Structural analysis of SARS-Cov-2 nonstructural protein 1 polymorphisms found in the Brazilian Amazon

The coronavirus disease COVID-19 has been the cause of millions of deaths worldwide. Among the SARS-CoV-2 proteins, the non-structural protein 1 (NSP1) has great importance during the virus infection process and is present in both alpha and beta-CoVs. Therefore, monitoring of NSP1 polymorphisms is crucial in order to understand their role during infection and virus-induced pathogenicity. Herein, we analyzed how mutations detected in the circulating SARS-CoV-2 in the population of the city of Manaus, Amazonas state, Brazil could modify the tertiary structure of the NSP1 protein. Three mutations were detected in the SARS-CoV-2 NSP1 gene: deletion of the amino acids KSF from positions 141 to 143 (delKSF), SARS-CoV-2, lineage B.1.195; and two substitutions, R29H and R43C, SARS-CoV-2 lineage B.1.1.28 and B.1.1.33, respectively. The delKSF was found in 47 samples, whereas R29H and R43C were found in two samples, one for each mutation. The NSP1 structures carrying the mutations R43C and R29H on the N-terminal portion (e.g. residues 10 to 127) showed minor backbone divergence compared to the Wuhan model. However, the NSP1 C-terminal region (residues 145 to 180) was severely affected in the delKSF and R29H mutants. The intermediate variable region (residues 144 to 148) leads to changes in the C-terminal region, particularly in the delKSF structure. New investigations must be carried out to analyze how these changes affect NSP1 activity during the infection. Our results reinforce the need for continuous genomic surveillance of SARS-CoV-2 to better understand virus evolution and assess the potential impact of the viral mutations on the approved vaccines and future therapies.

Possible Causes of Gender Differences in COVID-19 Infection Rate and Mortality

Purpose To study the causes of gender differences in infection rate and mortality of COVID-19.Methods According to the confirmed results so far, it was found that the expression of ACE2, TMPRSS2, NRP1 and FURIN genes were related to SARS-COV-2 virus infected cells; CD4, CD8 and NLRP3 genes were related to human immunological response; NSP1 gene was related to immunosuppression; IL6 (IL-6), IFNG (IFN-γ) and TNF (TNF-α) genes were related to the occurrence of cytokine storm. The differential expression of these genes between male and female were analyzed in normal and tumor patients, and further analyzed in different locations of normal and tumor tissues to find out risk factors affecting the infection rate and mortality. Results In our study, we identified that in the lung tissue, the expression level of ACE2, TMPRSS2, NRP1 and FURIN genes in male patients were higher than those in female patients. In all normal tissues of patients: NRP1, FURIN and NSP1 genes were significantly higher expressed in female. In all tumor tissues of patients: ACE2, FURIN and IL-6 genes were significantly higher expressed in male, while TMPRSS2, CD4, CD8, NLRP3, NSP1 and TNF genes were significantly higher expressed in female.Conclusions The significant differential expression of SARS-COV-2 receptor related genes and immune response related genes between male and female patients may be the reason for the difference in COVID-19 infection rate and mortality. The expression of COVID-19-related genes in normal and tumor patients were also significantly different, so clinical treatment should be treated differently.

SARS-CoV-2 genetic diversity in Venezuela: Predominance of D614G variants and analysis of one outbreak

SARS-CoV-2 is the new coronavirus responsible for COVID-19 disease. The first two cases of COVID-19 were detected in Venezuela on March 13, 2020. The aim of this study was the genetic characterization of Venezuelan SARS-CoV-2 isolates. A total of 7 full SARS-CoV-2 genome sequences were obtained by Sanger sequencing, from patients of different regions of Venezuela, mainly from the beginning of the epidemic. Ten out of 11 isolates (6 complete genomes and 4 partial spike genomic regions) belonged to lineage B, bearing the D614G mutation in the Spike protein. Isolates from the first outbreak that occurred in the Margarita Island harbored an in-frame deletion in its sequence, without amino acids 83–85 of the NSP1 of the ORF1. The search for deletions in 48,635 sequences showed that the NSP1 gene exhibit the highest frequency of deletions along the whole genome. Structural analysis suggests a change in the N-terminal domain with the presence of this deletion. In contrast, isolates circulating later in this island lacked the deletion, suggesting new introductions to the island after this first outbreak. In conclusion, a high diversity of SARS-CoV-2 isolates were found circulating in Venezuela, with predominance of the D614G mutation. The first small outbreak in Margarita Island seemed to be associated with a strain carrying a small deletion in the NSP1 protein, but these isolates do not seem to be responsible for the larger outbreak which started in July.

Rotavirus Species B Encodes a Functional Fusion-Associated Small Transmembrane Protein

ABSTRACT Rotavirus is an important cause of diarrheal disease in young mammals. Rotavirus species A (RVA) causes most human rotavirus diarrheal disease and primarily affects infants and young children. Rotavirus species B (RVB) has been associated with sporadic outbreaks of human adult diarrheal disease. RVA and RVB are predicted to encode mostly homologous proteins but differ significantly in the proteins encoded by the NSP1 gene. In the case of RVB, the NSP1 gene encodes two putative protein products of unknown function, NSP1-1 and NSP1-2. We demonstrate that human RVB NSP1-1 mediates syncytium formation in cultured human cells. Based on sequence alignment, NSP1-1 proteins from species B, G, and I contain features consistent with fusion-associated small transmembrane (FAST) proteins, which have previously been identified in other genera of the Reoviridae family. Like some other FAST proteins, RVB NSP1-1 is predicted to have an N-terminal myristoyl modification. Addition of an N-terminal FLAG peptide disrupts NSP1-1-mediated fusion. NSP1-1 from a human RVB mediates fusion of human cells but not hamster cells and, thus, may serve as a species tropism determinant. NSP1-1 also can enhance RVA replication in human cells, both in single-cycle infection studies and during a multicycle time course in the presence of fetal bovine serum, which inhibits rotavirus spread. These findings suggest potential yet untested roles for NSP1-1 in RVB species tropism, immune evasion, and pathogenesis. IMPORTANCE While species A rotavirus is commonly associated with diarrheal disease in young children, species B rotavirus has caused sporadic outbreaks of adult diarrheal disease. A major genetic difference between species A and B rotaviruses is the NSP1 gene, which encodes two proteins for species B rotavirus. We demonstrate that the smaller of these proteins, NSP1-1, can mediate fusion of cultured human cells. Comparison with viral proteins of similar function provides insight into NSP1-1 domain organization and fusion mechanism. These comparisons suggest that there is a fatty acid modification at the amino terminus of the protein, and our results show that an intact amino terminus is required for NSP1-1-mediated fusion. NSP1-1 from a human virus mediates fusion of human cells, but not hamster cells, and enhances species A rotavirus replication in culture. These findings suggest potential, but currently untested, roles for NSP1-1 in RVB host species tropism, immune evasion, and pathogenesis.

Group B rotavirus encodes a functional fusion-associated small transmembrane (FAST) protein

ABSTRACTRotavirus is an important cause of diarrheal disease in young mammals. Group A rotavirus (RVA) causes most human rotavirus diarrheal disease and primarily affects infants and young children. Group B rotavirus (RVB) has been associated with sporadic outbreaks of human adult diarrheal disease. RVA and RVB are predicted to encode mostly homologous proteins but differ significantly in the proteins encoded by the NSP1 gene. In the case of RVB, the NSP1 gene encodes two putative protein products of unknown function, NSP1-1 and NSP1-2. We demonstrate that human RVB NSP1-1 mediates syncytia formation in cultured human cells. Based on sequence alignment, NSP1-1 from groups B, G, and I contain features consistent with fusion-associated small transmembrane (FAST) proteins, which have previously been identified in other Reoviridae viruses. Like some other FAST proteins, RVB NSP1-1 is predicted to have an N-terminal myristoyl modification. Addition of an N-terminal FLAG peptide disrupts NSP1-1-mediated fusion, consistent with a role for this fatty-acid modification in NSP1-1 function. NSP1-1 from a human RVB mediates fusion of human cells but not hamster cells and, thus, may serve as a species tropism determinant. NSP1-1 also can enhance RVA replication in human cells, both in single-cycle infection studies and during a multi-cycle time course in the presence of fetal bovine serum, which inhibits rotavirus spread. These findings suggest potential yet untested roles for NSP1-1 in RVB species tropism, immune evasion, and pathogenesis.IMPORTANCEWhile group A rotavirus is commonly associated with diarrheal disease in young children, group B rotavirus has caused sporadic outbreaks of adult diarrheal disease. A major genetic difference between group A and B rotaviruses is the NSP1 gene, which encodes two proteins for group B rotavirus. We demonstrate that the smaller of these proteins, NSP1-1, can mediate fusion of cultured human cells. Comparison with viral proteins of similar function provides insight into NSP1-1 domain organization and fusion mechanism. Our findings are consistent with an important role for a fatty acid modification at the amino terminus of the protein in mediating its function. NSP1-1 from a human virus mediates fusion of human cells, but not hamster cells, and enhances rotavirus replication in culture. These findings suggest potential, but currently untested, roles for NSP1-1 in RVB species tropism, immune evasion, and pathogenesis.

Entirely plasmid-based reverse genetics system for rotaviruses

Rotaviruses (RVs) are highly important pathogens that cause severe diarrhea among infants and young children worldwide. The understanding of the molecular mechanisms underlying RV replication and pathogenesis has been hampered by the lack of an entirely plasmid-based reverse genetics system. In this study, we describe the recovery of recombinant RVs entirely from cloned cDNAs. The strategy requires coexpression of a small transmembrane protein that accelerates cell-to-cell fusion and vaccinia virus capping enzyme. We used this system to obtain insights into the process by which RV nonstructural protein NSP1 subverts host innate immune responses. By insertion into the NSP1 gene segment, we recovered recombinant viruses that encode split-green fluorescent protein–tagged NSP1 and NanoLuc luciferase. This technology will provide opportunities for studying RV biology and foster development of RV vaccines and therapeutics.

Identification of an avian group A rotavirus containing a novel VP4 gene with a close relationship to those of mammalian rotaviruses

Group A rotaviruses (RVAs) are an important cause of diarrhoeal illness in humans, as well as in mammalian and avian animal species. Previous sequence analyses indicated that avian RVAs are related only distantly to mammalian RVAs. Here, the complete genomes of RVA strain 03V0002E10 from turkey (Meleagris gallopavo) and RVA strain 10V0112H5 from pheasant (Phasianus colchicus) were analysed using a combination of 454 deep sequencing and Sanger sequencing technologies. An adenine-rich insertion similar to that found in the chicken RVA strain 02V0002G3, but considerably shorter, was found in the 3′ NCR of the NSP1 gene of the pheasant strain. Most genome segments of both strains were related closely to those of avian RVAs. The novel genotype N10 was assigned to the NSP2 gene of the pheasant RVA, which is related most closely to genotype N6 found in avian RVAs. However, this virus contains a VP4 gene of the novel genotype P[37], which is related most closely to RVAs from pigs, dogs and humans. This strain either may represent an avian/mammalian rotavirus reassortant, or it carries an unusual avian rotavirus VP4 gene, thereby broadening the potential genetic and antigenic variability among RVAs.

Genetic divergence and classification of non-structural protein 1 among porcine rotaviruses of species B

Porcine rotavirus B (RVB) has frequently been detected in diarrhoea of suckling and weaned pigs. Moreover, epidemiological studies using ELISA have demonstrated high antibody prevalence in sera from sows, indicating that RVB infections are widespread. Because it is difficult to propagate RVBs serially in cell culture, genetic analysis of RNA segments of porcine RVBs other than those encoding VP7 and NSP2 has been scarcely performed. We conducted sequence and phylogenetic analyses focusing on non-structural protein 1 (NSP1), using 15 porcine RVB strains isolated from diarrhoeic faeces collected around Japan. Sequence analysis showed that the porcine NSP1 gene contains two overlapping ORFs. Especially, peptide 2 of NSP1 retains highly conserved cysteine and histidine residues among RVBs. Comparison of NSP1 nucleotide and deduced amino acid sequences from porcine RVB strains demonstrated low identities to those from other RVB strains. Phylogenetic analysis of RVB NSP1 revealed the presence of murine, human, ovine, bovine and porcine clusters. Furthermore, the NSP1 genes of porcine RVBs were divided into three genotypes, suggesting the possibility that porcine species might be an original host of RVB infection. Of nine strains common to those used in our previous study, only one strain was classified into a different genotype from the others in the analysis of VP7, in contrast to the analysis of NSP1, where all belonged to the same cluster. This fact suggests the occurrence of gene reassortment among porcine RVBs. These findings should provide more beneficent information to understand the evolution and functions of RVBs.