for intensive care unit-acquired pneumonia. Identification of these bacteria is now based on determination of the 16S ribosomal DNA sequence. However, the 16S rRNA gene is not polymorphic enough to ensure discrimination of species defined by DNA-DNA relatedness. The complete rpoB sequences of 20 strains were first determined by both PCR and genome walking methods. The percentage of homology between different species ranged from 83 to 97% and was in all cases lower than that observed with the 16S rRNA gene; this was true even for species that differed in only one position. The taxonomy of Bosea and Afipia is discussed in light of these results. For strain identification that does not require the complete rpoB sequence (4,113 to 4,137 bp), we propose a simple computerized method that allows determination of nucleotide positions of high variability in the sequence that are bordered by conserved sequences and that could be useful for design of universal primers. A fragment of 740 to 752 bp that contained the most highly variable area (positions 408 to 420) was amplified and sequenced with these universal primers for 47 strains. The variability of this sequence allowed identification of all strains and correlated well with results of DNA-DNA relatedness. In the future, this method could be also used for the determination of variability "hot spots" in sets of housekeeping genes, not only for identification purposes but also for increasing the discriminatory power of sequence typing techniques such as multilocus sequence typing.

Legionella drancourtii sp. nov., a strictly intracellular amoebal pathogen.

A Legionella-like amoebal pathogen (LLAP), formerly named LLAP12(T), was characterized on the basis of microscopic appearance, staining characteristics, growth in Acanthamoeba polyphaga at different temperatures, DNA G+C content, serological cross-reactivity and 16S rRNA and macrophage infectivity potentiator (mip) gene sequence analysis. LLAP12(T) was found to be a motile, Gram-negative bacterium that grew within cytoplasmic vacuoles in infected amoebae. The infecting bacteria induced lysis of their amoebal hosts and time taken to do so was dependent on incubation temperature. Recovery of LLAP12(T) from amoebae onto axenic media could not be achieved. Phylogenetic analysis of LLAP12(T), based on 16S rRNA and mip gene sequence analysis, indicated that it lay within the radiation of the Legionellaceae and that it clustered specifically with Legionella lytica and Legionella rowbothamii. The divergence observed between LLAP12(T) and these two species was of a degree equal to, or greater than, that observed between other members of the family. In support of this delineation, LLAP12(T) was found not to cross-react serologically with any other Legionella species. The mip and 16S rRNA gene sequence-based analyses also indicated that LLAP12(T) was related very closely to two other previously identified LLAP isolates, LLAP4 and LLAP11. Taken together, these results support the proposal of LLAP12(T) as the type strain of Legionella drancourtii sp. nov.