Subgenomic replicons of the flavivirus Kunjin: construction and applications

Several Kunjin virus (KUN) subgenomic replicons containing large deletions in the structural region (C-prM-E) and in the 3' untranslated region (3'UTR) of the genome have been constructed. Replicon RNA Delta ME with 1,987 nucleotides deleted (from nucleotide 417 [in codon 108] in the C gene to nucleotide 2403 near the carboxy terminus of the E gene, inclusive) and replicon RNA C20rep with 2,247 nucleotides deleted (from nucleotide 157 [in codon 20] in C to nucleotide 2403) replicated efficiently in electroporated BHK21 cells. A further deletion from C20rep of 53 nucleotides, reducing the coding sequence in core protein to two codons (C2rep RNA), resulted in abolishment of RNA replication. Replicon Delta ME/76 with a deletion of 76 nucleotides in the 3'UTR of Delta ME RNA (nucleotides 10423 to 10498) replicated efficiently, whereas replicon Delta ME/352 with a larger deletion of 352 nucleotides (nucleotides 10423 to 10774), including two conserved sequences RCS3 and CS3, was significantly inhibited in RNA replication. To explore the possibility of using a reporter gene assay to monitor synthesis of the positive strand and the negative strand of KUN RNA, we inserted a chloramphenicol acetyltransferase (CAT) gene into the 3'UTR of Delta ME/76 RNA under control of the internal ribosomal entry site (IRES) of encephalomyelocarditis virus RNA in both plus (Delta ME/76CAT[+])- and minus (Delta ME/76CAT[-])-sense orientations. Although insertion of the IRES-CAT cassette in the plus-sense orientation resulted in a significant (10- to 20-fold) reduction of RNA replication compared to that of the parental Delta ME/76 RNA, CAT expression was readily detected in electroporated BHK cells. No CAT expression was detected after electroporation of RNA containing the IRES-CAT cassette inserted in the minus-sense orientation despite its apparently more efficient replication (similar to that of Delta ME/76 RNA); this result indicated that KUN negative-strand RNA was probably not released from its template after synthesis. Replacement of the CAT gene in the Delta ME/76CAT(+) RNA with the neomycin gene (Neo) enabled selection and recovery of a BHK cell culture in which the majority of cells were continuously expressing the replicon RNA for 41 days (nine passages) without apparent cytopathic effect. The constructed KUN replicons should provide valuable tools to study flavivirus RNA replication as well as providing possible vectors for a long-lasting and noncytopathic RNA virus expression system

Вплив полютантів на антиоксидантний захист видів роду Tilia на різних стадіях розвитку

The influence of transport fumes and industrial waste on the activity of catalase, benzidine-peroxidase and guaiacol-peroxidase was studied in the dormant buds, leaves and seeds of the following species of the genus Tilia:T. platyphyllos Scop., T. europaea L., T. amurensis Rupr. and T. begoniifolia Stev. We tested the hypothesis that the action of pollutants changes the state of antioxidant protection at different stages of tree development in contaminated phytocenoses. An increase in catalase activity was observed in leaves of all linden species, and the action of transport fumes caused excess over control level by 118, 118, 196, and 61% respectively for T. platyphyllos, T. europaea, T. amurensis and T. begoniifolia. The action of industrial waste was accompanied by a slight decrease in catalase activity in T. europaea leaves, and increase in activity in leaves of T. amurensis and T. begoniifolia (143% and 115%). Benzidine-peroxidase activity increased due to the influence of transport fumes on leaves of T. amurensis and T. begoniifolia (103% and 44%), but decreased due to the effect of industrial waste on leaves of T. europaea, T. amurensis and T. begoniifolia (46%, 30%, and 44% respectively), and was suppressed in the seeds of T. europaea, T. amurensis and T. begoniifolia both under the influence of transport (42%, 47% and 33% below control) and industrial emissions (19%, 19% and 45%), and was reduced in buds of T. platyphyllos, T. europaea and T. amurensis due to the effect of transport fumes (21%, 9% and 20% respectively). Guaiacol-peroxidase activity decreased due to the influence of transport fumes in buds of T. platyphyllos, T. europaea and T. amurensis (41%, 14% and 47% below control), while it increased in the seeds of T. platyphyllos and T. amurensis (104% and 83%), as well as in leaves of T. amurensis and T. begoniifolia both due to the effect of transport (129% and 144%) and of industrial emissions (respectively, 34% and 40% above control). The substantial restructuring of the antioxidant system components in leaves, dormant buds and seeds confirms the hypothesis that metabolic processes in Tilia trees adapt throughout all stages of their development in response to the polluted conditions in urban phytocenoses.Досліджено вплив транспортних вихлопів і промислових викидів на активність каталази, бензидин-пероксидази та гваякол-пероксидази у бруньках, листках і насінні Tilia platyphyllos Scop., T. europaea L., T. amurensis Rupr. і T. begoniifolia Stev. Перевірено гіпотезу про те, що вплив полютантів змінює стан антиоксидантного захисту на різних стадіях розвитку дерев у забруднених фітоценозах. Зростання активності каталази спостерігали у листках усіх видів лип. Вплив транспортних вихлопів спричинив збільшення контрольного рівня для T. platyphyllos на 118%, для T. europaea – на 118%, для T. amurensis – на 196% і для T. begoniifolia – на 61%. Вплив промислових викидів супроводжувався незначним зниженням активності каталази у листках T. europaea та зростанням активності у листках T. amurensis і T. begoniifolia (на 143% і 115% відповідно). Активність бензидин-пероксидази зростала за дії транспортних вихлопів у листках T. amurensis і T. begoniifolia (відповідно, на 103% і 44%), проте знижувалась за дії промислових викидів у листках T. europaea, T. amurensis і T. begoniifolia (на 46%, 30% і 44% відносно контролю відповідно), а також була пригнічена у насінні T. europaea, T. amurensis і T. begoniifolia як за дії транспортних (відповідно, на 42%, 47% і 33% від контролю), так і промислових емісій (на 19%, 19% і 45% від контролю, відповідно), та знижена у бруньках T. platyphyllos, T. europaea та T. amurensis за дії транспортних вихлопів (на 21%, 9% і 20% від контролю, відповідно). Активність гваякол-пероксидази за впливу транспортних вихлопів знижувалась у бруньках T. platyphyllos, T. europaea і T. amurensis (відповідно, на 41%, 14% і 47% відносно контролю), але збільшувалась у насінні T. platyphyllos і T. amurensis (на 104% і 83%, відповідно), а також зростала у листках T. amurensis і T. begoniifolia як за дії транспортних (відповідно, на 129% і 144%), так і промислових емісій (відповідно, на 34% і 40% відносно контролю). Суттєві перебудови компонентів антиоксидантної системи у листках, бруньках і насінні підтвердили припущення, що адаптація дерев роду Tilia до умов забруднених міських фітоценозів потребує змін метаболічних процесів упродовж усіх стадій розвитку. Досліджено вплив транспортних вихлопів і промислових викидів на активність каталази, бензидин-пероксидази та гваякол-пероксидази у бруньках, листках і насінні Tilia platyphyllos Scop., T. europaea L., T. amurensis Rupr. і T. begoniifolia Stev. Перевірено гіпотезу про те, що вплив полютантів змінює стан антиоксидантного захисту на різних стадіях розвитку дерев у забруднених фітоценозах. Зростання активності каталази спостерігали у листках усіх видів лип. Вплив транспортних вихлопів спричинив збільшення контрольного рівня для T. platyphyllos на 118%, для T. europaea – на 118%, для T. amurensis – на 196% і для T. begoniifolia – на 61%. Вплив промислових викидів супроводжувався незначним зниженням активності каталази у листках T. europaea та зростанням активності у листках T. amurensis і T. begoniifolia (на 143% і 115% відповідно). Активність бензидин-пероксидази зростала за дії транспортних вихлопів у листках T. amurensis і T. begoniifolia (відповідно, на 103% і 44%), проте знижувалась за дії промислових викидів у листках T. europaea, T. amurensis і T. begoniifolia (на 46%, 30% і 44% відносно контролю відповідно), а також була пригнічена у насінні T. europaea, T. amurensis і T. begoniifolia як за дії транспортних (відповідно, на 42%, 47% і 33% від контролю), так і промислових емісій (на 19%, 19% і 45% від контролю, відповідно), та знижена у бруньках T. platyphyllos, T. europaea та T. amurensis за дії транспортних вихлопів (на 21%, 9% і 20% від контролю, відповідно). Активність гваякол-пероксидази за впливу транспортних вихлопів знижувалась у бруньках T. platyphyllos, T. europaea і T. amurensis (відповідно, на 41%, 14% і 47% відносно контролю), але збільшувалась у насінні T. platyphyllos і T. amurensis (на 104% і 83%, відповідно), а також зростала у листках T. amurensis і T. begoniifolia як за дії транспортних (відповідно, на 129% і 144%), так і промислових емісій (відповідно, на 34% і 40% відносно контролю). Суттєві перебудови компонентів антиоксидантної системи у листках, бруньках і насінні підтвердили припущення, що адаптація дерев роду Tilia до умов забруднених міських фітоценозів потребує змін метаболічних процесів упродовж усіх стадій розвитку.

РАННЕПЕРМСКИЙ (300–270 МЛН ЛЕТ) МАГМАТИЗМ ВОСТОЧНОГО КАЗАХСТАНА КАК РЕЗУЛЬТАТ СОЧЕТАНИЯ ПЛЕЙТ- И ПЛЮМ-ТЕКТОНИЧЕСКИХ ФАКТОРОВ

The history of the Central Asian Orogenic Belt (CAOB) was marked by several major events of magmatism which produced large volumes of volcanic and intrusive (mafic-ultramafic and granitic) rocks within a relatively short time span (30–40 Ma) over a vast area. The magmatic activity postdated the orogenic stages of accretionary-collisional belts in Central Asia and likely resulted from the impact of mantle plumes that formed Large Igneous Provinces (LIPs). The formation of the Tarim–South Mongolia LIP at 300–270 Ma is the best known among the major Permian events of basaltic and granitic magmatism. Early Permian igneous rocks (volcanic, subvolcanic and intrusive suites that vary from ultramafic to felsic compositions) of the same age range (300 to 270 Ma) have been recently found also in Eastern Kazakhstan, within the late Paleozoic Altai collisional system. The compositions and ages of the rocks suggest that the Eastern Kazakhstan magmatism was the northward expansion of the Tarim LIP. The spread of the Tarim LIP was apparently facilitated by lithospheric extension after the Siberia-Kazakhstan collision. The extension led to rheological weakening of the lithosphere whereby deep mantle melts could penetrate to shallower depths. The early Permian history of Eastern Kazakhstan was controlled by the interplay of plate tectonic and plume processes: plate-tectonic accretion and collision formed the structural framework, and the Tarim mantle plume was a heat source maintaining voluminous magma generation.В истории развития крупнейшего Центрально-Азиатского складчатого пояса (ЦАСП) выявлены несколько периодов крупномасштабной эндогенной активности, характеризующихся проявлениями значительных объемов вулканических и интрузивных (как базитовых, так и гранитоидных) пород на обширных территориях в сравнительно короткие временные интервалы (30–40 млн лет). Эти вспышки магматической активности обычно происходят после завершения аккреционно-коллизионных процессов в складчатых системах и рассматриваются как результат воздействия мантийных плюмов на литосферу – крупные изверженные провинции. Одним из ярких примеров является Тарим-Южномонгольская крупная изверженная провинция (300–270 млн лет назад), характеризующаяся широким развитием базитового и гранитоидного магматизма в западной части ЦАСП. Исследования последних лет показали, что в Восточном Казахстане, в пределах Алтайской коллизионной системы герцинид, широко распространены как базитовые, так и гранитоидные комплексы раннепермского возраста (300–270 млн лет). В приведенном кратком обзоре показано, что особенности состава и условия формирования этих магматических ассоциаций позволяют рассматривать их как результат северо-западного распространения влияния Таримской крупной изверженной провинции. Распространение этого термического возмущения в литосфере,по-видимому, стало возможным благодаря пост-орогеническому растяжению после коллизии Сибирского и Казахстанского континентов. Реологическое ослабление литосферы позволило глубинным расплавам проникать в литосферную мантию, образовав крупные очаги базитовых магм. Таким образом, современный геологический облик и металлогеническая специфика территории Восточного Казахстана является результатом плейт-тектонических процессов посторогенического растяжения на фоне повышенного термического градиента в мантии, вызванного активностью Таримского мантийного плюма

The impact of pollutants on the antioxidant protection of species of the genus Tilia at different developmental stages

The influence of transport fumes and industrial waste on the activity of catalase, benzidine-peroxidase and guaiacol-peroxidase was studied in the dormant buds, leaves and seeds of the following species of the genus Tilia:T. platyphyllos Scop., T. europaea L., T. amurensis Rupr. and T. begoniifolia Stev. We tested the hypothesis that the action of pollutants changes the state of antioxidant protection at different stages of tree development in contaminated phytocenoses. An increase in catalase activity was observed in leaves of all linden species, and the action of transport fumes caused excess over control level by 118, 118, 196, and 61% respectively for T. platyphyllos, T. europaea, T. amurensis and T. begoniifolia. The action of industrial waste was accompanied by a slight decrease in catalase activity in T. europaea leaves, and increase in activity in leaves of T. amurensis and T. begoniifolia (143% and 115%). Benzidine-peroxidase activity increased due to the influence of transport fumes on leaves of T. amurensis and T. begoniifolia (103% and 44%), but decreased due to the effect of industrial waste on leaves of T. europaea, T. amurensis and T. begoniifolia (46%, 30%, and 44% respectively), and was suppressed in the seeds of T. europaea, T. amurensis and T. begoniifolia both under the influence of transport (42%, 47% and 33% below control) and industrial emissions (19%, 19% and 45%), and was reduced in buds of T. platyphyllos, T. europaea and T. amurensis due to the effect of transport fumes (21%, 9% and 20% respectively). Guaiacol-peroxidase activity decreased due to the influence of transport fumes in buds of T. platyphyllos, T. europaea and T. amurensis (41%, 14% and 47% below control), while it increased in the seeds of T. platyphyllos and T. amurensis (104% and 83%), as well as in leaves of T. amurensis and T. begoniifolia both due to the effect of transport (129% and 144%) and of industrial emissions (respectively, 34% and 40% above control). The substantial restructuring of the antioxidant system components in leaves, dormant buds and seeds confirms the hypothesis that metabolic processes in Tilia trees adapt throughout all stages of their development in response to the polluted conditions in urban phytocenoses

West Nile virus encodes a microRNA-like small RNA in the 3′ untranslated region which up-regulates GATA4 mRNA and facilitates virus replication in mosquito cells

West Nile virus (WNV) belongs to a group of medically important single-stranded, positive-sense RNA viruses causing deadly disease outbreaks around the world. The 3′ untranslated region (3′-UTR) of the flavivirus genome, in particular the terminal 3′ stem–loop (3′SL) fulfils multiple functions in virus replication and virus–host interactions. Using the Kunjin strain of WNV (WNVKUN), we detected a virally encoded small RNA, named KUN-miR-1, derived from 3′SL. Transcription of WNVKUN pre-miRNA (3′SL) in mosquito cells either from plasmid or Semliki Forest virus (SFV) RNA replicon resulted in the production of mature KUN-miR-1. Silencing of Dicer-1 but not Dicer-2 led to a reduction in the miRNA levels. Further, when a synthetic inhibitor of KUN-miR-1 was transfected into mosquito cells, replication of viral RNA was significantly reduced. Using cloning and bioinformatics approaches, we identified the cellular GATA4 mRNA as a target for KUN-miR-1. KUN-miR-1 produced in mosquito cells during virus infection or from plasmid DNA, SFV RNA replicon or mature miRNA duplex increased accumulation of GATA4 mRNA. Depletion of GATA4 mRNA by RNA silencing led to a significant reduction in virus RNA replication while a KUN-miR-1 RNA mimic enhanced replication of a mutant WNVKUN virus producing reduced amounts of KUN-miR-1, suggesting that GATA4-induction via KUN-miR-1 plays an important role in virus replication

Ancestral SARS-CoV-2, but not Omicron, replicates less efficiently in primary pediatric nasal epithelial cells

Children typically experience more mild symptoms of Coronavirus Disease 2019 (COVID-19) when compared to adults. There is a strong body of evidence that children are also less susceptible to Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) infection with the ancestral viral isolate. However, the emergence of SARS-CoV-2 variants of concern (VOCs) has been associated with an increased number of pediatric infections. Whether this is the result of widespread adult vaccination or fundamental changes in the biology of SARS-CoV-2 remain to be determined. Here, we use primary nasal epithelial cells (NECs) from children and adults, differentiated at an air-liquid interface to show that the ancestral SARS-CoV-2 replicates to significantly lower titers in the NECs of children compared to those of adults. This was associated with a heightened antiviral response to SARS-CoV-2 in the NECs of children. Importantly, the Delta variant also replicated to significantly lower titers in the NECs of children. This trend was markedly less pronounced in the case of Omicron. It is also striking to note that, at least in terms of viral RNA, Omicron replicated better in pediatric NECs compared to both Delta and the ancestral virus. Taken together, these data show that the nasal epithelium of children supports lower infection and replication of ancestral SARS-CoV-2, although this may be changing as the virus evolves.Peer reviewe

Ancestral SARS-CoV-2, but not Omicron, replicates less efficiently in primary pediatric nasal epithelial cells

Children typically experience more mild symptoms of Coronavirus Disease 2019 (COVID-19) when compared to adults. There is a strong body of evidence that children are also less susceptible to Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) infection with the ancestral viral isolate. However, the emergence of SARS-CoV-2 variants of concern (VOCs) has been associated with an increased number of pediatric infections. Whether this is the result of widespread adult vaccination or fundamental changes in the biology of SARS-CoV-2 remain to be determined. Here, we use primary nasal epithelial cells (NECs) from children and adults, differentiated at an air-liquid interface to show that the ancestral SARS-CoV-2 replicates to significantly lower titers in the NECs of children compared to those of adults. This was associated with a heightened antiviral response to SARS-CoV-2 in the NECs of children. Importantly, the Delta variant also replicated to significantly lower titers in the NECs of children. This trend was markedly less pronounced in the case of Omicron. It is also striking to note that, at least in terms of viral RNA, Omicron replicated better in pediatric NECs compared to both Delta and the ancestral virus. Taken together, these data show that the nasal epithelium of children supports lower infection and replication of ancestral SARS-CoV-2, although this may be changing as the virus evolves

A vaccinia-based single vector construct multi-pathogen vaccine protects against both Zika and chikungunya viruses

Zika and chikungunya viruses have caused major epidemics and are transmitted by Aedes aegypti and/or Aedes albopictus mosquitoes. The "Sementis Copenhagen Vector" (SCV) system is a recently developed vaccinia-based, multiplication-defective, vaccine vector technology that allows manufacture in modified CHO cells. Herein we describe a single-vector construct SCV vaccine that encodes the structural polyprotein cassettes of both Zika and chikungunya viruses from different loci. A single vaccination of mice induces neutralizing antibodies to both viruses in wild-type and IFNAR-/- mice and protects against (i) chikungunya virus viremia and arthritis in wild-type mice, (ii) Zika virus viremia and fetal/placental infection in female IFNAR-/- mice, and (iii) Zika virus viremia and testes infection and pathology in male IFNAR-/- mice. To our knowledge this represents the first single-vector construct, multi-pathogen vaccine encoding large polyproteins, and offers both simplified manufacturing and formulation, and reduced "shot burden" for these often co-circulating arboviruses.Natalie A. Prow, Liang Liu, Eri Nakayama, Tamara H. Cooper ... Kerrilyn R. Diener ... John D. Hayball .. et al

A New Model to Produce Infectious Hepatitis C Virus without the Replication Requirement

Numerous constraints significantly hamper the experimental study of hepatitis C virus (HCV). Robust replication in cell culture occurs with only a few strains, and is invariably accompanied by adaptive mutations that impair in vivo infectivity/replication. This problem complicates the production and study of authentic HCV, including the most prevalent and clinically important genotype 1 (subtypes 1a and 1b). Here we describe a novel cell culture approach to generate infectious HCV virions without the HCV replication requirement and the associated cell-adaptive mutations. The system is based on our finding that the intracellular environment generated by a West-Nile virus (WNV) subgenomic replicon rendered a mammalian cell line permissive for assembly and release of infectious HCV particles, wherein the HCV RNA with correct 5′ and 3′ termini was produced in the cytoplasm by a plasmid-driven dual bacteriophage RNA polymerase-based transcription/amplification system. The released particles preferentially contained the HCV-based RNA compared to the WNV subgenomic RNA. Several variations of this system are described with different HCV-based RNAs: (i) HCV bicistronic particles (HCVbp) containing RNA encoding the HCV structural genes upstream of a cell-adapted subgenomic replicon, (ii) HCV reporter particles (HCVrp) containing RNA encoding the bacteriophage SP6 RNA polymerase in place of HCV nonstructural genes, and (iii) HCV wild-type particles (HCVwt) containing unmodified RNA genomes of diverse genotypes (1a, strain H77; 1b, strain Con1; 2a, strain JFH-1). Infectivity was assessed based on the signals generated by the HCV RNA molecules introduced into the cytoplasm of target cells upon virus entry, i.e. HCV RNA replication and protein production for HCVbp in Huh-7.5 cells as well as for HCVwt in HepG2-CD81 cells and human liver slices, and SP6 RNA polymerase-driven firefly luciferase for HCVrp in target cells displaying candidate HCV surface receptors. HCV infectivity was inhibited by pre-incubation of the particles with anti-HCV antibodies and by a treatment of the target cells with leukocyte interferon plus ribavirin. The production of authentic infectious HCV particles of virtually any genotype without the adaptive mutations associated with in vitro HCV replication represents a new paradigm to decipher the requirements for HCV assembly, release, and entry, amenable to analyses of wild type and genetically modified viruses of the most clinically significant HCV genotypes

Direct Interaction between Two Viral Proteins, the Nonstructural Protein 2CATPase and the Capsid Protein VP3, Is Required for Enterovirus Morphogenesis

In spite of decades-long studies, the mechanism of morphogenesis of plus-stranded RNA viruses belonging to the genus Enterovirus of Picornaviridae, including poliovirus (PV), is not understood. Numerous attempts to identify an RNA encapsidation signal have failed. Genetic studies, however, have implicated a role of the non-structural protein 2CATPase in the formation of poliovirus particles. Here we report a novel mechanism in which protein-protein interaction is sufficient to explain the specificity in PV encapsidation. Making use of a novel “reporter virus”, we show that a quasi-infectious chimera consisting of the capsid precursor of C-cluster coxsackie virus 20 (C-CAV20) and the nonstructural proteins of the closely related PV translated and replicated its genome with wild type kinetics, whereas encapsidation was blocked. On blind passages, encapsidation of the chimera was rescued by a single mutation either in capsid protein VP3 of CAV20 or in 2CATPase of PV. Whereas each of the single-mutation variants expressed severe proliferation phenotypes, engineering both mutations into the chimera yielded a virus encapsidating with wild type kinetics. Biochemical analyses provided strong evidence for a direct interaction between 2CATPase and VP3 of PV and CAV20. Chimeras of other C-CAVs (CAV20/CAV21 or CAV18/CAV20) were blocked in encapsidation (no virus after blind passages) but could be rescued if the capsid and 2CATPase coding regions originated from the same virus. Our novel mechanism explains the specificity of encapsidation without apparent involvement of an RNA signal by considering that (i) genome replication is known to be stringently linked to translation, (ii) morphogenesis is known to be stringently linked to genome replication, (iii) newly synthesized 2CATPase is an essential component of the replication complex, and (iv) 2CATPase has specific affinity to capsid protein(s). These conditions lead to morphogenesis at the site where newly synthesized genomes emerge from the replication complex