Conserved Sequences in the 5' and 3' Untranslated Regions of Jingmenvirus Group Representatives.

The Jingmenvirus group (JVG), with members such as Jingmen tick virus (JMTV), Alongshan virus (ALSV), Yanggou tick virus (YGTV), and Takachi virus (TAKV), is drawing attention due to evidence of it causing disease in humans and its unique genome architecture. In the current work, complete untranslated regions (UTRs) of four strains of ALSV and eight strains of YGTV were obtained. An analysis of these sequences, as well as JVG sequences from GenBank, uncovered several regions within viral UTRs that were highly conserved for all the segments and viruses. Bioinformatics predictions suggested that the UTRs of all the segments of YGTV, ALSV, and JMTV could form similar RNA structures. The most notable feature of these structures was a stable stem-loop with one (5' UTR) or two (3' UTR) AAGU tetraloops on the end of a hairpin.

Pseudotyped Virus for Flaviviridae.

Members of Flaviviridae are enveloped single positive-stranded RNA viruses including hepacivirus, pestivirus, pegivirus, and mosquito-transmitted flavivirus, which are important pathogens of infectious diseases and pose serious threats to human health. Pseudotyped virus is an artificially constructed virus-like particle, which could infect host cells similar to a live virus but cannot produce infectious progeny virus. Therefore, pseudotyped virus has the advantages of a wide host range, high transfection efficiency, low biosafety risk, and accurate and objective quantification. It has been widely used in biological characteristics, drug screening, detection methods, and vaccine evaluation of Flaviviridae viruses like hepatitis C virus, Japanese encephalitis virus, dengue virus, and Zika virus.

[Structural Motifs and Spatial Structures of Helicase (NS3) and RNA-dependent RNA-polymerase (NS5) of a Flavi-like Kindia tick virus (unclassified Flaviviridae)].

INTRODUCTION: Kindia tick virus (KITV) is a novel segmented unclassified flavi-like virus of the Flaviviridae family. This virus is associated with ixodes ticks and is potentially pathogenic to humans. The main goal of this work was to search for structural motifs of viral polypeptides and to develop a 3D-structure for viral proteins of the flavi-like KITV. MATERIALS AND METHODS: The complete genome sequences for KITV, Zika, dengue, Japanese encephalitis, West Nile and yellow fever viruses were retrieved from GenBank. Bioinformatics analysis was performed using the different software packages. RESULTS: Analysis of the KITV structural proteins showed that they have no analogues among currently known viral proteins. Spatial models of NS3 and NS5 KITV proteins have been obtained. These models had a high level of topological similarity to the tick-borne encephalitis and dengue viral proteins. The methyltransferase and RNA-dependent RNA-polymerase domains were found in the NS5 KITV. The latter was represented by fingers, palm and thumb subdomains, and motifs A-F. The helicase domain and its main structural motifs IVI were identified in NS3 KITV. However, the protease domain typical of NS3 flaviviruses was not detected. The highly conserved amino acid motives were detected in the NS3 and NS5 KITV. Also, eight amino acid substitutions characteristic of KITV/2018/1 and KITV/2018/2 were detected, five of them being localized in alpha-helix and three in loops of nonstructural proteins. CONCLUSION: Nonstructural proteins of KITV have structural and functional similarities with unsegmented flaviviruses. This confirms their possible evolutionary and taxonomic relationships.

Virome diversity of ticks feeding on domestic mammals in China.

Ticks are considered the second most common pathogen vectors transmitting a broad range of vital human and veterinary viruses. From 2017 to 2018, 640 ticks were collected in eight different provinces in central and western China. Six species were detected, including H.longicornis, De.everestianus, Rh.microplus, Rh.turanicus, Rh.sanguineous, and Hy.asiaticum. Sixty-four viral metagenomic libraries were constructed on the MiSeq Illumina platform, resulting in 13.44 â€‹G (5.88 â€‹× â€‹107) of 250-bp-end reads, in which 2,437,941 are viral reads. We found 27 nearly complete genome sequences, including 16 genome sequences encoding entire protein-coding regions (lack of 3' or 5' end non-coding regions) and complete viral genomes, distributed in the arboviral family (Chuviridae, Rhabdoviridae, Nairoviridae, Phenuiviridae, Flaviviridae, Iflaviridae) as well as Parvoviridae and Polyomaviridae that cause disease in mammals and even humans. In addition, 13 virus sequences found in Chuviridae, Nairoviridae, Flaviviridae, Iflaviridae, Hepeviridae, Parvoviridae, and Polyomaviridae were identified as belonging to a new virus species in the identified viral genera. Besides, an epidemiological survey shows a high prevalence (9.38% and 15.63%) of two viruses (Ovine Copiparvovirus and Bovine parvovirus 2) in the tick cohort.

Piperazine-derived small molecules as potential Flaviviridae NS3 protease inhibitors. In vitro antiviral activity evaluation against Zika and Dengue viruses.

Since 2011 Direct Acting antivirals (DAAs) drugs targeting different non-structural (NS) viral proteins (NS3, NS5A or NS5B inhibitors) have been approved for clinical use in HCV therapies. However, currently there are not licensed therapeutics to treat Flavivirus infections and the only licensed DENV vaccine, Dengvaxia, is restricted to patients with preexisting DENV immunity. Similarly to NS5 polymerase, the NS3 catalytic region is evolutionarily conserved among the Flaviviridae family sharing strong structural similarity with other proteases belonging to this family and therefore is an attractive target for the development of pan-flavivirus therapeutics. In this work we present a library of 34 piperazine-derived small molecules as potential Flaviviridae NS3 protease inhibitors. The library was developed through a privileged structures-based design and then biologically screened using a live virus phenotypic assay to determine the half-maximal inhibitor concentration (IC50) of each compound against ZIKV and DENV. Two lead compounds, 42 and 44, with promising broad-spectrum activity against ZIKV (IC50 6.6 µM and 1.9 µM respectively) and DENV (IC50 6.7 µM and 1.4 µM respectively) and a good security profile were identified. Besides, molecular docking calculations were performed to provide insights about key interactions with residues in NS3 proteases' active sites.

[Development of a method for detection of specific antibodies to E protein of yellow fever virus (Flaviviridae: Flavivirus) by enzyme immunoassay].

INTRODUCTION: Yellow fever (YF) remains one of the most common natural focal infectious diseases in the world. In connection with the increasing tourist flow to countries endemic for YF, the discovery of stable populations of Aedes aegypti and Ae. albopictus which are the main vectors of the yellow fever virus (YFV), in the southern regions of Russia, and the fact that in medical institutions in our country it is possible to obtain a live attenuated vaccine against YF, but there is no way to evaluate the effectiveness of vaccination, the question arises of the development and implementation of diagnostic kits for detecting antibodies (AB) to the pathogen by enzyme immunoassay (ELISA).The aim of this study was to develop a method for detecting specific IgG antibodies to the E protein of YFV by ELISA and assessing its diagnostic characteristics. MATERIALS AND METHODS: A specific cDNA was synthesized by reverse transcription on an RNA template of YFV isolated on a cell culture of Aedes albopictus clone C6/36, and a fragment of the genome coding the YFV E protein was amplified and subsequently cloned into the plasmid pET160 (Thermo Fisher Scientific, USA). The resulting gene fragment was used as a DNA template to obtain a recombinant analog of the third domain of the YFV E protein in Escherichia coli cells (BL-21(DE3)). Next, the immunogenicity of the obtained antigen was evaluated and the analysis conditions were optimized. RESULTS: The optimal conditions for the production of the obtained recombinant E protein of YFV were determined, its specificity was confirmed by immunological methods (Western blot and ELISA), sorption buffers and blocking solutions were selected, and sensitivity and specificity of detection of antibodies to YFV using the recombinant antigen were assessed. CONCLUSION: A method for the detection of specific IgG antibodies to the YFV E protein by ELISA was developed. This diagnostic kit can be used both to study the protective properties of the YF vaccine and to detect imported cases of infection in non-endemic areas.

Proteochemometric Method for pIC50 Prediction of Flaviviridae.

Viruses remain an area of concern despite constant development of antiviral drugs and therapies. One of the contributors is the Flaviviridae family of viruses causing diseases that need attention. Among other anitviral methods, antiviral peptides are being studied as viable candidates. Although antiviral peptides (AVPs) are emerging as potential therapeutics, it is important to assess the efficacy of a given peptide in terms of its bioactivity. Experimental identification of the bioactivity of each potential peptide is an expensive and time consuming task. Computational methods like proteochemometric modeling (PCM) is a promising method for prediction of bioactivity (pIC50) based on peptide and target sequence pair. In this study, we propose a prediction of pIC50 of AVP against the Flaviviridae family that may help make the decision to choose a peptide with desired efficacy. The peptides data was collected from a public database and target sequences were manually curated from literature. Features are calculated using peptide and target sequence PCM descriptors which consist of individual and cross-term features of peptide and respective target. The resultant R 2 and MAPE values are 0.85 and 8.44%, respectively, for prediction of pIC50 value of AVPs.

The Second Human Pegivirus, a Non-Pathogenic RNA Virus with Low Prevalence and Minimal Genetic Diversity.

The second human pegivirus (HPgV-2) is a virus discovered in the plasma of a hepatitis C virus (HCV)-infected patient in 2015 belonging to the pegiviruses of the family Flaviviridae. HPgV-2 has been proved to be epidemiologically associated with and structurally similar to HCV but unrelated to HCV disease and non-pathogenic, but its natural history and tissue tropism remain unclear. HPgV-2 is a unique RNA virus sharing the features of HCV and the first human pegivirus (HPgV-1 or GBV-C). Moreover, distinct from most RNA viruses such as HCV, HPgV-1 and human immunodeficiency virus (HIV), HPgV-2 exhibits much lower genomic diversity, with a high global sequence identity ranging from 93.5 to 97.5% and significantly lower intra-host variation than HCV. The mechanisms underlying the conservation of the HPgV-2 genome are not clear but may include efficient innate immune responses, low immune selection pressure and, possibly, the unique features of the viral RNA-dependent RNA polymerase (RdRP). In this review, we summarize the prevalence, pathogenicity and genetic diversity of HPgV-2 and discuss the possible reasons for the uniformity of its genome sequence, which should elucidate the implications of RNA virus fidelity for attenuated viral vaccines.

Characterization of insect-specific Culex flavivirus (Flaviviridae) nucleotide sequences in mosquitoes from urban parks in São Paulo, Brazil.

BACKGROUND: Despite their worldwide occurrence, the distribution and role of insect-specific flaviviruses (ISFs) remain unclear. METHODS: We evaluated the presence of ISFs in mosquitoes collected in São Paulo, Brazil, using reverse transcription and semi-nested polymerase chain reaction (PCR). Some of the positive samples were subjected to nanopore sequencing. RESULTS: Twelve mosquito pools (2.8%) tested positive for flavivirus infection. Nanopore sequencing was successfully performed on six samples. Phylogenetic analysis grouped these sequences into genotype 2 of Culex flavivirus (CxFV). CONCLUSIONS: The identification of CxFV genotype 2 at new locations in São Paulo highlights the importance of understanding the role of ISFs in mosquito vector competence.

Flaviviridae Nonstructural Proteins: The Role in Molecular Mechanisms of Triggering Inflammation.

Members of the Flaviviridae family are posing a significant threat to human health worldwide. Many flaviviruses are capable of inducing severe inflammation in humans. Flaviviridae nonstructural proteins, apart from their canonical roles in viral replication, have noncanonical functions strongly affecting antiviral innate immunity. Among these functions, antagonism of type I IFN is the most investigated; meanwhile, more data are accumulated on their role in the other pathways of innate response. This review systematizes the last known data on the role of Flaviviridae nonstructural proteins in molecular mechanisms of triggering inflammation, with an emphasis on their interactions with TLRs and RLRs, interference with NF-κB and cGAS-STING signaling, and activation of inflammasomes.

[The analysis of immunoreactivity of individual B-cell epitopes of hepatitis C virus (Flaviviridae: Hepacivirus: Hepatitis С virus) NS4a antigen].

INTRODUCTION: Chronic viral hepatitis C (CHC) is a ubiquitous infectious disease, a significant limitation of which WHO attributes to the use of a new highly effective antiviral therapy. Previously, two B-cell epitopes were identified in NS4a antigen of the hepatitis C virus (HCV). It was shown that certain titers of antibodies (ABs) to the extended C-terminal epitope (1687-1718 a.a.) can predict a high probability of achieving a sustained virological response (SVR) to standard therapy with pegylated interferon-α and ribavirin.The aim of the work was to determine immunoreactivity of two B-cell epitopes (middle and C-terminal) of NS4a antigen, and to estimate a possible association of ABs to them with the achievement of SVR after standard interferon therapy and treatment with direct antiviral drugs (DAAs) daclatasvir and sofosbuvir (velpanat). MATERIALS AND METHODS: Blood serum samples of patients with CHC (n = 113), of which 55 participants received standard interferon therapy, 50 received velpanate treatment, the remaining 8 received no therapy were examined. The middle B-cell epitope (positions 24-34 a.a.) of NS4a was synthesized by the solid-phase method, while the C-terminal epitope (34-54 a.a.) was obtained using genetically engineered techniques. Enzyme immunoassay (ELISA) testing of the sera collected before treatment was performed for the two selected epitopes according to the conventional methods. RESULTS: The antibodies to the C-terminal epitope were detected significantly more frequently than those to the middle one (p = 0.01) when analyzing the blood sera of patients (n = 113). The presence of ABs to the C-terminal epitope in the serum samples of participants who completed standard interferon therapy was associated with the achievement of SVR (p = 0.0245). In the blood sera of participants who completed therapy with velpanate, an association of the presence of ABs to the C-terminal epitope with the achievement of SVR was also established (p < 0.0001). The presence of ABs to the middle B epitope was not associated with the achievement of SVR, regardless of the therapy used. DISCUSSION: The observed difference in the immunoreactivity of the two B-cell determinants may be associated with the localization of the nearest Th-epitopes, the sensitivity of NS4a antigen to proteolytic enzymes, and the peculiarities of epitope presentation by antigen-presenting cells. However, it should be noted that the immunoreactivity of the middle B-epitope is poorly studied. Although the association of ABs to the C-terminal epitope with the achievement of SVR has been shown by several scientific teams, the detailed molecular mechanism of their influence on the effectiveness of therapy is unclear. CONCLUSION: In CHC, ABs to the C-terminal epitope of NS4a are produced more frequently than those to the median epitope. The presence of ABs to the C-terminal epitope is a predictive marker of a high probability of achieving SVR, regardless of the type of therapy and antibody titer.

The epidemiology and disease burden of children hospitalized for viral infections within the family Flaviviridae in China: A national cross-sectional study.

BACKGROUND: Viruses of the family Flaviviridae, including Japanese encephalitis virus (JEV), dengue virus (DENV), yellow fever virus (YFV) and hepatitis C virus (HCV), are widely distributed worldwide. JEV, DENV and YFV belong to the genus Flavivirus, whereas HCV belongs to the genus Hepacivirus. Children's symptoms are usually severe. As a result, rates of hospitalization due to infection with these viruses are high. The epidemiology and disease burden of hospitalized children have rarely been described in detail to date. The objective of this study was to report the general epidemiological characteristics, clinical phenotype, length of stay (LOS), burden of disease, and potential risk factors for hospitalized children infected with JEV, DENV, YFV, or HCV in Chinese pediatric hospitals. METHODOLOGY: A cross-sectional study of epidemiology and disease burden of children hospitalized for Flaviviridae virus infections between December 2015 and December 2020 in China was performed. Face sheets of discharge medical records (FSMRs) were collected from 27 tertiary children's hospitals in the Futang Research Center of Pediatric Development and aggregated into FUTang Update medical REcords (FUTURE). Information on sociodemographic variables, clinical phenotype, and LOS as well as economic burden was included in FSMRs and compared using appropriate statistical tests. FINDINGS: The study described 490 children aged 0-15 years hospitalized for infections with Flaviviridae viruses. Japanese encephalitis (JE) cases are the highest, accounting for 92.65% of the total hospitalization cases caused by Flaviviridae virus infection. The incidence of JE peaked from July to October with a profile of a high proportion of severe cases (68.06%) and low mortality (0.44%). Rural children had a significantly higher incidence than urban children (91.63%). Most hospitalized dengue cases were reported in 2019 when dengue outbreaks occurred in many provinces of China, although only 14 dengue cases were collected during the study period. Yellow fever (YF) is still an imported disease in China. The hospitalizations for children with hepatitis C (HC) were not high, and mild chronic HC was the main clinical phenotype of patients. Among the four viral infections, JE had the highest disease burden (LOS and expenditure) for hospitalized children. CONCLUSION: First, the present study reveals that JE remains the most serious disease due to Flaviviridae virus infection and threatens children's health in China. Many pediatric patients have severe illnesses, but their mortality rate is lower, suggesting that existing treatment is effective. Both JEV vaccination and infection control of rural children should represent a focus of study. Second, although the dual risks of indigenous epidemics and imports of DENV still exist, the prevalence of DENV in children is generally manageable. Third, YFV currently shows no evidence of an epidemic in China. Finally, the proportion of children with chronic hepatitis C (CHC) is relatively large among hospitalized children diagnosed with HCV. Thus, early and effective intervention should be offered to children infected with HCV to ease the burden of CHC on public health.

Phylogenetic Characterisation of the Full Genome of a Bagaza Virus Isolate from Bird Fatalities in South Africa.

Bagaza virus (BAGV), a member of the Ntaya serogroup in the Flavivirus genus of the Flaviviridae, was isolated from the brain tissue of a Himalayan monal pheasant that died following neurological signs in Pretoria, South Africa in 2016. Next-generation sequencing was carried out on this isolate resulting in a genome sequence of 10980nt. The full genome sequence of this isolate, designated ZRU96-16, shared 98% nucleotide identity with a BAGV isolate found in Culex univitattus mosquitoes from Namibia and 97% nucleotide identity with a Spanish BAGV sequence isolated from an infected partridge. In total, seven amino acid variations were unique to ZRU96-16 after alignment with other BAGV and Israel turkey meningoencephalomyelitis (ITV) genomes. The 3'UTR sequence of ZRU96-16 was resolved with sufficient detail to be able to annotate the variable and conserved sequence elements within this region. Multiple sequence alignment of the 3'UTR suggested that it could be useful in lineage designation as more similar viruses carried similar mutations across this region, while also retaining certain unique sites. Maximum likelihood phylogenetic analysis revealed two clusters containing both BAGV and ITVs from Europe, the Middle East and Africa. Broadly, temporal clustering separated isolates into two groups, with one cluster representing viruses from the 1960-2000's and the other from 2010 onwards. This suggests that there is consistent exchange of BAGV and ITV between Europe and Africa. This investigation provides more information on the phylogenetics of an under-represented member of the Flaviviridae and provides an avenue for more extensive research on its pathogenesis and geographic expansion.

The envelope protein of Zika virus interacts with apolipoprotein E early in the infectious cycle and this interaction is conserved on the secreted viral particles.

BACKGROUND: Zika virus (ZIKV), a member of the Flaviviridae family, has caused massive outbreaks of infection in tropical areas over the last decade and has now begun spreading to temperate countries. Little is currently known about the specific host factors involved in the intracellular life cycle of ZIKV. Flaviviridae viruses interact closely with host-cell lipid metabolism and associated secretory pathways. Another Flaviviridae, hepatitis C virus, is highly dependent on apolipoprotein E (ApoE) for the completion of its infectious cycle. We therefore investigated whether ZIKV also interacted with this protein. METHODS: ZIKV infections were performed on both liver and microglia derived cell lines in order to proceed to colocalization analysis and immunoprecipitation assays of ApoE and Zika envelope glycoprotein (Zika E). Transmission electron microscopy combined to immunogold labeling was also performed on the infected cells and related supernatant to study the association of ApoE and Zika E protein in the virus-induced membrane rearrangements and secreted particles, respectively. Finally, the potential of neutralization of anti-ApoE antibodies on ZIKV particles was studied. RESULT: We demonstrated an interaction between ApoE and the Zika E protein. This specific interaction was observed in virus-induced host-cell membrane rearrangements, but also on newly formed intracellular particles. The partial neutralizing effect of anti-ApoE antibody and the immunogold labeling of the two proteins on secreted virions indicates that this interaction is conserved during ZIKV intracellular trafficking and release. CONCLUSIONS: These data suggest that another member of the Flaviviridae also interacts with ApoE, indicating that this could be a common mechanism for the viruses from this family.

Secretory glycoprotein NS1 plays a crucial role in the particle formation of flaviviruses.

Flaviviruses, which are globally distributed and cause a spectrum of potentially severe illnesses, pose a major threat to public health. Although Flaviviridae viruses, including flaviviruses, possess similar genome structures, only the flaviviruses encode the non-structural protein NS1, which resides in the endoplasmic reticulum (ER) and is secreted from cells after oligomerization. The ER-resident NS1 is known to be involved in viral genome replication, but the essential roles of secretory NS1 in the virus life cycle are not fully understood. Here we characterized the roles of secretory NS1 in the particle formation of flaviviruses. We first identified an amino acid residue essential for the NS1 secretion but not for viral genome replication by using protein-protein interaction network analyses and mutagenesis scanning. By using the recombinant flaviviruses carrying the identified NS1 mutation, we clarified that the mutant flaviviruses employed viral genome replication. We then constructed a recombinant NS1 with the identified mutation and demonstrated by physicochemical assays that the mutant NS1 was unable to form a proper oligomer or associate with liposomes. Finally, we showed that the functions of NS1 that were lost by the identified mutation could be compensated for by the in trans-expression of Erns of pestiviruses and host exchangeable apolipoproteins, which participate in the infectious particle formation of pestiviruses and hepaciviruses in the family Flaviviridae, respectively. Collectively, our study suggests that secretory NS1 plays a role in the particle formation of flaviviruses through its interaction with the lipid membrane.

Identification and molecular characterization of highly divergent RNA viruses in cattle, Uganda.

The risk for the emergence of novel viral zoonotic diseases in animals and humans in Uganda is high given its geographical location with high biodiversity. We aimed to identify and characterize viruses in 175 blood samples from cattle selected in Uganda using molecular approaches. We identified 8 viral species belonging to 4 families (Flaviviridae, Peribunyaviridae, Reoviridae and Rhabdoviridae) and 6 genera (Hepacivirus, Pestivirus, Orthobunyavirus, Coltivirus, Dinovernavirus and Ephemerovirus). Four viruses were highly divergent and tetantively named Zikole virus (Family: Flaviviridae), Zeboroti virus (Family: Reoviridae), Zebtine virus (Family: Rhabdoviridae) and Kokolu virus (Family: Rhabdoviridae). In addition, Bovine Hepacivirus, Obodhiang virus, Aedes pseudoscutellaris reovirus and Schmallenberg virus were identified for the first time in Ugandan cattle. We report 8 viral species belonging to 4 viral families including divergent ones in the blood of cattle in Uganda. Hence, cattle may be reservoir hosts for likely emergence of novel viruses with pathogenic potential to cause zoonotic diseases in different species with serious public health implications.

Clinical and molecular aspects of human pegiviruses in the interaction host and infectious agent.

BACKGROUND: Human pegivirus 1 (HPgV-1) is a Positive-sense single-stranded RNA (+ ssRNA) virus, discovered in 1995 as a Flaviviridae member, and the closest human virus linked to HCV. In comparison to HCV, HPgV-1 seems to be lymphotropic and connected to the viral group that infects T and B lymphocytes. HPgV-1 infection is not persuasively correlated to any known human disease; nevertheless, multiple studies have reported a connection between chronic HPgV-1 infection and improved survival in HPgV-1/HIV co-infected patients with a delayed and favorable impact on HIV infection development. While the process has not been thoroughly clarified, different mechanisms for these observations have been proposed. HPgV-1 is categorized into seven genotypes and various subtypes. Infection with HPgV-1 is relatively common globally. It can be transferred parenterally, sexually, and through vertical ways, and thereby its co-infection with HIV and HCV is common. In most cases, the clearance of HPgV-1 from the body can be achieved by developing E2 antibodies after infection. MAIN BODY: In this review, we thoroughly discuss the current knowledge and recent advances in understanding distinct epidemiological, molecular, and clinical aspects of HPgV-1. CONCLUSION: Due to the unique characteristics of the HPgV-1, so advanced research on HPgV-1, particularly in light of HIV co-infection and other diseases, should be conducted to explore the essential mechanisms of HIV clearance and other viruses and thereby suggest novel strategies for viral therapy in the future.

Occurrence and molecular characterization of human pegivirus-1 (HPgV-1) viremia in healthy volunteer blood donors from Northern Portugal.

Human pegivirus-1 (HPgV-1) is a member of the Flaviviridae family and the Pegivirus genus. Despite having been discovered 25 years ago, there is still much to know regarding HPgV-1 clinical impact, as this virus is currently not associated with any pathology. Yet, HPgV-1 prevalence and molecular characterization are still unknown in many countries, including Portugal. To fill in this knowledge gap, this study aimed to determine the occurrence and molecular characterization of HPgV-1 in a group of healthy blood donors from the north of Portugal. Blood samples from 465 Portuguese blood donors were collected from a major Hospital Center in the north of Portugal. RNA was extracted and an initial nested RT-PCR was performed targeting the conserved 5'-untranslated region  region of the HPgV-1 genome. A second nested RT-PCR targeting the E2 region was performed for genotyping. Only one sample tested positive for HPgV-1 RNA, resulting in a prevalence of approximately 0.22%. Phylogenetic analyses confirmed the characterization as genotype 2, the most prevalent in Europe.

[Genetic diversity and distribution of bovine pestiviruses (Flaviviridae: Pestivirus) in the world and in the Russian Federation].

The genus Pestivirus of the family Flaviviridae includes 11 species. Bovine pestiviruses are the causative agents of viral diarrhea/mucosal disease and include three genetically distinct species: pestivirus A (BVDV-1), B (BVDV-2), and H (BVDV-3). The number of BVDV-1 subtypes is 21, BVDV-2 - 4, and BVDV-3 - 4, which complicates the diagnosis of associated diseases, reduces the effectiveness of vaccination and control programs.We performed the search in the PubMed, Web of Science, Scopus, eLIBRARY.RU databases for articles published in 2000-2021.Pestivirus A is distributed everywhere, although the largest number of subtypes was found in cattle in Italy and China. The virus is widespread in the Central region of the Russia (subtypes 1a and 1m). In Siberia, eleven subtypes circulate among native and imported animals: 1a (5%), 1b (35%), 1c (5%), 1d (10%), 1f (20%), 1g, 1i (both 2.5%), 1j, 1k, 1p, and 1r (all for 5%). Pestivirus B subtype is more virulent, found less frequently and mainly in the North and South America, in some European countries, and in Asia. Three subtypes have been identified in Siberia: 2a (25%), 2b (10%), and 2c (5%). Pestivirus H circulates in Europe, Asia and South America. The main route of entry is contaminated biological products. In Russia, BVDV-3 of the Italian-Brazilian group (3a) was detected in 7 lots of fetal bovine serum.The role of the virus in the occurrence of respiratory diseases in calves, abortion, systemic infection and enteritis in calves and adult animals has been established. The source of the virus in such cases was a contaminated modified live vaccine.

The low-resolution structural models of hepatitis C virus RNA subdomain 5BSL3.2 and its distal complex with domain 3'X point to conserved regulatory mechanisms within the Flaviviridae family.

Subdomain 5BSL3.2 of hepatitis C virus RNA lies at the core of a network of distal RNA-RNA contacts that connect the 5' and 3' regions of the viral genome and regulate the translation and replication stages of the viral cycle. Using small-angle X-ray scattering and NMR spectroscopy experiments, we have determined at low resolution the structural models of this subdomain and its distal complex with domain 3'X, located at the 3'-terminus of the viral RNA chain. 5BSL3.2 adopts a characteristic 'L' shape in solution, whereas the 5BSL3.2-3'X distal complex forms a highly unusual 'Y'-shaped kissing junction that blocks the dimer linkage sequence of domain 3'X and promotes translation. The structure of this complex may impede an effective association of the viral polymerase with 5BSL3.2 and 3'X to start negative-strand RNA synthesis, contributing to explain the likely mechanism used by these sequences to regulate viral replication and translation. In addition, sequence and shape features of 5BSL3.2 are present in functional RNA motifs of flaviviruses, suggesting conserved regulatory processes within the Flaviviridae family.