Evaluation of the population heterogeneity of TBEV laboratory variants using high-throughput sequencing.

We studied minor variants within two tick-borne encephalitis virus (TBEV) populations with a common ancestor: the mouse brain-adapted variant EK-328c and the tick-adapted variant M. High-throughput sequencing with custom amplicons from RT-PCR viral RNA was performed on Illumina MiSeq 2*250 paired-end v2 chemistry. Using the LowFreq program (default settings) and Sanger-sequenced consensus as a reference, variants with an abundance of 1 % and above within the studied populations were identified. Using the obtained data in the context of our previous studies, we concluded that TBEV variants, which are different from the major population phenotype and can become a major part of the viral population under favourable environmental conditions, can exist at abundances of less than 1 % in the long-term. The comparison of our data with the literature allowed us to conclude that the laboratory variant EK-328c and variant M have similar SNV counts to TBEV variants from natural populations and some fast-evolving RNA viruses.

Evolutionary origins of hepatitis A virus in small mammals.

Hepatitis A virus (HAV) is an ancient and ubiquitous human pathogen recovered previously only from primates. The sole species of the genus Hepatovirus, existing in both enveloped and nonenveloped forms, and with a capsid structure intermediate between that of insect viruses and mammalian picornaviruses, HAV is enigmatic in its origins. We conducted a targeted search for hepatoviruses in 15,987 specimens collected from 209 small mammal species globally and discovered highly diversified viruses in bats, rodents, hedgehogs, and shrews, which by pairwise sequence distance comprise 13 novel Hepatovirus species. Near-complete genomes from nine of these species show conservation of unique hepatovirus features, including predicted internal ribosome entry site structure, a truncated VP4 capsid protein lacking N-terminal myristoylation, a carboxyl-terminal pX extension of VP1, VP2 late domains involved in membrane envelopment, and a cis-acting replication element within the 3D(pol) sequence. Antibodies in some bat sera immunoprecipitated and neutralized human HAV, suggesting conservation of critical antigenic determinants. Limited phylogenetic cosegregation among hepatoviruses and their hosts and recombination patterns are indicative of major hepatovirus host shifts in the past. Ancestral state reconstructions suggest a Hepatovirus origin in small insectivorous mammals and a rodent origin of human HAV. Patterns of infection in small mammals mimicked those of human HAV in hepatotropism, fecal shedding, acute nature, and extinction of the virus in a closed host population. The evolutionary conservation of hepatovirus structure and pathogenesis provide novel insight into the origins of HAV and highlight the utility of analyzing animal reservoirs for risk assessment of emerging viruses.

Close genetic relatedness of picornaviruses from European and Asian bats.

Our investigation of 1004 faecal specimens from European bats for picornaviruses by broadly reactive nested reverse transcription-PCR found picornaviral RNA in 28 samples (2.8 %). Phylogenetic analysis of the partial 3D genomic region suggested that one bat virus belonged to the species Enterovirus G (EV-G, formerly Porcine enterovirus B). Bat infection was supported by relatively high EV-G concentrations of 1.1×106 RNA copies per gram of faeces. All other bat viruses belonged either to the bat-associated genus Mischivirus, or to an unclassified Picornaviridae group distantly related to the genus Sapelovirus. Members of this unclassified sapelovirus-related group had RNA secondary structures in their 3'-nontranslated regions that were typical of enteroviruses and that resembled structures that occur in bat-associated coronaviruses, suggesting ancient recombination events. Based on sequence distances, several picornaviruses from European and Chinese bats were likely conspecific, suggesting connectivity of virus populations. Due to their high mutation rates and their diversity, picornaviruses may be useful tools for studies of bat and virus ecology.

A Cluster of Paralytic Poliomyelitis Cases Due to Transmission of Slightly Diverged Sabin 2 Vaccine Poliovirus.

UNLABELLED: Four cases of acute flaccid paralysis caused by slightly evolved (Sabin-like) vaccine polioviruses of serotype 2 were registered in July to August 2010 in an orphanage of Biysk (Altai Region, Russia). The Biysk cluster of vaccine-associated paralytic poliomyelitis (VAPP) had several uncommon, if not unique, features. (i) Until this outbreak, Sabin-like viruses (in distinction to more markedly evolved vaccine-derived polioviruses [VDPVs]) were reported to cause only sporadic cases of VAPP. Consequently, VAPP cases were not considered to require outbreak-type responses. However, the Biysk outbreak completely blurred the borderline between Sabin-like viruses and VDPVs in epidemiological terms. (ii) The outbreak demonstrated a very high disease/infection ratio, apparently exceeding even that reported for wild polioviruses. The viral genome structures did not provide any substantial hints as to the underlying reason(s) for such pathogenicity. (iii) The replacement of intestinal poliovirus lineages by other Sabin-like lineages during short intervals after the disease onsets was observed in two patients. Again, the sequences of the respective genomes provided no clues to explain these events. (iv) The polioviruses isolated from the patients and their contacts demonstrated a striking heterogeneity as well as rapid and uneven evolution of the whole genomes and their parts, apparently due to extensive interpersonal contacts in a relatively small closed community, multiple bottlenecking, and recombination. Altogether, the results demonstrate several new aspects of pathogenicity, epidemiology, and evolution of vaccine-related polioviruses and underscore several serious gaps in understanding these problems. IMPORTANCE: The oral poliovirus vaccine largely contributed to the nearly complete disappearance of poliovirus-caused poliomyelitis. Being generally safe, it can, in some cases, result in a paralytic disease. Two types of such outcomes are distinguished: those caused by slightly diverged (Sabin-like) viruses on the one hand and those caused by significantly diverged VDPVs on the other. This classification is based on the number of mutations in the viral genome region encoding a viral structural protein. Until now, only sporadic poliomyelitis cases due to Sabin-like polioviruses had been described, and in distinction from the VDPV-triggered outbreaks, they did not require broad-scale epidemiological responses. Here, an unusual outbreak of poliomyelitis caused by a Sabin-like virus is reported, which had an exceptionally high disease/infection ratio. This outbreak blurred the borderline between Sabin-like polioviruses and VDPVs both in pathogenicity and in the kind of responses required, as well as underscoring important gaps in understanding the pathogenicity, epidemiology, and evolution of vaccine-derived polioviruses.

Robustness against serum neutralization of a poliovirus type 1 from a lethal epidemic of poliomyelitis in the Republic of Congo in 2010.

In 2010, a large outbreak of poliomyelitis with unusual 47% lethality occurred in Pointe Noire, Republic of Congo. Vaccine-mediated immunity against the outbreak virus was never investigated. A wild poliovirus 1 (WPV1) isolated from a fatal case (termed PV1-RC2010) showed a previously unknown combination of amino acid exchanges in critical antigenic site 2 (AgS2, VP1 capsid protein positions 221SAAL → 221PADL). These exchanges were also detected in an additional 11 WPV1 strains from fatal cases. PV1-RC2010 escaped neutralization by three different mAbs relevant for AgS2. Virus neutralization was tested in sera from fatal cases, who died before supplementary immunization (n = 24), Gabonese recipients of recent oral polio vaccination (n = 12), routinely vaccinated German medical students (n = 34), and German outpatients tested for antipoliovirus immunity (n = 17) on Vero, human rhabdomyosarcoma, and human epidermoid carcinoma 2 cells. Fatal poliomyelitis cases gave laboratory evidence of previous trivalent vaccination. Neutralizing antibody titers against PV1-RC2010 were significantly lower than those against the vaccine strain Sabin-1, two genetically distinct WPV1s isolated in 1965 and 2010 and two genetically distinct vaccine-derived PV strains. Of German vaccinees tested according to World Health Organization protocols, 15-29% were unprotected according to their neutralization titers (<1:8 serum dilution), even though all were protected against Sabin-1. Phylogenetic analysis of the WPV1 outbreak strains suggested a recent introduction of virus progenitors from Asia with formation of separate Angolan and Congolese lineages. Only the latter carried both critical AgS2 mutations. Antigenetically variant PVs may become relevant during the final phase of poliomyelitis eradication in populations with predominantly vaccine-derived immunity. Sustained vaccination coverage and clinical and environmental surveillance will be necessary.

Highly divergent hepaciviruses from African cattle.

UNLABELLED: The hepatitis C virus (HCV; genus Hepacivirus) is a highly relevant human pathogen. Unique hepaciviruses (HV) were discovered recently in animal hosts. The direct ancestor of HCV has not been found, but the genetically most closely related animal HVs exist in horses. To investigate whether other peridomestic animals also carry HVs, we analyzed sera from Ghanaian cattle for HVs by reverse transcription-PCR (RT-PCR). Nine of 106 specimens from different sampling sites contained HV RNA (8.5%) at median viral loads of 1.6 × 10(5) copies/ml. Infection seemed unrelated to cattle age and gender. Near-full-genome sequencing of five representative viruses confirmed taxonomic classifications. Cattle HVs formed two distinct phylogenetic lineages that differed by up to 17.7% on the nucleotide level in the polyprotein-encoding region, suggesting cocirculation of different virus subtypes. A conserved microRNA122-binding site in the 5' internal ribosomal entry site suggested liver tropism of cattle HVs. Phylogenetic analyses suggested the circulation of HVs in cattle for several centuries. Cattle HVs were genetically highly divergent from all other HVs, including HCV. HVs from genetically related equine and bovine hosts were not monophyletic, corroborating host shifts during the evolution of the genus Hepacivirus. Similar to equine HVs, the genetic diversity of cattle HVs was low compared to that of HCV genotypes. This suggests an influence of the human-modified ecology of peridomestic animals on virus diversity. Further studies should investigate the occurrence of cattle HVs in other geographic areas and breeds, virus pathogenicity in cattle, and the potential exposure of human risk groups, such as farmers, butchers, and abattoir workers. IMPORTANCE: HCV (genus Hepacivirus) is a major human pathogen, causing liver failure and cancer. Unique hepaciviruses (HVs) were discovered over the last few years in animals, but the direct ancestor of HCV has not been found. The animal HV most closely related to HCV so far originated from horses, suggesting that other livestock animals also harbor HVs. Therefore, we investigated African cattle and discovered previously unknown HVs at high prevalence and viral loads. Because of the agricultural importance of cattle, it may be relevant to investigate HV pathogenicity. The frequent exposure of humans to cattle also may warrant investigations of the zoonotic potential of these viruses. Evolutionary analyses suggested that cattle HVs have existed for centuries. Despite the genetic relatedness of their animal hosts, HVs from cattle and horses were not phylogenetically related, corroborating frequent host shifts during the evolution of the genus Hepacivirus.

Evidence for novel hepaciviruses in rodents.

Hepatitis C virus (HCV) is among the most relevant causes of liver cirrhosis and hepatocellular carcinoma. Research is complicated by a lack of accessible small animal models. The systematic investigation of viruses of small mammals could guide efforts to establish such models, while providing insight into viral evolutionary biology. We have assembled the so-far largest collection of small-mammal samples from around the world, qualified to be screened for bloodborne viruses, including sera and organs from 4,770 rodents (41 species); and sera from 2,939 bats (51 species). Three highly divergent rodent hepacivirus clades were detected in 27 (1.8%) of 1,465 European bank voles (Myodes glareolus) and 10 (1.9%) of 518 South African four-striped mice (Rhabdomys pumilio). Bats showed anti-HCV immunoblot reactivities but no virus detection, although the genetic relatedness suggested by the serologic results should have enabled RNA detection using the broadly reactive PCR assays developed for this study. 210 horses and 858 cats and dogs were tested, yielding further horse-associated hepaciviruses but none in dogs or cats. The rodent viruses were equidistant to HCV, exceeding by far the diversity of HCV and the canine/equine hepaciviruses taken together. Five full genomes were sequenced, representing all viral lineages. Salient genome features and distance criteria supported classification of all viruses as hepaciviruses. Quantitative RT-PCR, RNA in-situ hybridisation, and histopathology suggested hepatic tropism with liver inflammation resembling hepatitis C. Recombinant serology for two distinct hepacivirus lineages in 97 bank voles identified seroprevalence rates of 8.3 and 12.4%, respectively. Antibodies in bank vole sera neither cross-reacted with HCV, nor the heterologous bank vole hepacivirus. Co-occurrence of RNA and antibodies was found in 3 of 57 PCR-positive bank vole sera (5.3%). Our data enable new hypotheses regarding HCV evolution and encourage efforts to develop rodent surrogate models for HCV.

Mutational robustness and resilience of a replicative cis-element of RNA virus: Promiscuity, limitations, relevance.

Since replication of RNA-viruses is generally a low-fidelity process, it would be advantageous, if specific interactions of their genomic cis-elements with dedicated ligands are relatively tolerant to mutations. The specificity/promiscuity trade-off of such interactions was addressed here by investigating structural requirements of the oriL (also known as the clover leaf-like element), of poliovirus RNA, a replicative cis-element containing a conserved essential tetraloop functionally interacting with the viral protein 3CD. The sequence of this tetraloop and 2 adjacent base-pairs was randomized in the viral genome, and viable viruses were selected in susceptible cells. Strikingly, each position of this octanucleotide in 62 investigated viable viruses could be occupied by any nucleotide (with the exception of one position, which lacked U), though with certain sequence preferences, confirmed by engineering mutant viral genomes whose phenotypic properties were found to correlate with the strength of the cis-element/ligand interaction. The results were compatible with a hypothesis that functional recognition by 3CD requires that this tetraloop should stably or temporarily adopt a YNMG-like (Y=U/C, N=any nucleotide, M=A/C) fold. The fitness of "weak" viruses could be increased by compensatory mutations "improving" the tetraloops. Otherwise, the recognition of "bad" tetraloops might be facilitated by alterations in the 3CD protein. The virus appeared to tolerate mutations in its cis-element relaying on either robustness (spatial structure degeneracy) or resilience (a combination of dynamic RNA folding, low-fidelity replication modifying the cis-element or its ligand, and negative selection). These mechanisms (especially resilience involving metastable low-fit intermediates) can also contribute to the viral evolvability.

Comprehensive Elucidation of the Role of L and 2A Security Proteins on Cell Death during EMCV Infection.

The EMCV L and 2A proteins are virulence factors that counteract host cell defense mechanisms. Both L and 2A exhibit antiapoptotic properties, but the available data were obtained in different cell lines and under incomparable conditions. This study is aimed at checking the role of these proteins in the choice of cell death type in three different cell lines using three mutants of EMCV lacking functional L, 2A, and both proteins together. We have found that both L and 2A are non-essential for viral replication in HeLa, BHK, and RD cell lines, as evidenced by the viability of the virus in the absence of both functional proteins. L-deficient infection led to the apoptotic death of HeLa and RD cells, and the necrotic death of BHK cells. 2A-deficient infection induced apoptosis in BHK and RD cells. Infection of HeLa cells with the 2A-deficient mutant was finalized with exclusive caspase-dependent death with membrane permeabilization, morphologically similar to pyroptosis. We also demonstrated that inactivation of both proteins, along with caspase inhibition, delayed cell death progression. The results obtained demonstrate that proteins L and 2A play a critical role in choosing the path of cell death during infection, but the result of their influence depends on the properties of the host cells.

Suppression of injuries caused by a lytic RNA virus (mengovirus) and their uncoupling from viral reproduction by mutual cell/virus disarmament.

Viruses often elicit cell injury (cytopathic effect [CPE]), a major cause of viral diseases. CPE is usually considered to be a prerequisite for and/or consequence of efficient viral growth. Recently, we proposed that viral CPE may largely be due to host defensive and viral antidefensive activities. This study aimed to check the validity of this proposal by using as a model HeLa cells infected with mengovirus (MV). As we showed previously, infection of these cells with wild-type MV resulted in necrosis, whereas a mutant with incapacitated antidefensive ("security") viral leader (L) protein induced apoptosis. Here, we showed that several major morphological and biochemical signs of CPE (e.g., alterations in cellular and nuclear shape, plasma membrane, cytoskeleton, chromatin, and metabolic activity) in cells infected with L(-) mutants in the presence of an apoptosis inhibitor were strongly suppressed or delayed for long after completion of viral reproduction. These facts demonstrate that the efficient reproduction of a lytic virus may not directly require development of at least some pathological alterations normally accompanying infection. They also imply that L protein is involved in the control of many apparently unrelated functions. The results also suggest that the virus-activated program with competing necrotic and apoptotic branches is host encoded, with the choice between apoptosis and necrosis depending on a variety of intrinsic and extrinsic conditions. Implementation of this defensive suicidal program could be uncoupled from the viral reproduction. The possibility of such uncoupling has significant implications for the pathogenesis and treatment of viral diseases.

Recombinant TBEV Protein E of the Siberian Subtype Is a Candidate Antigen in the ELISA Test System for Differential Diagnosis.

The tick-borne encephalitis virus (TBEV) is one of the most common members of the Orthoflavivirus genus, which comprises the causative agents of severe diseases in humans and animals. Due to the expanding areas of orthoflavivirus infection, its differential diagnosis is highly demanded. Commercial test kits based on inactivated TBEV may not provide reliable differentiation between flaviviruses because of serological crossover in this genus. Application of recombinant domains (sE and dIII) of the TBEV Sukhar-strain protein E as antigens in an ELISA test system allowed us to identify a wide range of antibodies specific to different TBEV strains. We tested 53 sera from human patients with confirmed TBE diagnosis (the efficacy of our test system based on sE protein was 98%) and 56 sera from patients with other orthoflavivirus infections in which no positive ones were detected using our ELISA test system, thus being indicative of its 100% specificity. We also tested mouse and rabbit sera containing antibodies specific to 17 TBEV strains belonging to different subtypes; this assay exhibited high efficacy and differentiation ability in detecting antibodies against TBEV from other orthoflaviviruses such as Omsk hemorrhagic fever, Powassan, yellow fever, dengue, West Nile, Zika, and Japanese encephalitis viruses.

Novel serotypes 105 and 116 are members of distinct subgroups of human enterovirus C.

The full coding sequences of two novel human enterovirus (HEV)-C serotypes 105 and 116, sampled in the Republic of the Congo in 2010 and in Russia in 2011, were identified in this study. Enterovirus (EV)-105 was closest to EV-104 in the 5' NTR and to EV-109 in the coding genome region. It had the same unconventional 5' NTR as EV-104 and EV-109. The non-cytopathogenic EV-116 was phylogenetically close to coxsackievirus (CV)-A1, CV-A19 and CV-A22, which also cannot be propagated in routinely used cell cultures. There were signs of recombination within this subgroup of HEV-C; however, recombination with conventional HEV-C was restricted, implying partial reproductive isolation. As there is also evidence of different permissive replication systems and distinct genetic properties of these subgroups, they may represent subspecies of the HEV-C species or different stages of speciation.

Enteroviruses Manipulate the Unfolded Protein Response through Multifaceted Deregulation of the Ire1-Xbp1 Pathway.

Many viruses are known to trigger endoplasmic reticulum (ER) stress in host cells, which in turn can develop a protective unfolded protein response (UPR). Depending on the conditions, the UPR may lead to either cell survival or programmed cell death. One of three UPR branches involves the upregulation of Xbp1 transcription factor caused by the unconventional cytoplasmic splicing of its mRNA. This process is accomplished by the phosphorylated form of the endoribonuclease/protein kinase Ire1/ERN1. Here, we show that the phosphorylation of Ire1 is up-regulated in HeLa cells early in enterovirus infection but down-regulated at later stages. We also find that Ire1 is cleaved in poliovirus- and coxsackievirus-infected HeLa cells 4-6 h after infection. We further show that the Ire1-mediated Xbp1 mRNA splicing is repressed in infected cells in a time-dependent manner. Thus, our results demonstrate the ability of enteroviruses to actively modulate the Ire1-Xbp1 host defensive pathway by inducing phosphorylation and proteolytic cleavage of the ER stress sensor Ire1, as well as down-regulating its splicing activity. Inactivation of Ire1 could be a novel mode of the UPR manipulation employed by viruses to modify the ER stress response in the infected cells.

Genomic features and evolutionary constraints in Saffold-like cardioviruses.

This study identified the complete genomic sequence of four type 2 and type 3 human Saffold-like cardioviruses (SLCVs) isolated in Germany and Brazil. The secondary structures of the SLCV internal ribosome entry sites (IRESs) were deduced based on RNA base-pairing conservation and co-variation, using an established Theiler's murine encephalomyelitis virus (TMEV) IRES structure as a reference. The SLCV IRES was highly similar to that of TMEV, but motifs critical in TMEV for binding of the polypyrimidine tract-binding protein (PTB) were disrupted. In TMEV, corresponding alterations have been associated with reduced neurovirulence in mice. In the non-structural genome region, there was evidence of multiple intertypic recombination events between different SLCV types. Between viruses of the same type, recombination also occurred in the capsid-encoding genome region. There were apparently no recombination events between mouse TMEV and human SLCV. In another genus of the family Picornaviridae, Enterovirus, natural recombination occurs strictly within species and can serve as an additional criterion for delimiting species. Accordingly, the results of this study suggest that SLCV and TMEV may represent distinct species within the genus Cardiovirus.

Antiapoptotic activity of the cardiovirus leader protein, a viral "security" protein.

Apoptosis is a common antiviral defensive mechanism that potentially limits viral reproduction and spread. Many viruses possess apoptosis-suppressing tools. Here, we show that the productive infection of HeLa cells with encephalomyocarditis virus (a cardiovirus) was not accompanied by full-fledged apoptosis (although the activation of caspases was detected late in infection) but rather elicited a strong antiapoptotic state, as evidenced by the resistance of infected cells to viral and nonviral apoptosis inducers. The development of the antiapoptotic state appeared to depend on a function(s) of the viral leader (L) protein, since its mutational inactivation resulted in the efflux of cytochrome c from mitochondria, the early activation of caspases, and the appearance of morphological and biochemical signs of apoptosis in a significant proportion of infected cells. Infection with both wild-type and L-deficient viruses induced the fragmentation of mitochondria, which in the former case was not accompanied with cytochrome c efflux. Although the exact nature of the antiapoptotic function(s) of cardioviruses remains obscure, our results suggested that it includes previously undescribed mechanisms operating upstream and possibly downstream of the mitochondrial level, and that L is involved in the control of these mechanisms. We propose that cardiovirus L belongs to a class of viral proteins, dubbed here security proteins, whose roles consist solely, or largely, in counteracting host antidefenses. Unrelated L proteins of other picornaviruses as well as their highly variable 2A proteins also may be security proteins. These proteins appear to be independent acquisitions in the evolution of picornaviruses, implying multiple cases of functional (though not structural) convergence.

Case of Poliomyelitis Caused by Significantly Diverged Derivative of the Poliovirus Type 3 Vaccine Sabin Strain Circulating in the Orphanage.

Significantly divergent polioviruses (VDPV) derived from the oral poliovirus vaccine (OPV) from Sabin strains, like wild polioviruses, are capable of prolonged transmission and neuropathology. This is mainly shown for VDPV type 2. Here we describe a molecular-epidemiological investigation of a case of VDPV type 3 circulation leading to paralytic poliomyelitis in a child in an orphanage, where OPV has not been used. Samples of feces and blood serum from the patient and 52 contacts from the same orphanage were collected twice and investigated. The complete genome sequencing was performed for five polioviruses isolated from the patient and three contact children. The level of divergence of the genomes of the isolates corresponded to approximately 9-10 months of evolution. The presence of 61 common substitutions in all isolates indicated a common intermediate progenitor. The possibility of VDPV3 transmission from the excretor to susceptible recipients (unvaccinated against polio or vaccinated with inactivated poliovirus vaccine, IPV) with subsequent circulation in a closed children's group was demonstrated. The study of the blood sera of orphanage residents at least twice vaccinated with IPV revealed the absence of neutralizing antibodies against at least two poliovirus serotypes in almost 20% of children. Therefore, a complete rejection of OPV vaccination can lead to a critical decrease in collective immunity level. The development of new poliovirus vaccines that create mucosal immunity for the adequate replacement of OPV from Sabin strains is necessary.

Genetic diversity of Kemerovo virus and phylogenetic relationships within the Great Island virus genetic group.

Kemerovo virus (KEMV) is a member of the Great Island virus genetic group, belonging to the tick-borne arboviruses of the genus Orbivirus within the family Reoviridae. Nine strains of KEMV, which were isolated from various locations in Russia, were sequenced by high-throughput sequencing to study their intraspecific diversity and the interspecific relationships of viruses within the Great Island genetic group. For the first time, multiple reassortment within KEMV was reliably demonstrated. Different types of independently emerged alternative reading frames in segment 9 and heterogeneity of the viral population in one of the KEMV strains were found. The hypothesis of the role of an alternative open reading frame (ORF) in segment 9 in KEMV cellular tropism was not confirmed in this study.

Isolation and Characterisation of Alongshan Virus in Russia.

In recent decades, many new flavi-like viruses have been discovered predominantly in different invertebrates and, as was recently shown, some of them may cause disease in humans. The Jingmenvirus (JMV) group holds a special place among flaviviruses and flavi-like viruses because they have a segmented ssRNA(+) genome. We detected Alongshan virus (ALSV), which is a representative of the JMV group, in ten pools of adult Ixodes persulcatus ticks collected in two geographically-separated Russian regions. Three of the ten strains were isolated in the tick cell line IRE/CTVM19. One of the strains persisted in the IRE/CTVM19 cells without cytopathic effect for three years. Most ALSV virions purified from tick cells were spherical with a diameter of approximately 40.5 nm. In addition, we found smaller particles of approximately 13.1 nm in diameter. We obtained full genome sequences of all four segments of two of the isolated ALSV strains, and partial sequences of one segment from the third strain. Phylogenetic analysis on genome segment 2 of the JMV group clustered our novel strains with other ALSV strains. We found evidence for the existence of a novel upstream open reading frame in the glycoprotein-coding segment of ALSV and other members of the JMV group.

Phlebovirus sequences detected in ticks collected in Russia: Novel phleboviruses, distinguishing criteria and high tick specificity.

Phlebovirus is an abundant and rather heterogeneous genus within the Phenuiviridae family (order Bunyavirales). The genus Phlebovirus is divided into two antigenic complexes, which also correspond to the main vector: sandflies/mosquitoes and ticks. Previously, only sandfly/mosquito-borne phleboviruses were associated with human disease, such as Rift Valley fever virus, Toscana virus, Sicilian and Naples Sandfly fever viruses and others. Until recently, tick-borne phleboviruses were not considered as human pathogens. After the discovery of severe fever with thrombocytopenia syndrome, interest to tick-borne phleboviruses has increased dramatically. In the last decade, many novel phleboviruses have been reported in different regions. Despite this, the diversity, ecology and pathogenicity of these viruses still remain obscure. The aim of this work was to study the diversity of phleboviruses in ticks collected in several regions of Russia. We used pan-phlebovirus RT-PCR assays based on multiple degenerate primers targeting the polymerase gene fragment. Arthropod specimens were collected from 2005 to 2018. A total of 5901 Ixodidae ticks combined into 1116 pools were screened. A total of 160 specific amplicons were produced. In three cases RT-PCR assays amplified two distinct viruses from same tick pools. Direct sequencing of amplicons and subsequent phylogenetic analysis revealed twelve representatives of divergent phlebovirus groups. Based on the distribution of pairwise nucleotide sequence identity values, a cut-off (88%) was suggested to distinguish tick-borne phleboviruses. According to this provisional criterion, two viruses found here could be termed novel, while ten viruses have been described in previous studies. Detected phleboviruses demonstrated almost perfect specificity to a tick species or, at least, a genus. The same pattern was observed for tick-borne phleboviruses found in different studies around the world. Viruses that grouped together on a phylogenetic tree and differed less than this sequence identity threshold suggested above were hosted by ticks from the same genus.

Characterization of Mutational Tolerance of a Viral RNA-Protein Interaction.

Replication of RNA viruses is generally markedly error-prone. Nevertheless, these viruses usually retain their identity under more or less constant conditions due to different mechanisms of mutation tolerance. However, there exists only limited information on quantitative aspects of the mutational tolerance of distinct viral functions. To address this problem, we used here as a model the interaction between a replicative cis-acting RNA element (oriL) of poliovirus and its ligand (viral protein 3CD). The mutational tolerance of a conserved tripeptide of 3CD, directly involved in this interaction, was investigated. Randomization of the relevant codons and reverse genetics were used to define the space of viability-compatible sequences. Surprisingly, at least 11 different amino acid substitutions in this tripeptide were not lethal. Several altered viruses exhibited wild-type-like phenotypes, whereas debilitated (but viable) genomes could increase their fitness by the acquisition of reversions or compensatory mutations. Together with our study on the tolerance of oriL (Prostova et al., 2015), the results demonstrate that at least 42 out of 51 possible nucleotide replacements within the two relevant genomic regions are viability-compatible. These results provide new insights into structural aspects of an important viral function as well as into the general problems of viral mutational robustness and evolution.