Genome Analysis of Epsilon CrAss-like Phages.

CrAss-like phages play an important role in maintaining ecological balance in the human intestinal microbiome. However, their genetic diversity and lifestyle are still insufficiently studied. In this study, a novel CrAssE-Sib phage genome belonging to the epsilon crAss-like phage genomes was found. Comparative analysis indicated that epsilon crAss-like phages are divided into two putative genera, which were proposed to be named Epsilonunovirus and Epsilonduovirus; CrAssE-Sib belongs to the former. The crAssE-Sib genome contains a diversity-generating retroelement (DGR) cassette with all essential elements, including the reverse transcriptase (RT) and receptor binding protein (RBP) genes. However, this RT contains the GxxxSP motif in its fourth domain instead of the usual GxxxSQ motif found in all known phage and bacterial DGRs. RBP encoded by CrAssE-Sib and other Epsilonunoviruses has an unusual structure, and no similar phage proteins were found. In addition, crAssE-Sib and other Epsilonunoviruses encode conserved prophage repressor and anti-repressors that could be involved in lysogenic-to-lytic cycle switches. Notably, DNA primase sequences of epsilon crAss-like phages are not included in the monophyletic group formed by the DNA primases of all other crAss-like phages. Therefore, epsilon crAss-like phage substantially differ from other crAss-like phages, indicating the need to classify these phages into a separate family.

Bacteriophage vB_SepP_134 and Endolysin LysSte_134_1 as Potential Staphylococcus-Biofilm-Removing Biological Agents.

Bacteria of the genus Staphylococcus are significant challenge for medicine, as many species are resistant to multiple antibiotics and some are even to all of the antibiotics we use. One of the approaches to developing new therapeutics to treat staphylococcal infections is the use of bacteriophages specific to these bacteria or the lytic enzymes of such bacteriophages, which are capable of hydrolyzing the cell walls of these bacteria. In this study, a new bacteriophage vB_SepP_134 (St 134) specific to Staphylococcus epidermidis was described. This podophage, with a genome of 18,275 bp, belongs to the Andhravirus genus. St 134 was able to infect various strains of 12 of the 21 tested coagulase-negative Staphylococcus species and one clinical strain from the Staphylococcus aureus complex. The genes encoding endolysin (LysSte134_1) and tail tip lysin (LysSte134_2) were identified in the St 134 genome. Both enzymes were cloned and produced in Escherichia coli cells. The endolysin LysSte134_1 demonstrated catalytic activity against peptidoglycans isolated from S. aureus, S. epidermidis, Staphylococcus haemolyticus, and Staphylococcus warneri. LysSte134_1 was active against S. aureus and S. epidermidis planktonic cells and destroyed the biofilms formed by clinical strains of S. aureus and S. epidermidis.

A Novel Podophage StenR_269 Suggests a New Family in the Class Caudoviricetes.

Stenotrophomonas rhizophila was first discovered in soil; it is associated with the rhizosphere and capable of both protecting roots and stimulating plant growth. Therefore, it has a great potential to be used in biocontrol. The study of S. rhizophila phages is important for a further evaluation of their effect on the fitness and properties of host bacteria. A novel phage StenR_269 and its bacterial host S. rhizophila were isolated from a soil sample in the remediation area of a coal mine. Electron microscopy revealed a large capsid (~Ø80 nm) connected with a short tail, which corresponds to the podovirus morphotype. The length of the genomic sequence of the StenR_269 was 66,322 bp and it contained 103 putative genes; 40 of them encoded proteins with predicted functions, 3 corresponded to tRNAs, and the remaining 60 were identified as hypothetical ones. Comparative analysis indicated that the StenR_269 phage had a similar genome organization to that of the unclassified Xanthomonas phage DES1, despite their low protein similarity. In addition, the signature proteins of StenR_269 and DES1 had low similarity and these proteins clustered far from the corresponding proteins of classified phages. Thus, the StenR_269 genome is orphan and the analyzed data suggest a new family in the class Caudoviricetes.

Tentaclins-A Novel Family of Phage Receptor-Binding Proteins That Can Be Hypermutated by DGR Systems.

Diversity-generating retroelements (DGRs) are prokaryotic systems providing rapid modification and adaptation of target proteins. In phages, the main targets of DGRs are receptor-binding proteins that are usually parts of tail structures and the variability of such host-recognizing structures enables phage adaptation to changes on the bacterial host surface. Sometimes, more than one target gene containing a hypermutated variable repeat (VR) can be found in phage DGRs. The role of mutagenesis of two functionally different genes is unclear. In this study, several phage genomes that contain DGRs with two target genes were found in the gut virome of healthy volunteers. Bioinformatics analysis of these genes indicated that they encode proteins with different topology; however, both proteins contain the C-type lectin (C-lec) domain with a hypermutated beta-hairpin on its surface. One of the target proteins belongs to a new family of proteins with a specific topology: N-terminal C-lec domain followed by one or more immunoglobulin domains. Proteins from the new family were named tentaclins after TENTACLe + proteIN. The genes encoding such proteins were found in the genomes of prophages and phages from the gut metagenomes. We hypothesized that tentaclins are involved in binding either to bacterial receptors or intestinal/immune cells.

Special Issue "Bacteriophage Genomics": Editorial.

Virus genomics as a separate branch of biology has emerged relatively recently [...].

StenM_174: A Novel Podophage That Infects a Wide Range of Stenotrophomonas spp. and Suggests a New Subfamily in the Family Autographiviridae.

Stenotrophomonas maltophilia was discovered as a soil bacterium associated with the rhizosphere. Later, S. maltophilia was found to be a multidrug-resistant hospital-associated pathogen. Lytic bacteriophages are prospective antimicrobials; therefore, there is a need for the isolation and characterization of new Stenotrophomonas phages. The phage StenM_174 was isolated from litter at a poultry farm using a clinical strain of S. maltophilia as the host. StenM_174 reproduced in a wide range of clinical and environmental strains of Stenotrophomonas, mainly S. maltophilia, and it had a podovirus morphotype. The length of the genomic sequence of StenM_174 was 42,956 bp, and it contained 52 putative genes. All genes were unidirectional, and 31 of them encoded proteins with predicted functions, while the remaining 21 were identified as hypothetical ones. Two tail spike proteins of StenM_174 were predicted using AlphaFold2 structural modeling. A comparative analysis of the genome shows that the Stenotrophomonas phage StenM_174, along with the phages Ponderosa, Pepon, Ptah, and TS-10, can be members of the new putative genus Ponderosavirus in the Autographiviridae family. In addition, the analyzed data suggest a new subfamily within this family.

An Update of Orthopoxvirus Molecular Evolution.

Although variola virus (VARV) has been eradicated through widespread vaccination, other orthopoxviruses pathogenic for humans circulate in nature. Recently, new orthopoxviruses, including some able to infect humans, have been found and their complete genomes have been sequenced. Questions about the orthopoxvirus mutation rate and the emergence of new threats to humankind as a result of the evolution of circulating orthopoxviruses remain open. Based on contemporary data on ancient VARV DNA and DNA of new orthopoxvirus species, an analysis of the molecular evolution of orthopoxviruses was carried out and the timescale of their emergence was estimated. It was calculated that the orthopoxviruses of the Old and New Worlds separated approximately 40,000 years ago; the recently discovered Akhmeta virus and Alaskapox virus separated from other orthopoxviruses approximately 10,000-20,000 years ago; the rest of modern orthopoxvirus species originated from 1700 to 6000 years ago, with the exception of VARV, which emerged in approximately 300 AD. Later, there was a separation of genetic variants of some orthopoxvirus species, so the monkeypox virus West African subtype originated approximately 600 years ago, and the VARV minor alastrim subtype emerged approximately 300 years ago.

Raoultella bacteriophage RP180, a new member of the genus Kagunavirus, subfamily Guernseyvirinae.

A novel lytic Raoultella phage, RP180, was isolated and characterized. The RP180 genome has 44,851 base pairs and contains 65 putative genes, 35 of them encoding proteins whose functions were predicted based on sequence similarity to known proteins. The RP180 genome possesses a gene synteny typical of members of the subfamily Guernseyvirinae. Phylogenetic analysis of the RP180 genome and similar phage genomes revealed that phage RP180 is the first member of the genus Kagunavirus, subfamily Guernseyvirinae, that is specific for Raoultella sp. The genome of RP180 encodes a putative protein with similarity to CRISPR-like Cas4 nucleases, which belong to the pfam12705/PDDEXK_1 family. Cas4-like proteins of this family have been shown to interfere with the bacterial host type II-C CRISPR-Cas system.

High Level of Identity of Calreticulin Gene Sequences of Ixodes persulcatus and Ixodes pavlovskyi Ticks.

In southern regions of Western Siberia, Ixodes persulcatus ticks co-exist with Ixodes pavlovskyi. Both tick species have similar morphology, and natural hybridization of I. persulcatus and I. pavlovskyi ticks has been observed. To investigate the role of I. pavlovskyi ticks and I. persulcatus/ I. pavlovskyi hybrids as vectors, correct identification of tick species is necessary. The nuclear crt gene might be useful in distinguishing I. persulcatus from I. pavlovskyi ticks. Genetic variability in this gene has been studied and substantial differences between crt gene sequences of I. persulcatus and I. pavlovskyi ticks has been demonstrated. In this study, maximum-likelihood analysis showed that sequences of I. persulcatus and I. pavlovskyi ticks were identical or highly homologous among themselves. These results were confirmed by a Bayesian phylogeny. We conclude that crt gene sequences of I. persulcatus and I. pavlovskyi cannot be used for distinguishing these tick species and that the conflicting results of prior studies reflect samples from incorrectly identified ticks rather than real genetic differences between I. persulcatus and I. pavlovskyi.

Occurrence and genetic variability of Kemerovo virus in Ixodes ticks from different regions of Western Siberia, Russia and Kazakhstan.

Kemerovo virus (KEMV), a member of the Reoviridae family, Orbivirus genus, is transmitted by Ixodes ticks and can cause aseptic meningitis and meningoencephalitis. Recently, this virus was observed in certain provinces of European part of Russia, Ural, and Western and Eastern Siberia. However, the occurrence and genetic diversity of KEMV in Western Siberia remain poorly studied. Therefore, the aim of this work was to investigate the prevalence and genetic variability of KEMV in Ixodes ticks from Western Siberia. A total of 1958 Ixodes persulcatus, I. pavlovskyi ticks and their hybrids from Novosibirsk and Omsk provinces, Altai Republic (Russia) and East Kazakhstan province (Kazakhstan) were analyzed for the presence of KEMV and tick-borne encephalitis virus (TBEV) RNA. It was observed that the KEMV distribution area in Western Siberia was wider than originally thought and included Northern and Northeastern Altai in addition to the Omsk and Novosibirsk provinces. For the first time, this virus was found in Kazakhstan. The occurrence of KEMV was statistically lower than TBEV in most locations in Western Siberia. KEMV was found both in I. persulcatus and I. pavlovskyi ticks and in their hybrids. Notably, KEMV variants observed in the 2010s were genetically different from those isolated in the 1960s, which indicated the ongoing process of evolution of the Kemerovo virus group. Moreover, the possibility of reassortment for KEMV was demonstrated for the first time.

The origin of the variola virus.

The question of the origin of smallpox, one of the major menaces to humankind, is a constant concern for the scientific community. Smallpox is caused by the agent referred to as the variola virus (VARV), which belongs to the genus Orthopoxvirus. In the last century, smallpox was declared eradicated from the human community; however, the mechanisms responsible for the emergence of new dangerous pathogens have yet to be unraveled. Evolutionary analyses of the molecular biological genomic data of various orthopoxviruses, involving a wide range of epidemiological and historical information about smallpox, have made it possible to date the emergence of VARV. Comparisons of the VARV genome to the genomes of the most closely related orthopoxviruses and the examination of the distribution their natural hosts' ranges suggest that VARV emerged 3000 to 4000 years ago in the east of the African continent. The VARV evolution rate has been estimated to be approximately 2 × 10-6 substitutions/site/year for the central conserved genomic region and 4 × 10-6 substitutions/site/year for the synonymous substitutions in the genome. Presumably, the introduction of camels to Africa and the concurrent changes to the climate were the particular factors that triggered the divergent evolution of a cowpox-like ancestral virus and thereby led to the emergence of VARV.

A study of the human bocavirus replicative genome structures.

The complete genomes of two human bocavirus 4 (HBoV4) isolates recovered in 2011 in Novosibirsk, Russia have been determined. A set of primers was designed based on the determined and previously published HBoV sequences; this primer pair was able to detect all possible HBoV replicative intermediates. This primer set was used to assay all HBoV genotypes and detected only those structures that correspond to an episomal form of this viral genome. Also, for the first time, head-to-tail nucleotide sequences have been determined for HBoV4. Secondary structures of the terminal noncoding regions (NCRs) of episomal forms have been computed for all HBoV genotypes, as well as for the canine bocavirus. Conserved secondary structures in episomal NCRs, which are likely to play an important part in the replication of bocaviruses, were found. NCR heterogeneity in the genomes of individual HBoV isolates has been shown for the first time.

Recombination in the evolution of human bocavirus.

Whole genome sequencing of Novosibirsk human bocavirus (HBoV) isolates has detected an isolate that emerged via recombination between HBoV3 and HBoV4 genotypes. The recombination site is located between regions with abnormally low and abnormally high GC contents in the genome. This site is a bocavirus recombination hotspot and coincides with one of two parvovirus recombination hotspots. The Novosibirsk recombinant isolate, which is similar to a previously studied isolate from Thailand, utilizes the strategy of borrowing ORF3, which encodes structural proteins, of a rare genotype HBoV4. The role of recombination in HBoV evolution is discussed.

Recombination analysis based on the HAstV-2 and HAstV-4 complete genomes.

Complete genome sequences of previously unstudied human astrovirus subgenotypes - HAstV-2a and HAstV-2c - and two isolates of a rare genotype HAstV-4 have been determined. These isolates were recovered from fecal samples of young children hospitalized with acute intestinal infections in Novosibirsk (Russia). Three of the four sequenced isolates (HAstV-2a, HAstV-2c, and HAstV-4) are recombinants. It has been shown that all known HAstV-2 genomes have emerged via recombination; the HAstV-1 and HAstV-4 genotypes contain both recombinant and non-recombinant isolates; and all HAstV-3, HAstV-5, and HAstV-6 whole-genome sequences display no reliable signs of recombination. The average mutation accumulation rate has been determined based on an extended ORF2 fragment and amounts to 1.0×10(-3) substitutions per site per year. The evolutionary chronology of current HAstV genotypes has been reconstructed.

Presence of aberrant VH6 domains in anti-interferon-γ autoantibodies in multiple sclerosis.

BACKGROUND: Anti-cytokine autoantibodies (auto-Abs) are ubiquitous both in patients suffering from infectious, inflammatory and autoimmune diseases and in healthy individuals. Particularly anti-IFN-γ auto-Abs are shown to be elevated in blood of multiple sclerosis (MS) patients. OBJECTIVE: The aim of present study was to investigate whether repertoires of anti-IFN-γ auto-Abs differ in MS patients and healthy donors. METHODS: Using phage display technique we have compared repertoires of the genes encoding anti-IFN-γ single-chain variable fragments selected from MS and naïve phage display libraries. RESULTS: The panel of anti-IFN-γ auto-Abs selected from MS library includes (i) 'fetal' auto-Abs, encoded by the VH6-1 gene segment and the combination proximal D segments with distal JH segments; (ii) naïve auto-Abs; (iii) affinity maturated antibodies; and (iv) abnormal single-domain antibodies. Meanwhile, the panel of anti-IFN-γ auto-Abs selected from naïve library mainly contains the naïve antibodies. Moreover, the overall antibody repertoire of MS library is skewed compared to the overall repertoire of naïve library and also contained the antibodies carrying a 'fetal' VH6 domain and the ratio of κ and λ chains was reversed. CONCLUSIONS: These results suggest existence of a special mechanism or trigger that provides for reconstitution of the immune system in MS.

Evolutionary time-scale of primate bocaviruses.

Human bocavirus (HBoV) is associated with acute gastroenteritis in humans, occurring mostly in young children and elderly people. Four bocavirus genotypes (HBoV1-HBoV4) have been found so far. Since there were no data on the contribution of HBoV to gastroenteritis in Russia, 1781 fecal samples collected from infants hospitalized with acute gastroenteritis in Novosibirsk, Russia during one year were tested for the presence of nucleic acids from HBoV and three major gastrointestinal viruses (rotavirus A, norovirus II, and astrovirus). HBoV was detected only in 1.9% of the samples: HBoV1 was detected in 0.6% and HBoV2, in 1.3%. Complete genome sequencing of three Novosibirsk isolates was performed. An evolutionary analysis of these sequences and the available sequences of human and great apes bocaviruses demonstrated that the current HBoV genotypes diverged comparatively recently, about 60-300years ago. The independent evolution of bocaviruses from chimpanzees and gorillas commenced at the same time period. This suggests that these isolates of great apes bocaviruses belong to separate genotypes within the species of human bocavirus, which is actually the primate bocavirus. The rate of mutation accumulation in the genome of primate bocaviruses has been estimated as approximately 9×10(-4)substitutions/site/year. It has been demonstrated that HBoV1 diverged from the ancestor common with chimpanzee bocavirus approximately 60-80years ago, while HBoV4 separated from great apes bocaviruses about 200-300years ago. The hypothesis postulating independent evolution of HBoV1 and HBoV4 genotypes from primate bocaviruses has been proposed.

A retrospective study of the orthopoxvirus molecular evolution.

The data on the structure of conserved genes of the Old and New World orthopoxviruses and unclassified Yoka poxvirus were used for a Bayesian dating of their independent evolution. This reconstruction estimates the time when an orthopoxvirus ancestor was transferred to the North American continent as approximately 50 thousand years ago (TYA) and allows for relation of this time interval with the global climate changes (with one of the short-term warmings during the Last Ice Age). The onset of the Yoka poxvirus evolution was assessed as approximately 90TYA. Availability of a large number of genome sequences of various cowpox virus strains provided for a comprehensive analysis of the orthopoxvirus evolutionary history. Such a study is especially topical in view of the postulated role of this virus in the evolution of various orthopoxviruses, namely, as an progenitor virus. The computations have demonstrated that the orthopoxviruses diverged from the ancestor virus to form the extant species about 10TYA, while the forbear of horsepox virus separated about 3TYA. An independent evolution of taterapox, camelpox, and variola viruses commenced approximately 3.5TYA. Study of the geographic distribution areas of the hosts of these three orthopoxviruses suggests the hypothesis on the region of their origin. It is likely that these viruses first emerged in Africa, in the region of the Horn of Africa, and that the introduction of camels to East Africa induced their divergent evolution.

High evolutionary rate of human astrovirus.

Human astrovirus is one of the etiological agents of acute gastroenteritis in humans, mostly in young children and elderly people. Complete genome sequencing of four human astrovirus strains isolated in Novosibirsk, Russia was performed. Analysis of these sequences and the sequences available in GenBank database has detected numerous potential recombination breakpoints. For the first time the rate of human astrovirus evolution was estimated based on the genome fragments without recombination breakpoints; the determined rate is typical of the RNA viruses with high evolutionary rate, amounting to approximately 3.7 × 10(-3) nucleotide substitutions per site per year, and for the synonymous changes, 2.8 × 10(-3) nucleotide substitutions per site per year.

Molecular dating in the evolution of vertebrate poxviruses.

OBJECTIVES: The goal of this work was to study the evolutionary history of the vertebrate poxviruses using the Bayesian relaxed clock and a large set of highly conserved vitally important viral genes. METHODS: Phylogenetic analysis was performed by the maximum likelihood method using the Paup program. The dating method of Bayes, realized in the Multidivtime, was made. RESULTS: The rate of poxviral evolution is estimated as 0.5-7 × 10(-6) nucleotide substitutions per site per year. We inferred that the modern viruses of the genus Avipoxvirus diverged from the ancestor nearly 249 ± 69 thousand years ago (Tya). The progenitor of the genus Orthopoxvirus separated approximately 166 ± 43 Tya. The separation of the forebear of the genus Leporipoxvirus took place about 137 ± 35 Tya. The next to diverge was the ancestor of the genus Yatapoxvirus. The progenitor of Capripoxvirus and Suipoxvirus diverged 111 ± 29 Tya. CONCLUSION: The evolutionary analysis based on the historical data and utilizing the Bayesian relaxed clock allowed us to determine the molecular evolution rates of the AT-rich genomes of the vertebrate poxviruses and assess the times of their emergences. Involvement of a large set of the conserved genes controlled by stabilizing selection allowed us to perform molecular dating of the vertebrate poxvirus history.

Real-time PCR system for detection of orthopoxviruses and simultaneous identification of smallpox virus.

A screening assay for real-time LightCycler (Roche Applied Science, Mannheim, Germany) PCR identification of smallpox virus DNA was developed and compiled in a kit system under good manufacturing practice conditions with standardized reagents. In search of a sequence region unique to smallpox virus, the nucleotide sequence of the 14-kDa fusion protein gene of each of 14 variola virus isolates of the Russian World Health Organization smallpox virus repository was determined and compared to published sequences. PCR primers were designed to detect all Eurasian-African species of the genus ORTHOPOXVIRUS: A single nucleotide mismatch resulting in a unique amino acid substitution in smallpox virus was used to design a hybridization probe pair with a specific sensor probe that allows reliable differentiation of smallpox virus from other orthopoxviruses by melting-curve analysis. The applicability was demonstrated by successful amplification of 120 strains belonging to the orthopoxvirus species variola, vaccinia, camelpox, mousepox, cowpox, and monkeypox virus. The melting temperatures (T(m)s) determined for 46 strains of variola virus (T(m)s, 55.9 to 57.8 degrees C) differed significantly (P = 0.005) from those obtained for 11 strains of vaccinia virus (T(m)s, 61.7 to 62.7 degrees C), 15 strains of monkeypox virus (T(m)s, 61.9 to 62.2 degrees C), 40 strains of cowpox virus (T(m)s, 61.3 to 63.7 degrees C), 8 strains of mousepox virus (T(m), 61.9 degrees C), and 8 strains of camelpox virus (T(m)s, 64.0 to 65.0 degrees C). As most of the smallpox virus samples were derived from infected cell cultures and tissues, smallpox virus DNA could be detected in a background of human DNA. By applying probit regression analysis, the analytical sensitivity was determined to be 4 copies of smallpox virus target DNA per sample. The DNAs of several human herpesviruses as well as poxviruses other than orthopoxviruses were not detected by this method. The assay proved to be a reliable technique for the detection of orthopoxviruses, with the advantage that it can simultaneously identify variola virus.