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EFSA Journal. European Food Safety... Apr 2024Two alternative methods for producing compost in a tunnel, from certain category (Cat.) 3 animal by-products (ABP) and other non-ABP material, were assessed. The first...
Two alternative methods for producing compost in a tunnel, from certain category (Cat.) 3 animal by-products (ABP) and other non-ABP material, were assessed. The first method proposed a minimum temperature of 55°C for 72 h and the second 60°C for 48 h, both with a maximum particle size of 200 mm. The assessment of the Panel on Biological Hazards (BIOHAZ) exclusively focused on Cat. 3 ABP materials (catering waste and processed foodstuffs of animal origin no longer intended for human consumption). The proposed composting processes were evaluated for their efficacy to achieve a reduction of at least 5 log of and Senftenberg (775W, HS negative) and at least 3 log of relevant thermoresistant viruses. The applicant provided a list of biological hazards that may enter the composting process and selected parvoviruses as the indicator of the thermoresistant viruses. The evidence provided by the applicant included: (a) literature data on thermal inactivation of biological hazards; (b) results from validation studies on the reduction of , Senftenberg 775W HS negative and canine parvovirus carried out in composting plants across Europe; (c) and experimental data from direct measurements of reduction of infectivity of murine parvovirus in compost material applying the time/temperature conditions of the two alternative methods. The evidence provided showed the capacity of the proposed alternative methods to reduce and Senftenberg 775W HS negative by at least 5 log, and parvoviruses by at least 3 log. The BIOHAZ Panel concluded that the two alternative methods under assessment can be considered to be equivalent to the processing method currently approved in the Commission Regulation (EU) No 142/2011.
PubMed: 38681740
DOI: 10.2903/j.efsa.2024.8745 -
Antiviral Research Jun 2024Immune tolerance to the hepatitis B virus (HBV) is crucial for developing chronic hepatitis B, and the HBV surface antigen (HBsAg) produced and secreted in high amounts...
Immune tolerance to the hepatitis B virus (HBV) is crucial for developing chronic hepatitis B, and the HBV surface antigen (HBsAg) produced and secreted in high amounts is regarded as a key contributor. HBsAg is expressed in HBV-infected hepatocytes and those carrying an HBV integration. Whether either HBsAg secretion or the high antigen amount expressed in the liver determines its immunomodulatory properties, however, remains unclear. We, therefore, developed a novel HBV animal model that allowed us to study the role of secreted HBsAg. We introduced a previously described HBs mutation, C65S, abolishing HBsAg secretion into a replication-competent 1.3-overlength HBV genome and used adeno-associated virus vectors to deliver it to the mouse liver. The AAV-HBV established a carrier state of wildtype and C65S mutant HBV, respectively. We investigated antiviral B- and T-cell immunity in the HBV-carrier mice after therapeutic vaccination. Moreover, we compared the effect of a lacking HBsAg secretion with that of an antiviral siRNA. While missing HBsAg secretion allowed for higher levels of detectable anti-HBs antibodies after therapeutic vaccination, it did neither affect antiviral T-cell responses nor intrahepatic HBV gene expression, irrespective of the starting level. A treatment with HBV siRNA restricting viral antigen expression within hepatocytes, however, improved the antiviral efficacy of therapeutic vaccination, irrespective of the ability of HBV to secrete HBsAg. Our data indicate that clearing HBsAg from blood cannot significantly impact HBV persistence or T-cell immunity. This indicates that a restriction of hepatic viral antigen expression will be required to break HBV immunotolerance.
Topics: Animals; Hepatitis B Surface Antigens; Hepatitis B virus; Mice; Disease Models, Animal; T-Lymphocytes; Liver; Hepatitis B, Chronic; Hepatitis B; Mutation; Mice, Inbred C57BL; Dependovirus; Hepatitis B Antibodies; Hepatocytes; Humans
PubMed: 38679167
DOI: 10.1016/j.antiviral.2024.105896 -
Scientific Reports Apr 2024Among the several animal models of α-synucleinopathies, the well-known viral vector-mediated delivery of wild-type or mutated (A53T) α-synuclein requires new tools to...
Among the several animal models of α-synucleinopathies, the well-known viral vector-mediated delivery of wild-type or mutated (A53T) α-synuclein requires new tools to increase the lesion in mice and follow up in vivo expression. To this end, we developed a bioluminescent expression reporter of the human A53T-α-synuclein gene using the NanoLuc system into an AAV2/9, embedded or not in a fibroin solution to stabilise its expression in space and time. We first verified the expression of the fused protein in vitro on transfected cells by bioluminescence and Western blotting. Next, two groups of C57Bl6Jr mice were unilaterally injected with the AAV-NanoLuc-human-A53T-α-synuclein above the substantia nigra combined (or not) with fibroin. We first show that the in vivo cerebral bioluminescence signal was more intense in the presence of fibroin. Using immunohistochemistry, we find that the human-A53T-α-synuclein protein is more restricted to the ipsilateral side with an overall greater magnitude of the lesion when fibroin was added. However, we also detected a bioluminescence signal in peripheral organs in both conditions, confirmed by the presence of viral DNA corresponding to the injected AAV in the liver using qPCR.
Topics: Animals; alpha-Synuclein; Dependovirus; Humans; Mice; Luminescent Measurements; Genetic Vectors; Fibroins; Mice, Inbred C57BL; Central Nervous System; Male; Luciferases
PubMed: 38678103
DOI: 10.1038/s41598-024-60613-6 -
Viruses Apr 2024Southern Africa Territories 2 (SAT2) foot-and-mouth disease (FMD) has crossed long-standing regional boundaries in recent years and entered the Middle East. However, the...
Chimeric Porcine Parvovirus VP2 Virus-like Particles with Epitopes of South African Serotype 2 Foot-and-Mouth Disease Virus Elicits Specific Humoral and Cellular Responses in Mice.
Southern Africa Territories 2 (SAT2) foot-and-mouth disease (FMD) has crossed long-standing regional boundaries in recent years and entered the Middle East. However, the existing vaccines offer poor cross-protection against the circulating strains in the field. Therefore, there is an urgent need for an alternative design approach for vaccines in anticipation of a pandemic of SAT2 Foot-and-mouth disease virus (FMDV). The porcine parvovirus (PPV) VP2 protein can embed exogenous epitopes into the four loops on its surface, assemble into virus-like particles (VLPs), and induce antibodies and cytokines to PPV and the exogenous epitope. In this study, chimeric porcine parvovirus VP2 VLPs (chimeric PPV-SAT2-VLPs) expressing the T-and/or B-cell epitopes of the structural protein VP1 of FMDV SAT2 were produced using the recombinant pFastBac™ Dual vector of baculoviruses in Sf9 and HF cells We used the Bac-to-Bac system to construct the recombinant baculoviruses. The VP2-VLP--SAT2 chimeras displayed chimeric T-cell epitope (amino acids 21-40 of VP1) and/or the B-cell epitope (amino acids 135-174) of SAT FMDV VP1 by substitution of the corresponding regions at the N terminus (amino acids 2-23) and/or loop 2 and/or loop 4 of the PPV VP2 protein, respectively. In mice, the chimeric PPV-SAT2-VLPs induced specific antibodies against PPV and the VP1 protein of SAT2 FMDV. The VP2-VLP-SAT2 chimeras induced specific antibodies to PPV and the VP1 protein specific epitopes of FMDV SAT2. In this study, as a proof-of-concept, successfully generated chimeric PPV-VP2 VLPs expressing epitopes of the structural protein VP1 of FMDV SAT2 that has a potential to prevent FMDV SAT2 and PPV infection in pigs.
Topics: Animals; Foot-and-Mouth Disease Virus; Mice; Foot-and-Mouth Disease; Capsid Proteins; Parvovirus, Porcine; Antibodies, Viral; Viral Vaccines; Vaccines, Virus-Like Particle; Swine; Immunity, Humoral; Immunity, Cellular; Epitopes, T-Lymphocyte; Epitopes, B-Lymphocyte; Serogroup; Mice, Inbred BALB C; Female; Epitopes; Sf9 Cells; Antibodies, Neutralizing; Antigens, Viral
PubMed: 38675963
DOI: 10.3390/v16040621 -
Viruses Apr 2024The higher-order structure (HOS) is a critical quality attribute of recombinant adeno-associated viruses (rAAVs). Evaluating the HOS of the entire rAAV capsid is...
The higher-order structure (HOS) is a critical quality attribute of recombinant adeno-associated viruses (rAAVs). Evaluating the HOS of the entire rAAV capsid is challenging because of the flexibility and/or less folded nature of the VP1 unique (VP1u) and VP1/VP2 common regions, which are structural features essential for these regions to exert their functions following viral infection. In this study, hydrogen/deuterium exchange mass spectrometry (HDX-MS) was used for the structural analysis of full and empty rAAV8 capsids. We obtained 486 peptides representing 85% sequence coverage. Surprisingly, the VP1u region showed rapid deuterium uptake even though this region contains the phospholipase A2 domain composed primarily of α-helices. The comparison of deuterium uptake between full and empty capsids showed significant protection from hydrogen/deuterium exchange in the full capsid at the channel structure of the 5-fold symmetry axis. This corresponds to cryo-electron microscopy studies in which the extended densities were observed only in the full capsid. In addition, deuterium uptake was reduced in the VP1u region of the full capsid, suggesting the folding and/or interaction of this region with the encapsidated genome. This study demonstrated HDX-MS as a powerful method for probing the structure of the entire rAAV capsid.
Topics: Dependovirus; Capsid Proteins; Capsid; Serogroup; Deuterium Exchange Measurement; Hydrogen Deuterium Exchange-Mass Spectrometry; Humans; Deuterium; Mass Spectrometry; Cryoelectron Microscopy; Models, Molecular
PubMed: 38675928
DOI: 10.3390/v16040585 -
Viruses Mar 2024Individuals chronically infected with hepatitis B virus (HBV) and hepatitis Delta virus (HDV) present an increased risk of developing cirrhosis and hepatocellular...
Individuals chronically infected with hepatitis B virus (HBV) and hepatitis Delta virus (HDV) present an increased risk of developing cirrhosis and hepatocellular carcinoma in comparison to HBV mono-infected individuals. Although HDV only replicates in individuals coinfected or superinfected with HBV, there is currently no in vitro model that can stably express both viruses simultaneously, mimicking the chronic infections seen in HBV/HDV patients. Here, we present the HepG2BD cell line as a novel in vitro culture system for long-term replication of HBV and HDV. HepG2BD cells derive from HepG2.2.15 cells in which a 2 kb HDV cDNA sequence was inserted into the adeno-associated virus safe harbor integration site 1 (AAVS1) using CRISPR-Cas9. A Tet-Off promoter was placed 5' of the genomic HDV sequence for reliable initiation/repression of viral replication and secretion. HBV and HDV replication were then thoroughly characterized. Of note, non-dividing cells adopt a hepatocyte-like morphology associated with an increased production of both HDV and HBV virions. Finally, HDV seems to negatively interfere with HBV in this model system. Altogether, HepG2BD cells will be instrumental to evaluate, in vitro, the fundamental HBV-HDV interplay during simultaneous chronic replication as well as for antivirals screening targeting both viruses.
Topics: Hepatitis Delta Virus; Humans; Virus Replication; Hepatitis B virus; Hep G2 Cells; Hepatocytes; Hepatitis D; CRISPR-Cas Systems; Dependovirus; Coinfection
PubMed: 38675875
DOI: 10.3390/v16040532 -
International Journal of Molecular... Apr 2024Polyglutamine (polyQ)-encoding CAG repeat expansions represent a common disease-causing mutation responsible for several dominant spinocerebellar ataxias (SCAs)....
Polyglutamine (polyQ)-encoding CAG repeat expansions represent a common disease-causing mutation responsible for several dominant spinocerebellar ataxias (SCAs). PolyQ-expanded SCA proteins are toxic for cerebellar neurons, with Purkinje cells (PCs) being the most vulnerable. RNA interference (RNAi) reagents targeting transcripts with expanded CAG reduce the level of various mutant SCA proteins in an allele-selective manner in vitro and represent promising universal tools for treating multiple CAG/polyQ SCAs. However, it remains unclear whether the therapeutic targeting of CAG expansion can be achieved in vivo and if it can ameliorate cerebellar functions. Here, using a mouse model of SCA7 expressing a mutant Atxn7 allele with 140 CAGs, we examined the efficacy of short hairpin RNAs (shRNAs) targeting CAG repeats expressed from PHP.eB adeno-associated virus vectors (AAVs), which were introduced into the brain via intravascular injection. We demonstrated that shRNAs carrying various mismatches with the CAG target sequence reduced the level of polyQ-expanded ATXN7 in the cerebellum, albeit with varying degrees of allele selectivity and safety profile. An shRNA named A4 potently reduced the level of polyQ-expanded ATXN7, with no effect on normal ATXN7 levels and no adverse side effects. Furthermore, A4 shRNA treatment improved a range of motor and behavioral parameters 23 weeks after AAV injection and attenuated the disease burden of PCs by preventing the downregulation of several PC-type-specific genes. Our results show the feasibility of the selective targeting of CAG expansion in the cerebellum using a blood-brain barrier-permeable vector to attenuate the disease phenotype in an SCA mouse model. Our study represents a significant advancement in developing CAG-targeting strategies as a potential therapy for SCA7 and possibly other CAG/polyQ SCAs.
Topics: Animals; Spinocerebellar Ataxias; Peptides; Disease Models, Animal; Dependovirus; Mice; Ataxin-7; Trinucleotide Repeat Expansion; RNA, Small Interfering; Phenotype; Genetic Vectors; Purkinje Cells; Mice, Transgenic; Cerebellum; Humans; Genetic Therapy; Alleles
PubMed: 38673939
DOI: 10.3390/ijms25084354 -
Biomolecules Apr 2024In this study, we introduce electrospun polydioxanone (PDO) nonwoven fabrics as a platform for the delivery of adeno-associated virus (AAV) vectors for transduction and...
In this study, we introduce electrospun polydioxanone (PDO) nonwoven fabrics as a platform for the delivery of adeno-associated virus (AAV) vectors for transduction and genome editing by adhering them to organ surfaces, including the heart. AAV vectors were loaded onto the PDO fabrics by soaking the fabrics in a solution containing AAV vectors. In vitro, the amount of AAV vectors loaded onto the fabrics could be adjusted by changing their concentration in the solution, and the number of cells expressing the green fluorescent protein (GFP) encoded by the AAV vectors increased in correlation with the increasing amount of loaded AAV vectors. In vivo, both transduction and genome editing resulted in the observation of GFP expression around AAV vector-loaded PDO fabrics attached to the surfaces of mouse hearts, indicating effective transduction and expression at the target site. These results demonstrate the great potential of electrospun PDO nonwoven fabrics carrying therapeutic AAV vectors for gene therapy.
Topics: Dependovirus; Animals; Genetic Vectors; Polydioxanone; Gene Editing; Mice; Humans; Transduction, Genetic; Green Fluorescent Proteins; HEK293 Cells; Genetic Therapy; Myocardium
PubMed: 38672522
DOI: 10.3390/biom14040506 -
Genome Biology Apr 2024Prime editing enables precise base substitutions, insertions, and deletions at targeted sites without the involvement of double-strand DNA breaks or exogenous donor DNA...
BACKGROUND
Prime editing enables precise base substitutions, insertions, and deletions at targeted sites without the involvement of double-strand DNA breaks or exogenous donor DNA templates. However, the large size of prime editors (PEs) hampers their delivery in vivo via adeno-associated virus (AAV) due to the viral packaging limit. Previously reported split PE versions provide a size reduction, but they require intricate engineering and potentially compromise editing efficiency.
RESULTS
Herein, we present a simplified split PE named as CC-PE, created through non-covalent recruitment of reverse transcriptase to the Cas9 nickase via coiled-coil heterodimers, which are widely used in protein design due to their modularity and well-understood sequence-structure relationship. We demonstrate that the CC-PE maintains or even surpasses the efficiency of unsplit PE in installing intended edits, with no increase in the levels of undesired byproducts within tested loci amongst a variety of cell types (HEK293T, A549, HCT116, and U2OS). Furthermore, coiled-coil heterodimers are used to engineer SpCas9-NG-PE and SpRY-PE, two Cas9 variants with more flexible editing scope. Similarly, the resulting NG-CC-PE and SpRY-CC-PE also achieve equivalent or enhanced efficiency of precise editing compared to the intact PE. When the dual AAV vectors carrying CC-PE are delivered into mice to target the Pcsk9 gene in the liver, CC-PE enables highly efficient precise editing, resulting in a significant reduction of plasma low-density lipoprotein cholesterol and total cholesterol.
CONCLUSIONS
Our innovative, modular system enhances flexibility, thus potentially facilitating the in vivo applicability of prime editing.
Topics: Gene Editing; Humans; Animals; Mice; CRISPR-Associated Protein 9; CRISPR-Cas Systems; HEK293 Cells; Dependovirus
PubMed: 38671524
DOI: 10.1186/s13059-024-03257-z -
Scientific Reports Apr 2024Parvovirus B19V (B19V) infection during pregnancy can be complicated by potentially life-threatening fetal hydrops, which can be managed by intrauterine transfusion...
Parvovirus B19V (B19V) infection during pregnancy can be complicated by potentially life-threatening fetal hydrops, which can be managed by intrauterine transfusion (IUT). This study investigates the long-term temporal patterns in the epidemiology of B19V and evaluates the impact on fetal hydrops, by combining data on B19V infections from the Dutch Sentinel Surveillance system in the period 1990 to 2023, Dutch blood banking data and hospital data on fetal hydrops. Using wavelet analysis, we identified annual epidemic cycles in the Netherlands in the period 1990-2019 and we identified superimposed multiannual cycles in the period 1990-2009. After 2009, no multiannual cycle could be identified, although the incidence fluctuated and correlates with number of IUT performed. As of 2020, weekly reports of B19V infection demonstrated a historically low incidence and B19V-DNA positive blood donors were nearly absent. From May 2020 to May 2023, no IUT for B19V-related hydrops was performed. In the spring of 2023, B19V infections re-emerged, reaching pre-pandemic epidemic levels. Due to the changes in B19V epidemiology over the last 30 years and the near-absence of B19V during the COVID-19 pandemic, the resulting low immunity levels may lead to rebound outbreaks. Alertness to severe complications such as fetal hydrops is warranted.
Topics: Humans; Netherlands; COVID-19; Parvovirus B19, Human; Female; Pregnancy; Hydrops Fetalis; Incidence; Parvoviridae Infections; Pregnancy Complications, Infectious; SARS-CoV-2; Pandemics; Erythema Infectiosum; Blood Transfusion, Intrauterine; Adult
PubMed: 38671058
DOI: 10.1038/s41598-024-59582-7