An entropy-based study on mutational trajectory of SARS-CoV-2 in India.

The pandemic of COVID-19 has been haunting us for almost the past two years. Although, the vaccination drive is in full swing throughout the world, different mutations of the SARS-CoV-2 virus are making it very difficult to put an end to the pandemic. The second wave in India, one of the worst sufferers of this pandemic, can be mainly attributed to the Delta variant i.e. B.1.617.2. Thus, it is very important to analyse and understand the mutational trajectory of SARS-CoV-2 through the study of the 26 virus proteins. In this regard, more than 17,000 protein sequences of Indian SARS-CoV-2 genomes are analysed using entropy-based approach in order to find the monthly mutational trajectory. Furthermore, Hellinger distance is also used to show the difference of the mutation events between the consecutive months for each of the 26 SARS-CoV-2 protein. The results show that the mutation rates and the mutation events of the viral proteins though changing in the initial months, start stabilizing later on for mainly the four structural proteins while the non-structural proteins mostly exhibit a more constant trend. As a consequence, it can be inferred that the evolution of the new mutative configurations will eventually reduce.

Antigenicity and Immunogenicity Analysis of the E. coli Expressed FMDV Structural Proteins; VP1, VP0, VP3 of the South African Territories Type 2 Virus.

An alternative vaccine design approach and diagnostic kits are highly required against the anticipated pandemicity caused by the South African Territories type 2 (SAT2) Foot and Mouth Disease Virus (FMDV). However, the distinct antigenicity and immunogenicity of VP1, VP0, and VP3 of FMDV serotype SAT2 are poorly understood. Similarly, the particular roles of the three structural proteins in novel vaccine design and development remain unexplained. We therefore constructed VP1, VP0, and VP3 encoding gene (SAT2:JX014256 strain) separately fused with His-SUMO (histidine-small ubiquitin-related modifier) inserted into pET-32a cassette to express the three recombinant proteins and separately evaluated their antigenicity and immunogenicity in mice. The fusion protein was successfully expressed and purified by the Ni-NTA resin chromatography. The level of serum antibody, spleen lymphocyte proliferation, and cytokines against the three distinct recombinant proteins were analyzed. Results showed that the anti-FMDV humoral response was triggered by these proteins, and the fusion proteins did enhance the splenocyte immune response in the separately immunized mice. We observed low variations among the three fusion proteins in terms of the antibody and cytokine production in mice. Hence, in this study, results demonstrated that the structural proteins of SAT2 FMDV could be used for the development of immunodiagnostic kits and subunit vaccine designs.

PhANNs, a fast and accurate tool and web server to classify phage structural proteins.

For any given bacteriophage genome or phage-derived sequences in metagenomic data sets, we are unable to assign a function to 50-90% of genes, or more. Structural protein-encoding genes constitute a large fraction of the average phage genome and are among the most divergent and difficult-to-identify genes using homology-based methods. To understand the functions encoded by phages, their contributions to their environments, and to help gauge their utility as potential phage therapy agents, we have developed a new approach to classify phage ORFs into ten major classes of structural proteins or into an "other" category. The resulting tool is named PhANNs (Phage Artificial Neural Networks). We built a database of 538,213 manually curated phage protein sequences that we split into eleven subsets (10 for cross-validation, one for testing) using a novel clustering method that ensures there are no homologous proteins between sets yet maintains the maximum sequence diversity for training. An Artificial Neural Network ensemble trained on features extracted from those sets reached a test F1-score of 0.875 and test accuracy of 86.2%. PhANNs can rapidly classify proteins into one of the ten structural classes or, if not predicted to fall in one of the ten classes, as "other," providing a new approach for functional annotation of phage proteins. PhANNs is open source and can be run from our web server or installed locally.

[Genetic evolution of VP1 of enterovirus type 71 in Shenzhen].

OBJECTIVE: Genetic evolution of VP1 of enterovirus type 71 in Shenzhen were analyzed. METHODS: All samples were tested by RT-PCR using EV71 specific primer. The VP1 of EV71 were amplified and sequenced. A phylogenetic tree was constructed by comparison of the sequences with subgenotype A, B and C using DNAStar, BioEdit and Mega 3.1 software. RESULTS: Among 35 strains, the homogeneity of the VP1 nucleotide sequence was between 92.1%-100%. The homogeneity of the VP1 nucleotide sequence with subgenotype A and B was between 81.4% -91.1%. The VP1 nucleotide sequence of 35 strains of Shenzhen shared between 93% -97.4% identity with cluster C4. The prevalence strains of EV71 were cluster C4b from 1998 to 2004, and gradually moved to C4a since 2003. All of EV71 were C4b from 2006 to 2008. Also, the homogeneity of the VP1 nucleotide sequence with Anhui FY23 EV71 strain were 94.5% -94.7%, 95.7% -95.8%, 96.2%, 95.4% -97.5%, 96.3% -99.2% from 2003 to 2008. It shows that the homogeneity was increased year by year. There was a mutation (A --> C) at No. 66 nucleotide of VP1 of EV71 that two strains were isolated in 2003 and 8 strains in 2008, that caused amino acid mutation (Q --> H) at No. 22 of VP1. CONCLUSION: EV71 C4b was gradually moved to C4a from 1998 to 2008. There was a missense mutation at No. 66 nucleotide of VP1.

[Entification of the Rubella virus genotype 1H in Western Siberia].

Molecular epidemiological study of novel strain of Rubella virus isolated during the outbreak in Western Siberia in 2004 was described. Detailed phylogenetic analysis performed based upon entire SP-region, which encodes all three Rubella structural proteins (C, E2, and E1), was implemented. This analysis provides characterization of this strain and classifies it as 1H genotype, thereby correcting previous classification of this strain based upon shorter nucleotide sequence, only encoding E1 protein. Therefore, this study identified the genotype of the Rubella virus not previously detected in Western Siberia (and even entire Russian Federation), which highlights the importance of more extensive characterization of genetic variability of the Rubella virus, especially with regard to potential influence of vaccination on the Rubella virus mutagenesis.

Identification of the nucleocapsid, tegument, and envelope proteins of the shrimp white spot syndrome virus virion.

The protein components of the white spot syndrome virus (WSSV) virion have been well established by proteomic methods, and at least 39 structural proteins are currently known. However, several details of the virus structure and assembly remain controversial, including the role of one of the major structural proteins, VP26. In this study, Triton X-100 was used in combination with various concentrations of NaCl to separate intact WSSV virions into distinct fractions such that each fraction contained envelope and tegument proteins, tegument and nucleocapsid proteins, or nucleocapsid proteins only. From the protein profiles and Western blotting results, VP26, VP36A, VP39A, and VP95 were all identified as tegument proteins distinct from the envelope proteins (VP19, VP28, VP31, VP36B, VP38A, VP51B, VP53A) and nucleocapsid proteins (VP664, VP51C, VP60B, VP15). We also found that VP15 dissociated from the nucleocapsid at high salt concentrations, even though DNA was still present. These results were confirmed by CsCl isopycnic centrifugation followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and liquid chromatography-nanoelectrospray ionization-tandem mass spectrometry, by a trypsin sensitivity assay, and by an immunogold assay. Finally, we propose an assembly process for the WSSV virion.

The product of human cytomegalovirus UL73 is a new polymorphic structural glycoprotein (gpUL73).

OBJECTIVES: This work focuses on human cytomegalovirus (HCMV) UL73, which encodes for a putative transmembrane glycoprotein that is highly conserved among herpesviruses. STUDY DESIGN: pUL73 expression was analyzed both in transiently transfected and in HCMV-infected cells using a pUL73-specific antiserum by immunoblot and immunofluorescence. Sequencing analysis from several clinical isolates and laboratory-adapted strains was also performed. RESULTS: pUL73 expressed in transiently transfected cells consists in a polypeptide of the expected size (15-18 kd) with cytoplasmic localization. In infected cells, pUL73 is expressed with true-late kinetics and localizes both in perinuclear granular formations and on the cell surface. A broad band (39-53 kd), sensitive to O-glycosidase digestion was detected in purified virus. In addition, sequence analysis showed that the N-terminal portion of pUL73 from clinical isolates is highly polymorphic. CONCLUSIONS: UL73 encodes for a new structural glycoprotein (gpUL73) expressed on the cell surface of infected cells and highly polymorphic among clinical isolates.

Sequence and phylogenetic analyses of highly virulent infectious bursal disease virus.

The nucleotide sequences of the genome segments A and B encoding the precursor polyprotein (NH2-VP2-VP4-VP3-COOH) and VP1 were determined for a highly virulent strain of infectious bursal disease virus (IBDV). The precursor polyprotein and VP1 coding regions of highly virulent OKYM strain consisted of 3039 nucleotides (1012 deduced amino acids) and 2640 nucleotides (879 deduced amino acids), respectively. Comparison of the deduced amino acid sequences of the highly virulent IBDV (HV-IBDV) with other serotype 1 and 2 sequences revealed 17 amino acid residues which were conserved only in the HV-IBDV. Among the 17 unique amino acid differences, 8 were in VP1, 4 were in VP2, 3 were in VP3 and 2 were in VP4. Although it is impossible to predict the effect of the unique amino acid residues without detailed knowledge of the three-dimensional structure and function of the proteins, they could affect the virulence of HV-IBDV. Alignment of the nucleic acid sequences of precursor polyprotein, VP1, VP2, VP3 and VP4 coding regions followed by distance analysis allowed the generation of phylogenetic trees. The same tree topology was obtained for the nucleotide sequence of precursor polyprotein, VP2, VP3 and VP4. On the other hand, the tree topology of VP1 was quite different from that obtained for the nucleotide sequence of precursor polyprotein, VP2, VP3 and VP4. These findings indicate that not a genetic recombination but a genetic reassortment may play an important role in the emergence of HV-IBDV.

Two-dimensional gel analysis of [35S]methionine labelled and phosphorylated proteins present in virions and light particles of herpes simplex virus type 1, and detection of potentially new structural proteins.

Cells infected with herpes simplex virus (HSV) synthesize both infectious viruses and non-infectious light particles (L-particles). The latter contain the envelope and tegument components of the virions, but lack virus capsid and DNA. Electrophoresis in SDS-polyacrylamide gels (SDS-PAGE) has been used extensively for analysis of structural proteins in virions and L-particles. Two-dimensional (2-D) gel electrophoresis, however has a markedly higher resolution, and in the present work we have used this technique to study both [35S]methionine labelled and phosphorylated structural proteins in virions and L-particles. Proteins were assigned to the tegument or the envelope by the analysis of L-particles. Localization of structural proteins was also determined by stepwise solubilization in the presence of the neutral detergent NP-40 and NaCl, and by isolation of capsids from nuclei of infected cells. Different steps in posttranslational modification can be detected by 2-D gel electrophoresis such that a single polypeptide may appear as several spots. This was most clearly observed for some of the HSV-encoded glycoproteins which were shown to exist in multiple forms in the virion. Some polypeptides apparently not identified previously were either capsid associated, or localized in the tegument or envelope. The degrees of phosphorylation in L-particles and virions are almost identical for some proteins, but markedly different for others. Thus, glycoprotein E of HSV-1 is for the first time shown to be phosphorylated, and most heavily so in virions. The IE VMW)110 protein represents a group of proteins which are more phosphorylated in L-particles than in virions. Attempts are made to correlate the proteins detected by 2-D analysis with those previously separated by SDS-PAGE.

Determinants of serotype specificity in transcription of vesicular stomatitis virus synthetic nucleocapsids.

Transcription of the nucleocapsid template of vesicular stomatitis virus (VSV) is serotype specific, since the viral RNA polymerase of the VSV Indiana (Ind) serotype transcribes the Ind nucleocapsid but not the nucleocapsid of the VSV New Jersey (NJ) serotype and, similarly, the NJ RNA polymerase transcribes only the NJ nucleocapsid. We have prepared synthetic nucleocapsid templates from various combinations of purified leader gene RNA and N protein of these two VSV serotypes for analysis of serotype specificity in vitro. In agreement with previous observations, in vitro transcription of synthetic homologous nucleocapsids with the 3' terminal leader RNA gene of either the (+) or (-) strand sense and the N protein of the same serotype is also serotype specific. We show with chimeric nucleocapsids, where the RNA and N protein are of different serotypes, that both components of the template are important for specificity, although the specifics depend on the serotype from which the RNA polymerase is derived. The Ind RNA polymerase will transcribe only the homologous nucleocapsids where both the RNA and N protein are of the Ind serotype, and not the chimeric nucleocapsids. In contrast, the NJ RNA polymerase is active not only on the homologous synthetic nucleocapsid, but also gives significant levels of transcription as long as one of the nucleocapsid components (N protein or RNA) is of the NJ serotype. The divergent RNA sequence of the distal portion of the leader gene (nt22 to 50/51) seems to be a major determinant for specificity, since transcription of synthetic nucleocapsids containing just the conserved proximal 1-22 nucleotides is significantly less serotype-restricted. Restriction appears to occur at the level of elongation of the product rather than initiation of RNA synthesis, since synthesis of template-length RNA, but not reiterated small initiation products, is inhibited. In addition, the serotype of the P protein subunit of the RNA polymerase also contributes to the serotype specificity of transcription, since the NJ P protein can bind equally to NJ and Ind nucleocapsids, while Ind P protein binds preferentially to Ind nucleocapsids.

RNA editing in Newcastle disease virus.

The co-transcriptional editing of the Newcastle disease virus (NDV) P gene has been studied by sequence analysis of cloned viral genomic RNA and mRNA. Evidence has been obtained for the specific insertion of non-templated G nucleotides, the consequence of which is the generation of three populations of P gene-derived mRNAs. The three populations encode proteins (P, V and W) which have a common N-terminal region, but which utilize three different reading frames at their C termini. Paradoxically, NDV edits its P gene mRNA by the insertion of non-templated G residues in a manner similar to Sendai and measles viruses (P-->V editing) despite its apparent closer evolutionary relationship to the simian virus type 5, mumps and related group of viruses which edit a V genomic sequence to generate an mRNA to encode a functional P protein (V-->P editing).

Classification of a new member of the TBE flavivirus subgroup by its immunological, pathogenetic and molecular characteristics: identification of subgroup-specific pentapeptides.

The antigenic, pathogenic and molecular characteristics of Turkish sheep encephalitis (TSE) virus, strain TTE80, were compared with other members of the tick-borne encephalitis (TBE) virus complex. Monoclonal antibodies with defined specificity for the flavivirus envelope glycoprotein distinguished TSE virus from louping ill (LI), western or far eastern TBE, Langat and Powassan virus in indirect immunofluorescence, haemagglutination-inhibition and neutralization tests. On the other hand, TSE virus, which produces an LI-like disease in sheep, resembled LI virus in mouse neurovirulence tests. Molecular homology data of all the structural genes of TSE virus compared with other tick-borne flaviviruses demonstrated that TSE virus is a distinct member in the TBE virus subgroup. The data are consistent with the conclusion that TSE virus has evolved by a separate evolutionary pathway as compared with the close antigenic relatives, western European, far eastern TBE viruses and LI virus. By aligning the encoded amino acids in the viral envelope glycoprotein of mosquito- and tick-borne flaviviruses, we have also identified subgroup-specific pentapeptide motifs for the tick-borne encephalitis, Japanese encephalitis and dengue subgroup viruses of the genus Flavivirus. These pentapeptides have important implications for the evolution, classification and diagnosis of flaviviruses.