Application prospects of the 2BS cell-adapted China fixed rabies virus vaccine strain 2aG4-B40.

BACKGROUND: Rabies is a fatal zoonotic disease whose pathogenesis has not been fully elucidated, and vaccination is the only effective method for protecting against rabies virus infection. Most inactivated vaccines are produced using Vero cells, which are African green monkey kidney cells, to achieve large-scale production. However, there is a potential carcinogenic risk due to nonhuman DNA contamination. Thus, replacing Vero cells with human diploid cells may be a safer strategy. In this study, we developed a novel 2BS cell-adapted rabies virus strain and analysed its sequence, virulence and immunogenicity to determine its application potential as a human diploid cell inactivated vaccine. METHODS AND RESULTS: The 2BS cell-adapted rabies virus strain 2aG4-B40 was established by passage for 40 generations and selection of plaques in 2BS cells. RNA sequence analysis revealed that mutations in 2BS cell-adapted strains were not located at key sites that regulate the production of neutralizing antibodies or virulence in the aG strain (GQ412744.1). The gradual increase in virulence (remaining above 7.0 logLD50/ml from the 40th to 55th generation) and antigen further indicated that these mutations may increase the affinity of the adapted strains for human diploid cells. Identification tests revealed that the 2BS cell-adapted virus strain was neutralized by anti-rabies serum, with a neutralization index of 19,952. PrEP and PEP vaccination and the NIH test further indicated that the vaccine prepared with the 2aG4-B40 strain had high neutralizing antibody levels (2.24 to 46.67 IU/ml), immunogenicity (protection index 270) and potency (average 11.6 IU/ml). CONCLUSIONS: In this study, a 2BS cell-adapted strain of the 2aG4 rabies virus was obtained by passage for 40 generations. The results of sequencing analysis and titre determination of the adapted strain showed that the mutations in the adaptive process are not located at key sequence regions of the virus, and these mutations may enhance the affinity of the adapted strain for human diploid cells. Moreover, vaccines made from the adapted strain 2aG4-B40 had high potency and immunogenicity and could be an ideal candidate rabies virus strain for inactivated vaccine preparation.

Therapeutic potential of Coumarin-polyphenolic acid hybrids in PD: Inhibition of α-Syn aggregation and disaggregation of preformed fibrils, leading to reduced neuronal inclusion formation.

This study focuses on the discovery of new potential drugs for treating PD by targeting the aggregation of α-Syn. A series of hybrids combining Coumarin and phenolic acid were designed and synthesized. Four particularly promising compounds were identified, showing strong inhibitory effects with IC50 values ranging from low micromolar to submicromolar concentrations, as low as 0.63 µM. These compounds exhibited a higher binding affinity to α-Syn residues and effectively hindered the entire aggregation process, maintaining the proteostasis conformation of α-Syn and preventing the formation of ß-sheet aggregates. This approach holds significant promise for PD prevention. Additionally, these candidate compounds demonstrated the ability to break down preformed α-Syn oligomers and fibrils, resulting in the formation of smaller aggregates and monomers. Moreover, the candidate compounds showed impressive effectiveness in inhibiting α-Syn aggregation within nerve cells, thereby reducing the likelihood of α-Syn inclusion formation resembling Lewy bodies, which highlights their potential for treating PD.

Multiplex genome editing of mammalian cells for producing recombinant heparin.

Heparin is an essential anticoagulant used for treating and preventing thrombosis. However, the complexity of heparin has hindered the development of a recombinant source, making its supply dependent on a vulnerable animal population. In nature, heparin is produced exclusively in mast cells, which are not suitable for commercial production, but mastocytoma cells are readily grown in culture and make heparan sulfate, a closely related glycosaminoglycan that lacks anticoagulant activity. Using gene expression profiling of mast cells as a guide, a multiplex genome engineering strategy was devised to produce heparan sulfate with high anticoagulant potency and to eliminate contaminating chondroitin sulfate from mastocytoma cells. The heparan sulfate purified from engineered cells grown in chemically defined medium has anticoagulant potency that exceeds porcine-derived heparin and confers anticoagulant activity to the blood of healthy mice. This work demonstrates the feasibility of producing recombinant heparin from mammalian cell culture as an alternative to animal sources.

Hybrids of polyphenolic acids and xanthone, the potential preventive and therapeutic effects on PD: Design, synthesis, in vitro anti-aggregation of α-synuclein, and disaggregation against the existed α-synuclein oligomer and fibril.

The misfolding and aggregation of α-Syn are the central mechanism linking and facilitating the other pathological mechanisms of PD. Maintaining α-Syn proteostasis by suitable inhibitors is an effective means to prevent PD. Disintegrating the neurotoxic oligomers and fibrils into the normal functional α-Syn by inhibitors is a more efficient way for PD treatment. This work synthesized two series hybrids of polyphenolic acids and xanthone. The hybrids possess a sheet-like conjugated skeleton and higher binding energies with α-Syn residues. Some compounds present well α-Syn aggregation inhibitory activities in vitro (IC50 down to 2.58 µM). The inhibitory action goes throughout the aggregation process from lag to the stationary phase by stabilizing α-Syn proteostasis conformation and preventing ß-sheets aggregation. The candidate compounds with appropriate LogP values (2.02-3.11) present good disintegration abilities against the existed α-Syn oligomers and fibrils. The preliminary mechanism studies suggest that the inhibitors could quickly and randomly bind to the specific site closed to the ß-sheet domain in the fibril, resulting in unstable and collapse of the protein fibril, yielding a complex system with aggregates of different sizes and monomers.

Room-Temperature Transport Properties of Graphene with Defects Derived from Oxo-Graphene.

In recent years, graphene oxide has been considered as a soluble precursor of graphene for electronic applications. However, the performance lags behind that of graphene due to lattice defects. Here, the relation between the density of defects in the range of 0.2 % and 1.5 % and the transport properties is quantitatively studied. Therefore, the related flakes of monolayers of graphene were prepared from oxo-functionalized graphene (oxo-G). The morphologic structure of oxo-G was imaged by atomic force microscopy (AFM) and scanning tunneling microscopy (STM). Field-effect mobility values were determined to range between 0.3 cm2 V-1 s-1 and 33.2 cm2 V-1 s-1 , which were inversely proportional to the density of defects. These results provide the first quantitative description of the density of defects and transport properties, which plays an important role for potential applications.

Evidence for Electron Transfer between Graphene and Non-Covalently Bound π-Systems.

Hybridizing graphene and molecules possess a high potential for developing materials for new applications. However, new methods to characterize such hybrids must be developed. Herein, the wet-chemical non-covalent functionalization of graphene with cationic π-systems is presented and the interaction between graphene and the molecules is characterized in detail. A series of tricationic benzimidazolium salts with various steric demand and counterions was synthesized, characterized and used for the fabrication of graphene hybrids. Subsequently, the doping effects were studied. The molecules are adsorbed onto graphene and studied by Raman spectroscopy, XPS as well as ToF-SIMS. The charged π-systems show a p-doping effect on the underlying graphene. Consequently, the tricationic molecules are reduced through a partial electron transfer process from graphene, a process which is accompanied by the loss of counterions. DFT calculations support this hypothesis and the strong p-doping could be confirmed in fabricated monolayer graphene/hybrid FET devices. The results are the basis to develop sensor applications, which are based on analyte/molecule interactions and effects on doping.

Identification of Semiconductive Patches in Thermally Processed Monolayer Oxo-Functionalized Graphene.

The thermal decomposition of graphene oxide (GO) is a complex process at the atomic level and not fully understood. Here, a subclass of GO, oxo-functionalized graphene (oxo-G), was used to study its thermal disproportionation. We present the impact of annealing on the electronic properties of a monolayer oxo-G flake and correlated the chemical composition and topography corrugation by two-probe transport measurements, XPS, TEM, FTIR and STM. Surprisingly, we found that oxo-G, processed at 300 °C, displays C-C sp3 -patches and possibly C-O-C bonds, next to graphene domains and holes. It is striking that those C-O-C/C-C sp3 -separated sp2 -patches a few nanometers in diameter possess semiconducting properties with a band gap of about 0.4 eV. We propose that sp3 -patches confine conjugated sp2 -C atoms, which leads to the local semiconductor properties. Accordingly, graphene with sp3 -C in double layer areas is a potential class of semiconductors and a potential target for future chemical modifications.

Mast Cell Degranulation and Fibroblast Activation in the Morphine-induced Spinal Mass: Role of Mas-related G Protein-coupled Receptor Signaling.

BACKGROUND: As the meningeally derived, fibroblast-rich, mass-produced by intrathecal morphine infusion is not produced by all opiates, but reduced by mast cell stabilizers, the authors hypothesized a role for meningeal mast cell/fibroblast activation. Using the guinea pig, the authors asked: (1) Are intrathecal morphine masses blocked by opiate antagonism?; (2) Do opioid agonists not producing mast cell degranulation or fibroblast activation produce masses?; and (3) Do masses covary with Mas-related G protein-coupled receptor signaling thought to mediate mast cell degranulation? METHODS: In adult male guinea pigs (N = 66), lumbar intrathecal catheters connected to osmotic minipumps (14 days; 0.5 µl/h) were placed to deliver saline or equianalgesic concentrations of morphine sulfate (33 nmol/h), 2',6'-dimethyl tyrosine-(Tyr-D-Arg-Phe-Lys-NH2) (abbreviated as DMT-DALDA; 10 pmol/h; µ agonist) or PZM21 (27 nmol/h; biased µ agonist). A second pump delivered subcutaneous naltrexone (25 µg/h) in some animals. After 14 to 16 days, animals were anesthetized and perfusion-fixed. Drug effects on degranulation of human cultured mast cells, mouse embryonic fibroblast activation/migration/collagen formation, and Mas-related G protein-coupled receptor activation (PRESTO-Tango assays) were determined. RESULTS: Intrathecal infusion of morphine, DMT-DALDA or PZM21, but not saline, comparably increased thermal thresholds for 7 days. Spinal masses proximal to catheter tip, composed of fibroblast/collagen type I (median: interquartile range, 0 to 4 scale), were produced by morphine (2.3: 2.0 to 3.5) and morphine plus naltrexone (2.5: 1.4 to 3.1), but not vehicle (1.2: 1.1 to 1.5), DMT-DALDA (1.0: 0.6 to 1.3), or PZM21 (0.5: 0.4 to 0.8). Morphine in a naloxone-insensitive fashion, but not PZM21 or DMT-DALDA, resulted in mast cell degranulation and fibroblast proliferation/collagen formation. Morphine-induced fibroblast proliferation, as mast cell degranulation, is blocked by cromolyn. Mas-related G protein-coupled receptor activation was produced by morphine and TAN67 (∂-opioid agonist), but not by PZM21, TRV130 (mu biased ligand), or DMT-DALDA. CONCLUSIONS: Opiates that activate Mas-related G protein-coupled receptor will degranulate mast cells, activate fibroblasts, and result in intrathecal mass formation. Results suggest a mechanistically rational path forward to safer intrathecal opioid therapeutics.

Lipocalin 2: A New Antimicrobial in Mast Cells.

Mast cells (MCs) play a significant role in the innate immune defense against bacterial infection through the release of cytokines and antimicrobial peptides. However, their antimicrobial function is still only partially described. We therefore hypothesized that MCs express additional antimicrobial peptides. In this study, we used FANTOM 5 transcriptome data to identify for the first time that MCs express lipocalin 2 (LCN2), a known inhibitor of bacterial growth. Using MCs derived from mice which were deficient in LCN2, we showed that this antimicrobial peptide is an important component of the MCs' antimicrobial activity against Escherichia coli (E. coli). Since sphingosine-1-phosphate receptors (S1PRs) on MCs are known to regulate their function during infections, we hypothesized that S1P could activate LCN2 production in MCs. Using an in vitro assay, we demonstrated that S1P enhances MCs antimicrobial peptide production and increases the capacity of MCs to directly kill S. aureus and E. coli via an LCN2 release. In conclusion, we showed that LCN2 is expressed by MCs and plays a role in their capacity to inhibit bacterial growth.

Selective Functionalization of Graphene at Defect-Activated Sites by Arylazocarboxylic tert-Butyl Esters.

The development of versatile functionalization concepts for graphene is currently in the focus of research. Upon oxo-functionalization of graphite, the full surface of graphene becomes accessible for C-C bond formation to introduce out-of-plane functionality. Herein, we present the arylation of graphene with arylazocarboxylic tert-butyl esters, which generates aryl radicals after activation with an acid. Surprisingly, the degree of functionalization is related to the concentration of lattice vacancy defects in the graphene material. Consequently, graphene materials that are free from lattice defects are not reactive. The reaction can be applied to graphene dispersed in solvents and leads to bitopic functionalization as well as monotopic functionalization when the graphene is deposited on surfaces. As the arylazocarboxylic tert-butyl ester moiety can be attached to various molecules, the presented method paves the way to functional graphene derivatives, with the density of defects determining the degree of functionalization.

Effects of opioid and nonopioid analgesics on canine wheal formation and cultured human mast cell degranulation.

Mast cell (MC) degranulation has been implicated in the side effect profile of a variety of clinically useful agents. Thus, after intrathecal delivery, formation of space-occupying, meningeally-derived masses may be related to local MC degranulation. We systematically characterized degranulating effects of opioid and nonopioid analgesics on cutaneous flares in the dog and in primary human MC (hMC) cultures. METHODS: Dogs were anesthetized with IV propofol and received intradermal (ID) injections (50µL). Flare diameters were measured at 30min. Drugs showing flare responses were tested after intramuscular (IM) cromolyn (10mg/kg), a MC stabilizer. Human primary MCs (human cord blood CD34+/CD45+ cells) were employed and ß-hexosaminidase in cell-free supernatants were measured to assess degranulation. RESULTS: A significant skin flare for several classes of agents was observed including opioids, ziconotide, ketamine, ST-91, neostigmine, adenosine, bupivacaine, lidocaine, MK-801 and 48/80. Tizanidine, fentanyl, alfentanil, gabapentin and baclofen produced no flare. Flare produced by all ID agents, except adenosine, bupivacaine and lidocaine, was reduced by cromolyn. Naloxone had no effect upon opiate or 48/80 evoked flares. In hMC studies, 48/80 resulted in a concentration-dependent release of ß-hexosaminidase. The rank order of drug-induced hMC ß-hexosaminidase release was similar to that for flares. CONCLUSIONS: A variety of therapeutically useful drugs degranulate MCs. This action may account for side effects such as the intrathecal granuloma resulting from spinally-delivered opioids. This degranulating effect may be useful in predicting potential intrathecal toxicity in the development of novel agents.

Skin commensal bacteria Staphylococcus epidermidis promote survival of melanocytes bearing UVB-induced DNA damage, while bacteria Propionibacterium acnes inhibit survival of melanocytes by increasing apoptosis.

BACKGROUND/PURPOSE: Skin commensal bacteria have been described to help orchestrate skin homeostasis, signaling through innate immunity pathways. This study for the first time aimed at studying the relationship between skin commensals and melanocytes after UVB exposure. METHODS: An in vitro UVB radiation model with normal human epidermal melanocytes (NHMs) and skin commensal bacteria supernatant from Staphylococcus epidermidis and Propionibacterium acnes was established. Melanocytes DNA damage, cyclobutane pyrimidine dimers (CPD), and cellular proliferation marker Ki-67 were measured by ELISA and immunofluorescence staining. Cell apoptosis was assessed by flow cytometry and PCR array and RT-qPCR. RESULTS: Normal human epidermal melanocytes are able to survive and proliferate while bearing DNA damage after UVB radiation. Skin commensal bacteria S. epidermidis and its by-product LTA promote melanocytes survival by inducing upregulation of TRAF1, CASP14, CASP5, and TP73. On the other hand, P. acnes can inhibit UVB-irradiated melanocytes survival by increasing apoptosis. CONCLUSION: Our studies show different aspects of commensal activity on melanocytes during irradiation. The possible balance achieved by the different skin commensal can influence NHM potential to become cancer cells.

Skin microbiome promotes mast cell maturation by triggering stem cell factor production in keratinocytes.

BACKGROUND: Mast cell (MC) progenitors leave the bone marrow, enter the circulation, and settle in the skin and other tissues. Their maturation in tissues is influenced by the surrounding microenvironment. OBJECTIVE: We tested the hypothesis that environmental factors play a role in MC maturation in the skin. METHODS: MCs were numerically, phenotypically, and functionally compared between germ-free (GF), specific pathogen-free, and GF mice reconstituted with microbiota. The maturity of MCs was then correlated with skin levels of stem cell factor (SCF), a critical MC differentiation factor, and lipoteichoic acid (LTA), a Toll-like receptor 2 ligand. MCs were also evaluated in mice with keratinocyte-specific deletion of Scf. RESULTS: We found that GF mice express abnormally low amounts of SCF, a critical MC differentiation factor, and contain MCs that are largely undifferentiated. Reconstituting the GF microbiota reverted this MC phenotype to normal, indicating that the phenotype is related to ongoing interactions of the microbiota and skin. Consistent with the immaturity of GF MCs, degranulation-provoking compound 48/80 induced less edema in the skin of GF mice than in conventional mice. Our results show that the skin microbiome drives SCF production in keratinocytes, which triggers the differentiation of dermal MCs. Because the skin microbiome is a rich source of LTA, a Toll-like receptor 2 ligand, we mimicked the GF microbiome's effect on MCs by applying LTA to the skin of GF mice. We also demonstrated that MC migration within the skin depends exclusively on keratinocyte-produced SCF. CONCLUSION: This study has revealed a novel mechanism by which the skin microbiota signals the recruitment and maturation of MCs within the dermis through SCF production by LTA-stimulated keratinocytes.

Invasive micropapillary carcinoma of the breast had poor clinical characteristics but showed no difference in prognosis compared with invasive ductal carcinoma.

BACKGROUND: It is controversial for prognosis of invasive micropapillary carcinoma (IMPC) compared with invasive ductal carcinoma (IDC) of the breast. To better understand the difference between IMPC and IDC prognoses, we conducted this retrospective study. METHODS: Data from 33 patients with IMPC were retrospectively reviewed, and the clinicopathologic characteristics and survival status were compared with those of 347 patients with IDC who were treated during the same period. RESULTS: The IMPC cases were of larger tumor size, greater proportion of nodal involvement, and an increased incidence of lymphovascular invasion compared with IDC cases. The overall survival (OS), local relapse-free survival (LRFS), distant metastasis-free survival (DMFS), and failure-free survival (FFS) rates were not significantly different between IMPC and IDC. The 3-year OS rate was 97 vs 94.2 % for the IMPC and IDC patients, respectively. The 3-year FFS rate was 87.9 vs 86.2 % for the IMPC and IDC patients, respectively. For IMPC patients, the 3-year LRFS rate was 93.9 % and in IDC patients was 89.0 %. The 3-year DMFS rates of IMPC patients was 90.9 % and IDC patients was 89 %. CONCLUSIONS: IMPC had poor clinical characteristics, but it showed no difference in OS, FFS, LRFS, and DMFS compare with IDC.

Prearrest hypothermia improved defibrillation and cardiac function in a rabbit ventricular fibrillation model.

BACKGROUND: Hypothermia when cardiopulmonary resuscitation begins may help achieve defibrillation and return of spontaneous circulation (ROSC), but few data are available. OBJECTIVE: The objective of this study was to determine whether prearrest hypothermia improved defibrillation and cardiac function in a rabbit ventricular fibrillation (VF) model. RESULTS: Thirty-six New Zealand rabbits were randomized equally to receive normothermia (Norm) (~39°C), post-ROSC hypothermia (~33°C), or prearrest hypothermia (~33°C). Ventricular fibrillation was induced by alternating current. After 4 minutes of VF, rabbits were defibrillated and given cardiopulmonary resuscitation until ROSC or no response (≥30 minutes). Hemodynamics and electrocardiogram were monitored; N-terminal pro-brain natriuretic peptideand troponin I were determined by enzyme-linked immunosorbent assay. Myocardial histology and echocardiographic data were evaluated. First-shock achievement of perfusion rhythm was more frequent in prearrest than normothermic animals (7/12 vs 1/12; P=.027). After ROSC, dp/dtmax was higher in prearrest than normothermic animals (P<.001). Left ventricular end-systolic pressure was higher in prearrest than normothermic animals (P=.001). At 240 minutes after ROSC, troponin I and N-terminal pro-brain natriuretic peptide were lower in prearrest than normothermic animals (15.74±2.26 vs 25.09±1.85 ng/mL and 426±23 vs 284±45 pg/mL, respectively), the left ventricular ejection fraction and cardiac output were lower in the Norm group than other 2 groups (P<.01). Myocardial histology was more disturbed in normothermic than post-ROSC and prearrest animals, but was not different in the latter 2 groups. CONCLUSIONS: Induction of hypothermia before VF led to improved cardiac function in a rabbit VF model through improving achievement of perfusing rhythm by first-shock defibrillation and facilitating resuscitation.

Transcriptome profiling of Vitis amurensis, an extremely cold-tolerant Chinese wild Vitis species, reveals candidate genes and events that potentially connected to cold stress.

Vitis amurensis Rupr. is an exceptional wild-growing Vitis (grape) species that can safely survive a wide range of cold conditions, but the underlying cold-adaptive mechanism associated with gene regulation is poorly investigated. We have analyzed the physiochemical and transcriptomic changes caused by cold stress in a cold-tolerant accession, 'Heilongjiang seedling', of Chinese wild V. amurensis. We statistically determined that a total of 6,850 cold-regulated transcripts were involved in cold regulation, including 3,676 up-regulated and 3,174 down-regulated transcripts. A global survey of messenger RNA revealed that skipped exon is the most prevalent form of alternative spicing event. Importantly, we found that the total splicing events increased with the prolonged cold stress. We also identified thirty-eight major TF families that were involved in cold regulation, some of which were previously unknown. Moreover, a large number of candidate pathways for the metabolism or biosynthesis of secondary metabolites were found to be regulated by cold, which is of potential importance in coordinating cold tolerance with growth and development. Several heat shock proteins and heat shock factors were also detected to be intensively cold-regulated. Furthermore, we validated the expression profiles of 16 candidates using qRT-PCR to further confirm the accuracy of the RNA-seq data. Our results provide a genome-wide view of the dynamic changes in the transcriptome of V. amurensis, in which it is evident that various structural and regulatory genes are crucial for cold tolerance/adaptation. Moreover, our robust dataset advances our knowledge of the genes involved in the complex regulatory networks of cold stress and leads to a better understanding of cold tolerance mechanisms in this extremely cold-tolerant Vitis species.

Commensal bacteria lipoteichoic acid increases skin mast cell antimicrobial activity against vaccinia viruses.

Mast cells (MCs) are considered sentinels in the skin and mucosa. Their ability to release antimicrobial peptides, such as cathelicidin, protects against bacterial infections when the epithelial barrier is breached. We recently described that MCs defend against bacterial and viral infections through the release of cathelicidin during degranulation. In this study, we hypothesize that cathelicidin expression is induced in MCs by the activation of TLR2 from bacterial products (lipoteichoic acid) produced by commensal bacteria at the epithelial surface. Our research shows that signaling through TLR2 increases the production and expression of cathelicidin in mast cells, thereby enhancing their capacity to fight vaccinia virus. MCs deficient in cathelicidin were less efficient in killing vaccinia virus after lipoteichoic acid stimulation than wild-type cells. Moreover, the activation of TLR2 increases the MC recruitment at the skin barrier interface. Taken together, our findings reveal that the expression and control of antimicrobial peptides and TLR signaling on MCs are key in fighting viral infection. Our findings also provide new insights into the pathogenesis of skin infections and suggest potential roles for MCs and TLR2 ligands in antiviral therapy.

Skin mast cells protect mice against vaccinia virus by triggering mast cell receptor S1PR2 and releasing antimicrobial peptides.

Mast cells (MCs) are well-known effectors of allergic reactions and are considered sentinels in the skin and mucosa. In addition, through their production of cathelicidin, MCs have the capacity to oppose invading pathogens. We therefore hypothesized that MCs could act as sentinels in the skin against viral infections using antimicrobial peptides. In this study, we demonstrate that MCs react to vaccinia virus (VV) and degranulate using a membrane-activated pathway that leads to antimicrobial peptide discharge and virus inactivation. This finding was supported using a mouse model of viral infection. MC-deficient (Kit(wsh-/-)) mice were more susceptible to skin VV infection than the wild type animals, whereas Kit(wsh-/-) mice reconstituted with MCs in the skin showed a normal response to VV. Using MCs derived from mice deficient in cathelicidin antimicrobial peptide, we showed that antimicrobial peptides are one important antiviral granule component in in vivo skin infections. In conclusion, we demonstrate that MC presence protects mice from VV skin infection, MC degranulation is required for protecting mice from VV, neutralizing Ab to the L1 fusion entry protein of VV inhibits degranulation apparently by preventing S1PR2 activation by viral membrane lipids, and antimicrobial peptide release from MC granules is necessary to inactivate VV infectivity.