Evolution of SARS-CoV-2 in the Rhine-Neckar/Heidelberg Region 01/2021 - 07/2023.

In January 2021, the monitoring of circulating variants of SARS-CoV-2 was initiated in Germany under the Corona Surveillance Act, which was discontinued after July 2023. This initiative aimed to enhance pandemic containment, as specific amino acid changes, particularly in the spike protein, were associated with increased transmission and reduced vaccine efficacy. Our group conducted whole genome sequencing using the ARTIC protocol (currently V4) on Illumina's NextSeq 500 platform (and, starting in May 2023, on the MiSeq DX platform) for SARS-CoV-2 positive specimen from patients at Heidelberg University Hospital, associated hospitals, and the public health office in the Rhine-Neckar/Heidelberg region. In total, we sequenced 26,795 SARS-CoV-2-positive samples between January 2021 and July 2023. Valid sequences, meeting the requirements for upload to the German electronic sequencing data hub (DESH) operated by the Robert Koch Institute (RKI), were determined for 24,852 samples, and the lineage/clade could be identified for 25,912 samples. The year 2021 witnessed significant dynamics in the circulating variants in the Rhine-Neckar/Heidelberg region, including A.27.RN, followed by the emergence of B.1.1.7 (Alpha), subsequently displaced by B.1.617.2 (Delta), and the initial occurrences of B.1.1.529 (Omicron). By January 2022, B.1.1.529 had superseded B.1.617.2, dominating with over 90%. The years 2022 and 2023 were then characterized by the dominance of B.1.1.529 and its sublineages, particularly BA.5 and BA.2, and more recently, the emergence of recombinant variants like XBB.1.5. Since the global dominance of B.1.617.2, the identified variant distribution in our local study, apart from a time delay in the spread of new variants, can be considered largely representative of the global distribution. om a time delay in the spread of new variants, can be considered largely representative of the global distribution.

AAV vectors displaying bispecific DARPins enable dual-control targeted gene delivery.

Precise delivery of genes to therapy-relevant cells is crucial for in vivo gene therapy. Receptor-targeting as prime strategy for this purpose is limited to cell types defined by a single cell-surface marker. Many target cells are characterized by combinations of more than one marker, such as the HIV reservoir cells. Here, we explored the tropism of adeno-associated viral vectors (AAV2) displaying designed ankyrin repeat proteins (DARPins) mono- and bispecific for CD4 and CD32a. Cryo-electron tomography revealed an unaltered capsid structure in the presence of DARPins. Surprisingly, bispecific AAVs transduced CD4/CD32a double-positive cells at much higher efficiencies than single-positive cells, even if present in low amounts in cell mixtures or human blood. This preference was confirmed when vector particles were systemically administered into mice. Cell trafficking studies revealed an increased cell entry rate for bispecific over monospecific AAVs. When equipped with an HIV genome-targeting CRISPR/Cas cassette, the vectors prevented HIV replication in T cell cultures. The data provide proof-of-concept for high-precision gene delivery through tandem-binding regions on AAV. Reminiscent of biological products following Boolean logic AND gating, the data suggest a new option for receptor-targeted vectors to improve the specificity and safety of in vivo gene therapy.

Enhanced Transduction of P2X7-Expressing Cells with Recombinant rAAV Vectors.

Adeno-associated viruses (AAV) are useful vectors for transducing cells in vitro and in vivo. Targeting of specific cell subsets with AAV is limited by the broad tropism of AAV serotypes. Nanobodies are single immunoglobulin variable domains from heavy chain antibodies that naturally occur in camelids. Their small size and high solubility allow easy reformatting into fusion proteins. In this chapter we provide protocols for inserting a P2X7-specific nanobody into a surface loop of the VP1 capsid protein of AAV2. Such nanobody-displaying recombinant AAV allow 50- to 500-fold stronger transduction of P2X7-expressing cells than the parental AAV. We provide protocols for monitoring the transduction of P2X7-expressing cells by nanobody-displaying rAAV by flow cytometry and fluorescence microscopy.

Identification of adeno-associated virus variants for gene transfer into human neural cell types by parallel capsid screening.

Human brain cells generated by in vitro cell programming provide exciting prospects for disease modeling, drug discovery and cell therapy. These applications frequently require efficient and clinically compliant tools for genetic modification of the cells. Recombinant adeno-associated viruses (AAVs) fulfill these prerequisites for a number of reasons, including the availability of a myriad of AAV capsid variants with distinct cell type specificity (also called tropism). Here, we harnessed a customizable parallel screening approach to assess a panel of natural or synthetic AAV capsid variants for their efficacy in lineage-related human neural cell types. We identified common lead candidates suited for the transduction of directly converted, early-stage induced neural stem cells (iNSCs), induced pluripotent stem cell (iPSC)-derived later-stage, radial glia-like neural progenitors, as well as differentiated astrocytic and mixed neuroglial cultures. We then selected a subset of these candidates for functional validation in iNSCs and iPSC-derived astrocytes, using shRNA-induced downregulation of the citrate transporter SLC25A1 and overexpression of the transcription factor NGN2 for proofs-of-concept. Our study provides a comparative overview of the susceptibility of different human cell programming-derived brain cell types to AAV transduction and a critical discussion of the assets and limitations of this specific AAV capsid screening approach.

Ex vivo and in vivo suppression of SARS-CoV-2 with combinatorial AAV/RNAi expression vectors.

Despite rapid development and deployment of vaccines against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), clinically relevant modalities to curb the pandemic by directly attacking the virus on a genetic level remain highly desirable and are urgently needed. Here we comprehensively illustrate the capacity of adeno-associated virus (AAV) vectors co-expressing a cocktail of three short hairpin RNAs (shRNAs; RNAi triggers) directed against the SARS-CoV-2 RdRp and N genes as versatile and effective antiviral agents. In cultured monkey cells and human gut organoids, our most potent vector, SAVIOR (SARS virus repressor), suppressed SARS-CoV-2 infection to background levels. Strikingly, in control experiments using single shRNAs, multiple SARS-CoV-2 escape mutants quickly emerged from infected cells within 24-48 h. Importantly, such adverse viral adaptation was fully prevented with the triple-shRNA AAV vector even during long-term cultivation. In addition, AAV-SAVIOR efficiently purged SARS-CoV-2 in a new model of chronically infected human intestinal cells. Finally, intranasal AAV-SAVIOR delivery using an AAV9 capsid moderately diminished viral loads and/or alleviated disease symptoms in hACE2-transgenic or wild-type mice infected with human or mouse SARS-CoV-2 strains, respectively. Our combinatorial and customizable AAV/RNAi vector complements ongoing global efforts to control the coronavirus disease 2019 (COVID-19) pandemic and holds great potential for clinical translation as an original and flexible preventive or therapeutic antiviral measure.

Lentiviral and adeno-associated vectors efficiently transduce mouse T lymphocytes when targeted to murine CD8.

Preclinical studies on gene delivery into mouse lymphocytes are often hampered by insufficient activity of lentiviral (LV) and adeno-associated vectors (AAVs) as well as missing tools for cell type selectivity when considering in vivo gene therapy. Here, we selected designed ankyrin repeat proteins (DARPins) binding to murine CD8. The top-performing DARPin was displayed as targeting ligand on both vector systems. When used on engineered measles virus (MV) glycoproteins, the resulting mCD8-LV transduced CD8+ mouse lymphocytes with near-absolute (>99%) selectivity. Despite its lower functional titer, mCD8-LV achieved 4-fold higher gene delivery to CD8+ cells than conventional VSV-LV when added to whole mouse blood. Addition of mCD8-LV encoding a chimeric antigen receptor (CAR) specific for mouse CD19 to splenocytes resulted in elimination of B lymphocytes and lymphoma cells. For display on AAV, the DARPin was inserted into the GH2-GH3 loop of the AAV2 capsid protein VP1, resulting in a DARPin-targeted AAV we termed DART-AAV. Stocks of mCD8-AAV contained similar genome copies as AAV2 but were >20-fold more active in gene delivery in mouse splenocytes, while exhibiting >99% specificity for CD8+ cells. These results suggest that receptor targeting can overcome blocks in transduction of mouse splenocytes.

Single-Use Capture Purification of Adeno-Associated Viral Gene Transfer Vectors by Membrane-Based Steric Exclusion Chromatography.

We present membrane-based steric exclusion chromatography (SXC) as a universal capture step for purification of adeno-associated virus (AAV) gene transfer vectors independent of their serotype and surface characteristics. SXC is performed by mixing an unpurified cell culture supernatant containing AAV particles with polyethylene glycol (PEG) and feeding the mixture onto a chromatography filter unit. The purified AAV particles are recovered by flushing the unit with a solution lacking PEG. SXC is an inexpensive single-use method that permits to concentrate, purify, and re-buffer AAV particles with yields >95% and >80% impurity clearance. SXC could theoretically be employed at industrial scales with units of nearly 20 m2.

Cone-shaped HIV-1 capsids are transported through intact nuclear pores.

Human immunodeficiency virus (HIV-1) remains a major health threat. Viral capsid uncoating and nuclear import of the viral genome are critical for productive infection. The size of the HIV-1 capsid is generally believed to exceed the diameter of the nuclear pore complex (NPC), indicating that capsid uncoating has to occur prior to nuclear import. Here, we combined correlative light and electron microscopy with subtomogram averaging to capture the structural status of reverse transcription-competent HIV-1 complexes in infected T cells. We demonstrated that the diameter of the NPC in cellulo is sufficient for the import of apparently intact, cone-shaped capsids. Subsequent to nuclear import, we detected disrupted and empty capsid fragments, indicating that uncoating of the replication complex occurs by breaking the capsid open, and not by disassembly into individual subunits. Our data directly visualize a key step in HIV-1 replication and enhance our mechanistic understanding of the viral life cycle.

SARS-CoV-2 RNA Extraction Using Magnetic Beads for Rapid Large-Scale Testing by RT-qPCR and RT-LAMP.

Rapid large-scale testing is essential for controlling the ongoing pandemic of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The standard diagnostic pipeline for testing SARS-CoV-2 presence in patients with an ongoing infection is predominantly based on pharyngeal swabs, from which the viral RNA is extracted using commercial kits, followed by reverse transcription and quantitative PCR detection. As a result of the large demand for testing, commercial RNA extraction kits may be limited and, alternatively, non-commercial protocols are needed. Here, we provide a magnetic bead RNA extraction protocol that is predominantly based on in-house made reagents and is performed in 96-well plates supporting large-scale testing. Magnetic bead RNA extraction was benchmarked against the commercial QIAcube extraction platform. Comparable viral RNA detection sensitivity and specificity were obtained by fluorescent and colorimetric reverse transcription loop-mediated isothermal amplification (RT-LAMP) using a primer set targeting the N gene, as well as RT-qPCR using a primer set targeting the E gene, showing that the RNA extraction protocol presented here can be combined with a variety of detection methods at high throughput. Importantly, the presented diagnostic workflow can be quickly set up in a laboratory without access to an automated pipetting robot.

Pre-arrayed Pan-AAV Peptide Display Libraries for Rapid Single-Round Screening.

Display of short peptides on the surface of adeno-associated viruses (AAVs) is a powerful technology for the generation of gene therapy vectors with altered cell specificities and/or transduction efficiencies. Following its extensive prior use in the best characterized AAV serotype 2 (AAV2), recent reports also indicate the potential of other AAV isolates as scaffolds for peptide display. In this study, we systematically explored the respective capacities of 13 different AAV capsid variants to tolerate 27 peptides inserted on the surface followed by production of reporter-encoding vectors. Single-round screening in pre-arrayed 96-well plates permitted rapid and simple identification of superior vectors in >90 cell types, including T cells and primary cells. Notably, vector performance depended not only on the combination of capsid, peptide, and cell type, but also on the position of the inserted peptide and the nature of flanking residues. For optimal data availability and accessibility, all results were assembled in a searchable online database offering multiple output styles. Finally, we established a reverse-transduction pipeline based on vector pre-spotting in 96- or 384-well plates that facilitates high-throughput library panning. Our comprehensive illustration of the vast potential of alternative AAV capsids for peptide display should accelerate their in vivo screening and application as unique gene therapy vectors.

Nanobody-Enhanced Targeting of AAV Gene Therapy Vectors.

A limiting factor for the use of adeno-associated viruses (AAVs) as vectors in gene therapy is the broad tropism of AAV serotypes, i.e., the parallel infection of several cell types. Nanobodies are single immunoglobulin variable domains from heavy chain antibodies that naturally occur in camelids. Their small size and high solubility allow easy reformatting into fusion proteins. Herein we show that a membrane protein-specific nanobody can be inserted into a surface loop of the VP1 capsid protein of AAV2. Using three structurally distinct membrane proteins-a multispan ion channel, a single-span transmembrane protein, and a glycosylphosphatidylinositol (GPI)-anchored ectoenzyme-we show that this strategy can dramatically enhance the transduction of specific target cells by recombinant AAV2. Moreover, we show that the nanobody-VP1 fusion of AAV2 can be incorporated into the capsids of AAV1, AAV8, and AAV9 and thereby effectively redirect the target specificity of other AAV serotypes. Nanobody-mediated targeting provides a highly efficient AAV targeting strategy that is likely to open up new avenues for genetic engineering of cells.

Cell-specific CRISPR-Cas9 activation by microRNA-dependent expression of anti-CRISPR proteins.

The rapid development of CRISPR-Cas technologies brought a personalized and targeted treatment of genetic disorders into closer reach. To render CRISPR-based therapies precise and safe, strategies to confine the activity of Cas(9) to selected cells and tissues are highly desired. Here, we developed a cell type-specific Cas-ON switch based on miRNA-regulated expression of anti-CRISPR (Acr) proteins. We inserted target sites for miR-122 or miR-1, which are abundant specifically in liver and cardiac muscle cells, respectively, into the 3'UTR of Acr transgenes. Co-expressing these with Cas9 and sgRNAs resulted in Acr knockdown and released Cas9 activity solely in hepatocytes or cardiomyocytes, while Cas9 was efficiently inhibited in off-target cells. We demonstrate control of genome editing and gene activation using a miR-dependent AcrIIA4 in combination with different Streptococcus pyogenes (Spy)Cas9 variants (full-length Cas9, split-Cas9, dCas9-VP64). Finally, to showcase its modularity, we adapted our Cas-ON system to the smaller and more target-specific Neisseria meningitidis (Nme)Cas9 orthologue and its cognate inhibitors AcrIIC1 and AcrIIC3. Our Cas-ON switch should facilitate cell-specific activity of any CRISPR-Cas orthologue, for which a potent anti-CRISPR protein is known.

Publisher Correction: Immune genes are primed for robust transcription by proximal long noncoding RNAs located in nuclear compartments.

In the version of this article initially published, '+' and '-' labels were missing from the graph keys at the bottom of Fig. 8d. The error has been corrected in the HTML and PDF versions of the article.

HIV-1 nuclear import in macrophages is regulated by CPSF6-capsid interactions at the nuclear pore complex.

Nuclear entry of HIV-1 replication complexes through intact nuclear pore complexes is critical for successful infection. The host protein cleavage-and-polyadenylation-specificity-factor-6 (CPSF6) has been implicated in different stages of early HIV-1 replication. Applying quantitative microscopy of HIV-1 reverse-transcription and pre-integration-complexes (RTC/PIC), we show that CPSF6 is strongly recruited to nuclear replication complexes but absent from cytoplasmic RTC/PIC in primary human macrophages. Depletion of CPSF6 or lack of CPSF6 binding led to accumulation of HIV-1 subviral complexes at the nuclear envelope of macrophages and reduced infectivity. Two-color stimulated-emission-depletion microscopy indicated that under these circumstances HIV-1 complexes are retained inside the nuclear pore and undergo CA-multimer dependent CPSF6 clustering adjacent to the nuclear basket. We propose that nuclear entry of HIV-1 subviral complexes in macrophages is mediated by consecutive binding of Nup153 and CPSF6 to the hexameric CA lattice.

Impact of the Assembly-Activating Protein on Molecular Evolution of Synthetic Adeno-Associated Virus Capsids.

Over the last decade, the role of the assembly-activating protein (AAP) has begun to be dissected for the formation of adeno-associated virus (AAV) capsids based on different viral serotypes. Recently, the authors' group has specifically studied AAP's relevance during production of AAV gene therapy vectors in mammalian or insect cells, and AAP was found to be essential for capsid protein stabilization and generation of functional vector particles. Here, the lingering question is additionally addressed of whether molecular AAV evolution via DNA family shuffling of viral capsid genes would perturb AAP functionality due to concurrent and inadvertent recombination of the AAP open reading frame. To this end, a battery of complementary experiments was conducted in which: (1) the ability of chimeric AAP from AAVDJ, a hybrid of serotypes 2, 8, and 9, was tested to rescue AAP knockouts in the three parental serotypes; (2) the functionality of 60 chimeric AAPs extracted from five shuffled, unselected capsid libraries was measured; (3) whether production of different shuffled libraries, 10 wild-type serotypes or 25 individual chimeric capsids, can be enhanced by overexpression of AAP cocktails was assessed; and (4) the activity of 12 chimeric AAPs isolated from a shuffled library that was iteratively selected in vivo in mouse livers was studied. Collectively, the data demonstrate a remarkable tolerance of AAP for recombination via DNA family shuffling, evidenced by the findings that (1) all chimeric AAPs studied here retained at least partial activity, even in cases where the cognate hybrid capsid may be non-functional, and that (2) ectopic AAP overexpression did not enhance production of shuffled AAV chimeras or libraries, implying that the inherently encoded hybrid AAP variants are sufficiently active. Together, this work provides compelling evidence that AAP is not rate limiting during AAV capsid shuffling and thereby relieves a major concern in the field of AAV vector evolution.

Immune genes are primed for robust transcription by proximal long noncoding RNAs located in nuclear compartments.

Accumulation of trimethylation of histone H3 at lysine 4 (H3K4me3) on immune-related gene promoters underlies robust transcription during trained immunity. However, the molecular basis for this remains unknown. Here we show three-dimensional chromatin topology enables immune genes to engage in chromosomal contacts with a subset of long noncoding RNAs (lncRNAs) we have defined as immune gene-priming lncRNAs (IPLs). We show that the prototypical IPL, UMLILO, acts in cis to direct the WD repeat-containing protein 5 (WDR5)-mixed lineage leukemia protein 1 (MLL1) complex across the chemokine promoters, facilitating their H3K4me3 epigenetic priming. This mechanism is shared amongst several trained immune genes. Training mediated by ß-glucan epigenetically reprograms immune genes by upregulating IPLs in manner dependent on nuclear factor of activated T cells. The murine chemokine topologically associating domain lacks an IPL, and the Cxcl genes are not trained. Strikingly, the insertion of UMLILO into the chemokine topologically associating domain in mouse macrophages resulted in training of Cxcl genes. This provides strong evidence that lncRNA-mediated regulation is central to the establishment of trained immunity.

Detailed Characterization of Early HIV-1 Replication Dynamics in Primary Human Macrophages.

Macrophages are natural target cells of human immunodeficiency virus type 1 (HIV-1). Viral replication appears to be delayed in these cells compared to lymphocytes; however, little is known about the kinetics of early post-entry events. Time-of-addition experiments using several HIV-1 inhibitors and the detection of reverse transcriptase (RT) products with droplet digital PCR (ddPCR) revealed that early replication was delayed in primary human monocyte-derived macrophages of several donors and peaked late after infection. Direct imaging of reverse-transcription and pre-integration complexes (RTC/PIC) by click-labeling of newly synthesized DNA further confirmed our findings and showed a concomitant shift to the nuclear stage over time. Altering the entry pathway enhanced infectivity but did not affect kinetics of viral replication. The addition of viral protein X (Vpx) enhanced productive infection and accelerated completion of reverse transcription and nuclear entry. We propose that sterile alpha motif (SAM) and histidine/aspartate (HD) domain-containing protein 1 (SAMHD1) activity lowering deoxyribonucleotide triphosphate (dNTP) pools is the principal factor delaying early HIV-1 replication in macrophages.

Engineered anti-CRISPR proteins for optogenetic control of CRISPR-Cas9.

Anti-CRISPR proteins are powerful tools for CRISPR-Cas9 regulation; the ability to precisely modulate their activity could facilitate spatiotemporally confined genome perturbations and uncover fundamental aspects of CRISPR biology. We engineered optogenetic anti-CRISPR variants comprising hybrids of AcrIIA4, a potent Streptococcus pyogenes Cas9 inhibitor, and the LOV2 photosensor from Avena sativa. Coexpression of these proteins with CRISPR-Cas9 effectors enabled light-mediated genome and epigenome editing, and revealed rapid Cas9 genome targeting in human cells.

Lipidomimetic Compounds Act as HIV-1 Entry Inhibitors by Altering Viral Membrane Structure.

The envelope of Human Immunodeficiency Virus type 1 (HIV-1) consists of a liquid-ordered membrane enriched in raft lipids and containing the viral glycoproteins. Previous studies demonstrated that changes in viral membrane lipid composition affecting membrane structure or curvature can impair infectivity. Here, we describe novel antiviral compounds that were identified by screening compound libraries based on raft lipid-like scaffolds. Three distinct molecular structures were chosen for mode-of-action studies, a sterol derivative (J391B), a sphingosine derivative (J582C) and a long aliphatic chain derivative (IBS70). All three target the viral membrane and inhibit virus infectivity at the stage of fusion without perturbing virus stability or affecting virion-associated envelope glycoproteins. Their effect did not depend on the expressed envelope glycoproteins or a specific entry route, being equally strong in HIV pseudotypes carrying VSV-G or MLV-Env glycoproteins. Labeling with laurdan, a reporter of membrane order, revealed different membrane structure alterations upon compound treatment of HIV-1, which correlated with loss of infectivity. J582C and IBS70 decreased membrane order in distinctive ways, whereas J391B increased membrane order. The compounds' effects on membrane order were reproduced in liposomes generated from extracted HIV lipids and thus independent both of virion proteins and of membrane leaflet asymmetry. Remarkably, increase of membrane order by J391B required phosphatidylserine, a lipid enriched in the HIV envelope. Counterintuitively, mixtures of two compounds with opposite effects on membrane order, J582C and J391B, did not neutralize each other but synergistically inhibited HIV infection. Thus, altering membrane order, which can occur by different mechanisms, constitutes a novel antiviral mode of action that may be of general relevance for enveloped viruses and difficult to overcome by resistance development.

MAP3K7 is recurrently deleted in pediatric T-lymphoblastic leukemia and affects cell proliferation independently of NF-κB.

BACKGROUND: Deletions of 6q15-16.1 are recurrently found in pediatric T-cell acute lymphoblastic leukemia (T-ALL). This chromosomal region includes the mitogen-activated protein kinase kinase kinase 7 (MAP3K7) gene which has a crucial role in innate immune signaling and was observed to be functionally and prognostically relevant in different cancer entities. Therefore, we correlated the presence of MAP3K7 deletions with clinical parameters in a cohort of 327 pediatric T-ALL patients and investigated the function of MAP3K7 in the T-ALL cell lines CCRF-CEM, Jurkat and MOLT-4. METHODS: MAP3K7 deletions were detected by multiplex ligation-dependent probe amplification (MLPA). T-ALL cell lines were transduced with adeno-associated virus (AAV) vectors expressing anti-MAP3K7 shRNA or a non-silencing shRNA together with a GFP reporter. Transduction efficiency was measured by flow cytometry and depletion efficiency by RT-PCR and Western blots. Induction of apoptosis was measured by flow cytometry after staining with PE-conjugated Annexin V. In order to assess the contribution of NF-κB signaling to the effects of MAP3K7 depletion, cells were treated with TNF-α and cell lysates analyzed for components of the NF-κB pathway by Western blotting and for expression of the NF-κB target genes BCL2, CMYC, FAS, PTEN and TNF-α by RT-PCR. RESULTS: MAP3K7 is deleted in approximately 10% and point-mutated in approximately 1% of children with T-ALL. In 32 of 33 leukemias the deletion of MAP3K7 also included the adjacent CASP8AP2 gene. MAP3K7 deletions were associated with the occurrence of SIL-TAL1 fusions and a mature immunophenotype, but not with response to treatment and outcome. Depletion of MAP3K7 expression in T-ALL cell lines by shRNAs slowed down proliferation and induced apoptosis, but neither changed protein levels of components of NF-κB signaling nor NF-κB target gene expression after stimulation with TNF-α. CONCLUSIONS: This study revealed that the recurrent deletion of MAP3K7/CASP8AP2 is associated with SIL-TAL1 fusions and a mature immunophenotype, but not with response to treatment and risk of relapse. Homozygous deletions of MAP3K7 were not observed, and efficient depletion of MAP3K7 interfered with viability of T-ALL cells, indicating that a residual expression of MAP3K7 is indispensable for T-lymphoblasts.