Clinical characteristics and risk factors for COVID-19 infection and disease severity: A nationwide observational study in Estonia.

BACKGROUND: COVID-19 pandemic has led to overloading of health systems all over the world. For reliable risk stratification, knowledge on factors predisposing to SARS-CoV-2 infection and to severe COVID-19 disease course is needed for decision-making at the individual, provider, and government levels. Data to identify these factors should be easily obtainable. METHODS AND FINDINGS: Retrospective cohort study of nationwide e-health databases in Estonia. We used longitudinal health records from 66,295 people tested positive for SARS-CoV-2 RNA from 26 February 2020 to 28 February 2021 and 254,958 randomly selected controls from the reference population with no known history of SARS-CoV-2 infection or clinical COVID-19 diagnosis (case to control ratio 1:4) to predict risk factors of infection and severe course of COVID-19. We analysed sociodemographic and health characteristics of study participants. The SARS-CoV-2 infection risk was slightly higher among women, and was higher among those with comorbid conditions or obesity. Dementia (RRR 3.77, 95%CI 3.30⎼4.31), renal disease (RRR 1.88, 95%CI 1.56⎼2.26), and cerebrovascular disease (RRR 1.81, 95%CI 1.64⎼2.00) increased the risk of infection. Of all SARS-CoV-2 infected people, 92% had a non-severe disease course, 4.8% severe disease (requiring hospitalisation), 1.7% critical disease (needing intensive care), and 1.5% died. Male sex, increasing age and comorbid burden contributed significantly to more severe COVID-19, and the strength of association for male sex increased with the increasing severity of COVID-19 outcome. The strongest contributors to critical illness (expressed as RRR with 95% CI) were renal disease (7.71, 4.71⎼12.62), the history of previous myocardial infarction (3.54, 2.49⎼5.02) and obesity (3.56, 2.82⎼4.49). The strongest contributors to a lethal outcome were renal disease (6.48, 3.74⎼11.23), cancer (3.81, 3.06⎼4.75), liver disease (3.51, 1.36⎼9.02) and cerebrovascular disease (3.00, 2.31⎼3.89). CONCLUSIONS: We found divergent effect of age and gender on infection risk and severity of COVID-19. Age and gender did not contribute substantially to infection risk, but did so for the risk of severe disease Co-morbid health conditions, especially those affecting renin-angiotensin system, had an impact on both the risk of infection and severe disease course. Age and male sex had the most significant impact on the risk of severe COVID-19. Taking into account the role of ACE2 receptors in the pathogenesis of SARS-CoV-2 infection, as well as its modulating action on the renin-angiotensin system in cardiovascular and renal diseases, further research is needed to investigate the influence of hormonal status on ACE2 expression in different tissues, which may be the basis for the development of COVID-19 therapies.

Semliki Forest Virus Chimeras with Functional Replicase Modules from Related Alphaviruses Survive by Adaptive Mutations in Functionally Important Hot Spots.

Alphaviruses (family Togaviridae) include both human pathogens such as chikungunya virus (CHIKV) and Sindbis virus (SINV) and model viruses such as Semliki Forest virus (SFV). The alphavirus positive-strand RNA genome is translated into nonstructural (ns) polyprotein(s) that are precursors for four nonstructural proteins (nsPs). The three-dimensional structures of nsP2 and the N-terminal 2/3 of nsP3 reveal that these proteins consist of several domains. Cleavage of the ns-polyprotein is performed by the strictly regulated protease activity of the nsP2 region. Processing results in the formation of a replicase complex that can be considered a network of functional modules. These modules work cooperatively and should perform the same task for each alphavirus. To investigate functional interactions between replicase components, we generated chimeras using the SFV genome as a backbone. The functional modules corresponding to different parts of nsP2 and nsP3 were swapped with their counterparts from CHIKV and SINV. Although some chimeras were nonfunctional, viruses harboring the CHIKV N-terminal domain of nsP2 or any domain of nsP3 were viable. Viruses harboring the protease part of nsP2, the full-length nsP2 of CHIKV, or the nsP3 macrodomain of SINV required adaptive mutations for functionality. Seven mutations that considerably improved the infectivity of the corresponding chimeric genomes affected functionally important hot spots recurrently highlighted in previous alphavirus studies. These data indicate that alphaviruses utilize a rather limited set of strategies to survive and adapt. Furthermore, functional analysis revealed that the disturbance of processing was the main defect resulting from chimeric alterations within the ns-polyprotein. IMPORTANCE Alphaviruses cause debilitating symptoms and have caused massive outbreaks. There are currently no approved antivirals or vaccines for treating these infections. Understanding the functions of alphavirus replicase proteins (nsPs) provides valuable information for both antiviral drug and vaccine development. The nsPs of all alphaviruses consist of similar functional modules; however, to what extent these are independent in functionality and thus interchangeable among homologous viruses is largely unknown. Homologous domain swapping was used to study the functioning of modules from nsP2 and nsP3 of other alphaviruses in the context of Semliki Forest virus. Most of the introduced substitutions resulted in defects in the processing of replicase precursors that were typically compensated by adaptive mutations that mapped to determinants of polyprotein processing. Understanding the principles of virus survival strategies and identifying hot spot mutations that permit virus adaptation highlight a route to the rapid development of attenuated viruses as potential live vaccine candidates.

Timeliness of Proteolytic Events Is Prerequisite for Efficient Functioning of the Alphaviral Replicase.

Polyprotein processing has an important regulatory role in the life cycle of positive-strand RNA viruses. In the case of alphaviruses, sequential cleavage of the nonstructural polyprotein (ns-polyprotein) at three sites eventually yields four mature nonstructural proteins (nsPs) that continue working in complex to replicate viral genomic RNA and transcribe subgenomic RNA. Recognition of cleavage sites by viral nsP2 protease is guided by short sequences upstream of the scissile bond and, more importantly, by the spatial organization of the replication complex. In this study, we analyzed the consequences of the artificially accelerated processing of the Semliki Forest virus ns-polyprotein. It was found that in mammalian cells, not only the order but also the correct timing of the cleavage events is essential for the success of viral replication. Analysis of the effects of compensatory mutations in rescued viruses as well as in vitro translation and trans-replicase assays corroborated our findings and revealed the importance of the V515 residue in nsP2 for recognizing the P4 position in the nsP1/nsP2 cleavage site. We also extended our conclusions to Sindbis virus by analyzing the properties of the hyperprocessive variant carrying the N614D mutation in nsP2. We conclude that the sequence of the nsP1/nsP2 site in alphaviruses is under selective pressure to avoid the presence of sequences that are recognized too efficiently and would otherwise lead to premature cleavage at this site before completion of essential tasks of RNA synthesis or virus-induced replication complex formation. Even subtle changes in the ns-polyprotein processing pattern appear to lead to virus attenuation.IMPORTANCE The polyprotein expression strategy is a cornerstone of alphavirus replication. Three sites within the ns-polyprotein are recognized by the viral nsP2 protease and cleaved in a defined order. Specific substrate targeting is achieved by the recognition of the short sequence upstream of the scissile bond and a correct macromolecular assembly of ns-polyprotein. Here, we highlighted the importance of the timeliness of proteolytic events, as an additional layer of regulation of efficient virus replication. We conclude that, somewhat counterintuitively, the cleavage site sequences at the nsP1/nsP2 and nsP2/nsP3 junctions are evolutionarily selected to be recognized by protease inefficiently, to avoid premature cleavages that would be detrimental for the assembly and functionality of the replication complex. Understanding the causes and consequences of viral polyprotein processing events is important for predicting the properties of mutant viruses and should be helpful for the development of better vaccine candidates and understanding potential mechanisms of resistance to protease inhibitors.

A human genome-wide loss-of-function screen identifies effective chikungunya antiviral drugs.

Chikungunya virus (CHIKV) is a globally spreading alphavirus against which there is no commercially available vaccine or therapy. Here we use a genome-wide siRNA screen to identify 156 proviral and 41 antiviral host factors affecting CHIKV replication. We analyse the cellular pathways in which human proviral genes are involved and identify druggable targets. Twenty-one small-molecule inhibitors, some of which are FDA approved, targeting six proviral factors or pathways, have high antiviral activity in vitro, with low toxicity. Three identified inhibitors have prophylactic antiviral effects in mouse models of chikungunya infection. Two of them, the calmodulin inhibitor pimozide and the fatty acid synthesis inhibitor TOFA, have a therapeutic effect in vivo when combined. These results demonstrate the value of loss-of-function screening and pathway analysis for the rational identification of small molecules with therapeutic potential and pave the way for the development of new, host-directed, antiviral agents.

Effects of an In-Frame Deletion of the 6k Gene Locus from the Genome of Ross River Virus.

UNLABELLED: The alphaviral6kgene region encodes the two structural proteins 6K protein and, due to a ribosomal frameshift event, the transframe protein (TF). Here, we characterized the role of the6kproteins in the arthritogenic alphavirus Ross River virus (RRV) in infected cells and in mice, using a novel6kin-frame deletion mutant. Comprehensive microscopic analysis revealed that the6kproteins were predominantly localized at the endoplasmic reticulum of RRV-infected cells. RRV virions that lack the6kproteins 6K and TF [RRV-(Δ6K)] were more vulnerable to changes in pH, and the corresponding virus had increased sensitivity to a higher temperature. While the6kdeletion did not reduce RRV particle production in BHK-21 cells, it affected virion release from the host cell. Subsequentin vivostudies demonstrated that RRV-(Δ6K) caused a milder disease than wild-type virus, with viral titers being reduced in infected mice. Immunization of mice with RRV-(Δ6K) resulted in a reduced viral load and accelerated viral elimination upon secondary infection with wild-type RRV or another alphavirus, chikungunya virus (CHIKV). Our results show that the6kproteins may contribute to alphaviral disease manifestations and suggest that manipulation of the6kgene may be a potential strategy to facilitate viral vaccine development. IMPORTANCE: Arthritogenic alphaviruses, such as chikungunya virus (CHIKV) and Ross River virus (RRV), cause epidemics of debilitating rheumatic disease in areas where they are endemic and can emerge in new regions worldwide. RRV is of considerable medical significance in Australia, where it is the leading cause of arboviral disease. The mechanisms by which alphaviruses persist and cause disease in the host are ill defined. This paper describes the phenotypic properties of an RRV6kdeletion mutant. The absence of the6kgene reduced virion release from infected cells and also reduced the severity of disease and viral titers in infected mice. Immunization with the mutant virus protected mice against viremia not only upon exposure to RRV but also upon challenge with CHIKV. These findings could lead to the development of safer and more immunogenic alphavirus vectors for vaccine delivery.

Presentation overrides specificity: probing the plasticity of alphaviral proteolytic activity through mutational analysis.

Semliki Forest virus (genus Alphavirus) is an important model for studying regulated nonstructural (ns) polyprotein processing. In this study, we evaluated the strictness of the previously outlined cleavage rules, accounting for the timing and outcome of each of three cleavages within the ns polyprotein P1234, and assessed the significance of residues P6 to P4 within the cleavage sites using an alanine scanning approach. The processing of the 1/2 and 3/4 sites was most strongly affected following changes in residues P5 and P4, respectively. However, none of the mutations had a detectable effect on the processing of the 2/3 site. An analysis of recombinant viruses bearing combinations of mutations in cleavage sites revealed tolerance toward the cooccurrence of native and mutated cleavage sites within the same polyprotein, suggesting a remarkable plasticity of the protease recognition pocket. Even in a virus in which all of the cleavage sequences were replaced with alanines in the P6, P5, and P4 positions, the processing pattern was largely preserved, without leading to reversion of cleavage site mutations. Instead, the emergence of second-site mutations was identified, among which Q706R/L in nsP2 was confirmed to be associated with the recognition of the P4 position within the modified cleavage sites. Our results imply that the spatial arrangement of the viral replication complex inherently contributes to scissile-site presentation for the protease, alleviating stringent sequence recognition requirements yet ensuring the precision and the correct order of processing events. Obtaining a proper understanding of the consequences of cleavage site manipulations may provide new tools for taming alphaviruses.

The infection of mammalian and insect cells with SFV bearing nsP1 palmitoylation mutations.

Semliki Forest virus (SFV), an alphavirus, replicates in vertebrate host and mosquito vector cells. The virus-specific part of the replicase complex constitutes nonstructural proteins 1-4 (nsP1-nsP4) and is bound to cytoplasmic membranes by an amphipathic helix inside of nsP1 and through the palmitoylation of cysteine residues in nsP1. In mammalian cells, defects in these viral functions result in a nonviable phenotype or the emergence of second-site compensatory mutations that have a positive impact on SFV infection. In most cases, these second-site compensatory mutations were found to compensate for the defect caused by the absence of palmitoylation in mosquito cells (C6/36). In C6/36 cells, however, all palmitoylation-defective viruses had severely reduced synthesis of subgenomic RNA; at the same time, several of them had very efficient formation of defective interfering genomes. Analysis of C6/36 cells that individually expressed either wild type (wt) or palmitoylation-deficient nsP1 forms revealed that similar to mammalian cells, the wt nsP1 localized predominantly to the plasma membrane, whereas its mutant forms localized to the cytoplasm. In contrast to transfected mammalian cells, all forms of nsP1 induced the formation of filopodia-like structures on some, but not all, transfected mosquito cells. These findings indicate that the plasma membrane and associated host factors may have different roles in alphavirus replicase complex formation in mammalian and mosquito cells. In general, the lack of nsP1 palmitoylation had a less severe effect on the function of the replication complex in mammalian cells when compared with that in mosquito cells.

Properties and use of novel replication-competent vectors based on Semliki Forest virus.

BACKGROUND: Semliki Forest virus (SFV) has a positive strand RNA genome and infects different cells of vertebrates and invertebrates. The 5' two-thirds of the genome encodes non-structural proteins that are required for virus replication and synthesis of subgenomic (SG) mRNA for structural proteins. SG-mRNA is generated by internal initiation at the SG-promoter that is located at the complementary minus-strand template. Different types of expression systems including replication-competent vectors, which represent alphavirus genomes with inserted expression units, have been developed. The replication-competent vectors represent useful tools for studying alphaviruses and have potential therapeutic applications. In both cases, the properties of the vector, such as its genetic stability and expression level of the protein of interest, are important. RESULTS: We analysed 14 candidates of replication-competent vectors based on the genome of an SFV4 isolate that contained a duplicated SG promoter or an internal ribosomal entry site (IRES)-element controlled marker gene. It was found that the IRES elements and the minimal -21 to +5 SG promoter were non-functional in the context of these vectors. The efficient SG promoters contained at least 26 residues upstream of the start site of SG mRNA. The insertion site of the SG promoter and its length affected the genetic stability of the vectors, which was always higher when the SG promoter was inserted downstream of the coding region for structural proteins. The stability also depended on the conditions used for vector propagation. A procedure based on the in vitro transcription of ligation products was used for generation of replication-competent vector-based expression libraries that contained hundreds of thousands of different genomes, and maintained genetic diversity and the ability to express inserted genes over five passages in cell culture. CONCLUSION: The properties of replication-competent vectors of alphaviruses depend on the details of their construction. In the case of SFV4, such vectors should contain the SG promoter with structural characteristics for this isolate. The main factor for instability of SFV4-based replication-competent vectors was the deletion of genes of interest, since the resulting shorter genomes had a growth advantage over the original vector.

Mutations at the palmitoylation site of non-structural protein nsP1 of Semliki Forest virus attenuate virus replication and cause accumulation of compensatory mutations.

The replicase of Semliki Forest virus (SFV) consists of four non-structural proteins, designated nsP1-4, and is bound to cellular membranes via an amphipathic peptide and palmitoylated cysteine residues of nsP1. It was found that mutations preventing nsP1 palmitoylation also attenuated virus replication. The replacement of these cysteines by alanines, or their deletion, abolished virus viability, possibly due to disruption of interactions between nsP1 and nsP4, which is the catalytic subunit of the replicase. However, during a single infection cycle, the ability of the virus to replicate was restored due to accumulation of second-site mutations in nsP1. These mutations led to the restoration of nsP1-nsP4 interaction, but did not restore the palmitoylation of nsP1. The proteins with palmitoylation-site mutations, as well as those harbouring compensatory mutations in addition to palmitoylation-site mutations, were enzymically active and localized, at least in part, on the plasma membrane of transfected cells. Interestingly, deletion of 7 aa including the palmitoylation site of nsP1 had a relatively mild effect on virus viability and no significant impact on nsP1-nsP4 interaction. Similarly, the change of cysteine to alanine at the palmitoylation site of nsP1 of Sindbis virus had only a mild effect on virus replication. Taken together, these findings indicate that nsP1 palmitoylation as such is not the factor determining the ability to bind to cellular membranes and form a functional replicase complex. Instead, these abilities may be linked to the three-dimensional structure of nsP1 and the capability of nsP1 to interact with other components of the viral replicase complex.

CuZn-SOD suppresses the bovine papillomavirus-induced proliferation of fibroblasts.

Eukaryotic cells continuously produce reactive oxygen species (ROS) and have mechanisms to control ROS levels. ROS have been shown to mediate cell proliferation and transformation. We studied the effect of CuZn-superoxide dismutase (CuZnSOD) on the focus-forming ability of bovine papillomavirus (BPV-1) wtDNA and hypertransforming mutant of its major oncoprotein E5, E5-17S. We found that CuZnSOD suppresses the focus-forming ability of BPV-1 wtDNA and E5 oncoprotein. Significantly fewer foci were detected in pCGCuZnSOD- and BPV-1 DNA-cotransfected cell culture compare to BPV-1 DNA-transfected cell culture (p<0.001). CuZnSOD decreases the rate of cell proliferation in both non-transformed C127 and BPV-1- and E5-transformed cell lines. CuZnSOD decelerates cell entry into the S phase of the cell cycle and has a suppressing effect on the actively dividing cells. As the transformed cells proliferate faster than normal cells when confluent, CuZnSOD inhibits the growth of foci. These results indicate that superoxide radicals may be involved in signaling for cell proliferation and that SOD suppresses cell proliferation.