Like other alpha herpesviruses, pseudorabies virus (PRV) establishes lifelong latency in trigeminal ganglionic (TG) neurons. Upon stress, the latent viruses in the TG neurons reactivate and are transported anterograde from the neuron cell bodies to the nerve endings in the nasal mucosa, where they replicate and are discharged in the nasal and oral secretions. Consequently, the virus is transmitted to other naïve animals. This cycle of latency and reactivation continues until the animal dies or is slaughtered. We have constructed a PRV triple mutant virus (PRVtmv) and used it as a live subunit vaccine vector against porcine circovirus 2b (PCV2b) and classical swine fever virus (CSFV) (PRVtmv+). We compared the latency reactivation properties of PRVtmv+ with its parent wild-type (wt) Becker strain following intranasal infection. The results showed that PRV wt and PRVtmv+ established latency in the TG neurons. Based on nasal virus shedding, immediate early  and late genes, MCP (major capsid protein) and gC (glycoprotein C) transcriptions, and viral DNA copy numbers in the TGs of latently infected and dexamethasone (Dex)-treated pigs, both PRV wt and PRVtmv+ reactivated from latency. We noticed that PRV wt virus replicated productively in the terminally differentiated, postmitotic TG neurons, but PRVtmv+ failed to replicate and, therefore, there was no virus production in the TG. In addition, we found that only the PRV wt virus was shed in the nasal secretions following the Dex-induced reactivation. Our results demonstrated that the PRVtmv+ is safe as a live viral subunit vaccine vector without the possibility of productive replication in the TG upon reactivation from latency and without subsequent nasal virus shedding. This property of PRVtmv+ precludes the possibility of vaccine virus circulation in pigs and the risk of reversion to virulence. Pseudorabies, or Aujeszky’s disease (AD), caused by the pseudorabies virus (PRV), is an economically significant viral disease of pigs ,2. FolloWe recently constructed a PRV triple mutant virus (PRVtmv) by deleting the genes coding for envelope proteins gE and gG, and the non-structural protein thymidine kinase (TK) . Both gEIn the present study, our goal was to investigate whether the PRVtmv+ established latency in the TG neurons, reactivated upon dexamethasone (Dex)-induced reactivation and shed in the nasal secretions. The results demonstrated that both PRV wt and PRVtmv+ established latency in the TG neurons and reactivated upon Dex treatments. We noticed that only the wt virus replicated in the TG neurons, transported to the nerve endings in the nasal mucosa, and shed in the nasal secretions. The study was performed in accordance with protocols approved by the LSU Institutional Animal Care and Use Committee, Louisiana State University .®, Manassas, VA, USA) and Madin Darby bovine kidney cells  were propagated in Dulbecco’s modified eagle medium  supplemented with 10% heat-inactivated fetal bovine serum  and 1 × antibiotic-antimycotic solution  (growth medium). Swine kidney . Five 8-week-old healthy Yorkshire pigs were purchased from a PRV- and PCV2b-free supplier . Before inclusion in the study, pigs were tested for bovine viral diarrhea (BVD)-free status by the serological assay as described previously . After a6 PFUs/nostril . The pigs in the PRVtmv+ vaccine group were inoculated IN with 4 × 106 PFUs per nostril  and subcutaneously (S/C) with filtered (0.2 µm pore size) 4 × 106 PFUs. On the day of infection, each pig received Noromycin® 300 LA I/M injection  to prevent secondary bacterial infections. At 28 days post-infection (dpi), pigs in both groups received the dexamethasone (Dex) intravenously (I/V) (0.5 mg/kg), followed by two more S/C Dex injections (0.25 mg/kg) on 29 and 30 dpi. Each pig in the PRV wt infection group 1 was intranasally (IN) infected with total 2 × 10Pigs were routinely monitored for obvious clinical illness, feed intake, and water intake. Bodyweight and temperature were recorded. Clinical assessment included coughing, sneezing, nasal discharge, depression, respiratory difficulties, and other systemic illnesses .Nasal swabs were collected in 2 mL of DMEM, supplemented with 3× antibiotic-antimycotic solution and 2% FBS. Nasal swab samples were aliquoted and stored at −80 °C until use. Blood samples collected for sera were processed, aliquoted, and stored at −80 °C.® . At necropsy, TGs were collected for histopathology , virus isolation, and qPCR assays (dry ice). Formalin-fixed tissues were paraffinized, sectioned, and processed for histopathology (H&E staining).At five days post-dexamethasone treatment (5 dp-Dex), pigs were euthanized with xylazine and EuthasolPRV-specific neutralizing antibody titers in sera were determined by a standard plaque reduction assay  as described previously [® DNA mini kit . Before DNA isolation from TG, tissues were homogenized using 2.8 mm ceramic beads  in Pre-cellys 24 homogenizer . To determine the transcription and quantification of the targeted immediate early (infected cell protein 0 - ICP0) and late (major capsid protein - MCP and glycoprotein C - gC) PRV genes in the TG neurons, RNA was isolated. Briefly, after the homogenization of TG, RNA was isolated using an RNeasy mini kit . DNA contamination in RNA samples was removed by RNase-Free DNase Set . Finally, cDNA synthesis was performed using the Verso cDNA synthesis kit . To determine the PRV genome copies in the nasal swabs and in the TG, total DNA was isolated as described previously  using th1 to 106 copies per reaction).PRVtmv+ genomic copies were determined by TaqMan probe-based real-time qPCR in ABI PRISM™ 7900HT Sequence Detection System , using targeted genes ICP0-, MCP-, and gC-specific primers and probes , in the same cells. The mean copies of the PRV-MCP gene per one million cells were then plotted. For the PRVtmv+ latency control, we used the TG samples collected and stored at −80 °C from our previous vaccination experiment . To demo® software version 5.04 . The two-way analysis of variance (ANOVA) followed by Bonferroni post-tests to compare replicate means by row were performed. A value of p ˂ 0.05 was considered statistically significant. All data are expressed as means ± standard deviation. Statistical analyses were performed using GraphPad PRISMOn 3 dpi, PRV wt-infected pigs started to show moderate fever (40.6 °C) and mild to moderate respiratory signs  . The cli2 5 × 102 genome copies) persisted until 21 dpi in the PRV wt-infected pigs but was cleared by 28 dpi. Following primary intranasal infection of pigs, both PRV wt and PRVtmv+ replicated in the nasal mucosa and shed in nasal secretions. As shown in On 28 dpi, nasal virus shedding was no longer detectable by qPCR, indicating that the virus established latency in the TG neurons. Therefore, starting at 28 dpi, we began administering Dex to all five pigs to induce the reactivation of latent PRVtmv+ and PRV wt viruses. Nasal swabs of pigs collected on 3 and 5 dp-Dex were tested by qPCR for virus shedding. The results presented in To demonstrate that PRVtmv+ was transported retrogradely to the TG neurons following intranasal infection and established latency in the neuron cell bodies, we determined viral genome copies in TGs of wt and PRVtmv+-infected/vaccinated pigs by qPCR, targeting the PRV MCP gene. Specifically, we used the TGs collected from pigs at 53 days post-PRVtmv+ vaccination/21 days post-PCV2b challenge from a recent PRVtmv+ vaccination– PCV2b challenge study [To confirm that latent PRVtmv+ reactivated in the TG neurons following the Dex treatments, we investigated the transcription of the viral immediate early gene, ICP0, during latency  and at 5 days post-Dex treatment (of PRVtmv+ and PRV wt-infected pigs). The results depicted in The postmitotic neuron does not have a complementary TK enzyme as in the nasal epithelium. Therefore, we expected that PRVtmv+ lacking the viral TK could not replicate in the TG neurons. To prove that PRVtmv+ did not replicate, we investigated transcription levels of the DNA replication-dependent late γ genes, MCP and gC, in the TG neurons during latency (of PRVtmv+-infected pigs) and at 5 days post-Dex treatment (of PRVtmv+ and PRV wt-infected pigs). The rationale was that detecting the MCP and gC transcriptions, or no transcriptions, following the Dex treatment would indirectly indicate that the virus replicated or failed to replicate (abortive replication), respectively. The results depicted in Following latency reactivation in the TG, PRV and other alpha herpesviruses replicate initially in the TG neurons at a low level. Subsequently, progeny viruses travel anterogradely from neuron cell bodies down the axon to nerve endings in the nasal mucosa and replicate there, resulting in nasal virus shedding. Since the pigs were pre-exposed to PRV, there was a B cell response and generation of memory B cells. Consequently, upon virus reactivation in the TG and replication in the nasal epithelium, memory B cells are expected to undergo a recall immune response resulting in a rapid rise in SN antibody titers. Therefore, a drastic increase in the PRV-specific SN antibody titers due to memory immune response following Dex-induced latency reactivation is indirect evidence of latent virus reactivation in the TG, followed by virus replication in the nasal epithelium, and completion of all the steps in between noted above. As shown in Previously, we characterized the PRVtmv+ subunit vaccine vector against PCV2b and CSFV. In PRVtmv+, three viral genes, namely TK, gG, and gE, were deleted and replaced with three chimeric genes, PCV2b Cap, CSFV Erns-GM-CSF, and CSFV E2, respectively . In cellIt is well-established and documented knowledge that all animal alpha herpesviruses establish latency in the sensory neurons of the trigeminal ganglia upon primary intranasal infection. Further, it is also well known that envelope proteins gE, gG, and TK genes are not necessary for virus entry into the nerve termini of the trigeminal nerve and retrograde transport to the cell bodies in the TG neurons where both wt and PRVtmv+  viruses establish latency ,20,21,22Further, except for latency-associated transcript (LAT), there is no other gene transcription or translation and no productive virus replication during latency. Upon stress or dexamethasone-induced reactivation of the latent herpesvirus, the latent virus reactivates and sheds in the nasal secretion . In the We designed the PRVtmv with the following hypotheses: (i) it can be used as a live subunit vaccine vector for immunization against other swine viruses because the virus will replicate in the nasal epithelium and induce mucosal, humoral, and cellular immune responses; (ii) the intranasal vaccination route with a live virus vector would be less affected by pre-existing immune interference; (iii) after primary infection of the nasal epithelium, the virus would enter the nerve endings of the trigeminal nerve and be transported to the cell bodies in the TG neurons; (iv) the vaccine virus will establish latency in the TG neurons, but under no circumstances can the virus be shed in the nasal secretions, and therefore, the vaccine virus will not be transmitted to other pigs or maintained in the pig population; and (v) finally, the vaccinated animals can be distinguished from the PRV wt-infected pigs based on the DIVA (distinguishable from the field wild-type PRV) property, which will be critical if the vaccine needs to be used in PRV-free countries. We have already characterized PRVtmv+ in vitro and in vivo in pigs and determined its vaccine efficacy against PCV2b  and CSFVOur experimental design for this study was based on the following dogmas. (i) Neurons are postmitotic and, as such, do not code for TK enzyme; therefore, the TK minus viruses do not replicate in the neurons . (ii) TKBased on the above discussion, we never questioned the establishment of latency in the TGs of both PRV wt and PRVtmv+ after primary intranasal infection. Moreover, for PRV wt, Dex-induced latent virus reactivation and nasal virus shedding were expected. Thus, we used only two pigs as a positive control for PRV wt. For PRVtmv+, we expected the virus not to replicate in the TG neurons, even if the latent virus was reactivated. To prove that the PRVtmv+ reactivated in the TGs of Dex-treated pigs, we tested the transcription of the immediate early gene, ICP0, which would be transcribed immediately after reactivation regardless of whether the virus expressed TK or not. Our results demonstrate the ICP0 transcription in the TGs of all three Dex-treated pigs. To prove that PRVtmv+ failed to replicate in the same TG neurons, we compared the number of PRVtmv+ genomic copies between the TGs of latently infected- and Dex-treated pigs. As expected, the genomic copy numbers in both cases were similar. The final proof that PRVtmv+ did not replicate in the TG neurons of Dex-treated pigs was that the DNA replication-dependent gamma 2 (late gene) gC transcription was not detectable in all three pigs. Additionally, the leaky late , major capsid protein VP5 gene was not transcribed. On the contrary, both gC and VP5 transcripts were readily detectable in the TGs of Dex-treated wt-infected pigs (positive control). We have confirmed in two ways that after Dex-induced latency reactivation of PRVtmv+, there was no nasal virus shedding because PRVtmv+ replication in the TG neurons was aborted. Our data revealed that PRVtmv DNA was present in the TG neurons of latently infected pigs. There was ICP0 gene transcription in the TG neurons upon Dex-induced reactivation. Still, there were no transcriptions of the DNA replication-dependent, late viral genes gC and MCP in the TG neurons . Second,This rapid increase in SN titers in the PRV wt-infected pigs, despite the Dex-induced immunosuppression, could be explained by the following factors. First, the pigs were pre-exposed to PRV, resulting in the generation of memory B cells. Second, upon wt virus, latency reactivation due to immunosuppression, and subsequent replication in the nasal epithelium, the memory B cells underwent a secondary recall immune response resulting in a rapid memory B cell proliferation, differentiation to plasma cells, and antibody production. Third, the proliferating memory B cells and differentiated plasma cells have decreased susceptibility to dexamethasone-induced apoptosis. These factors correlate well with the rapid rise in neutralizing antibody titers of wt-infected pigs by 5 dp-Dex, a classical recall response . These fPrevious studies by other investigators revealed that pre-colonization of TG with a latent PRV prevented the latent infection in the TG with a superinfecting PRV strain . Even th


Herpes simplex virus 1 and 2 infections cause high unmet disease burdens worldwide. Mainly HSV-2 causes persistent sexually transmitted disease, fatal neonatal disease and increased transmission of HIV/AIDS. Thus, there is an urgent requirement to develop effective vaccines. We developed nucleic acid vaccines encoding a novel virus entry complex stabilising cell membrane fusion, ‘virus-like membranes’, VLM. Two dose intramuscular immunisations using DNA expression plasmids in a guinea pig model gave 100% protection against acute disease and significantly reduced virus replication after virus intravaginal challenge. There was also reduced establishment of latency within the dorsal root ganglia and spinal cord, but recurrent disease and recurrent virus shedding remained. To increase cellular immunity and protect against recurrent disease, cDNA encoding an inhibitor of chemokine receptors on T regulatory cells was added and compared to chemokine CCL5 effects. Immunisation including this novel human chemokine gene, newly defined splice variant from an endogenous virus genome, ‘virokine immune therapeutic’, VIT, protected most guinea pigs from recurrent disease and reduced recurrent virus shedding distinct from a gD protein vaccine similar to that previously evaluated in clinical trials. All DNA vaccines induced significant neutralising antibodies and warrant evaluation for new therapeutic treatments. Herpes simplex virus, HSV-1 and 2, like all human herpesvirus can establish latent infection that persist leading to life-long recurrences. Disease from recurrences range from mild to severe lesions and can be associated with fatal disseminated infections in neonates or immunocompromised patients . HSV-2 iEffective drugs have been developed to treat or prevent recurrent episodes of disease, but they do not prevent future recurrences when drug is discontinued, and infected persons can still transmit. Furthermore, HIV co-morbidities persist . MoreoveMuch of the focus for HSV vaccines has been on using envelope glycoproteins which are targets of neutralising antibody. These include mainly the glycoproteins gD and gB, and often in truncated, membrane-free secreted forms which can be purified as recombinantly produced protein. Clinical trial of vaccination using truncated gD and gB proteins with MF59 adjuvant did not protect against HSV-2 . HoweverIt is well established that the virus entry complex in the membrane direct binding and cellular fusion. The minimal requirements for in vitro cellular assays for cellular fusion, include a binding component, gD in HSV, and the conserved fusion complex of the fusogen gB, together with the fusion regulator, the gH/gL complex, interacting sequentially to mediate cell fusion ,14. TherImmunisation using nucleic acid provides significant advantages in induction of cellular immunity . The genExpressing the translated glycoproteins within the lipid membrane can increase innate immune responses as membrane fusion is detected by innate sensors through multiple mechanisms. For example, individual fusion mediating glycoproteins interacting with integrin can be sensed by TLR2 ,17. WhilTo further improve DNA vaccines immunogenicity, a direct immune modulator was additionally evaluated. DNA immunisation can be effective for inducing cellular immunity as the pathogen gene is expressed endogenously and presented via Class I MHC to CD4+ or CD8+ T cells. In order to enhance this process chemoattraction for specific cellular subsets were investigated. Earlier studies had combined chemokine genes, such as CCL5, CCL19 or CCL28 with gD DNA vaccines which improved responses in mice, but had not been tested in the guinea pig model for HSV-2, which can evaluate effects on recurrences ,21. CCL5These new approaches to DNA immunisation were tested here in comparison to the original clinically trialed adjuvanted gD protein vaccine in the guinea pig model of HSV-2 infection, which can assess both acute and recurrent disease as well as recurrent virus shedding.Spodoptera frugiperda) cells (GIBCO BRL), , p = 0.42, no significant difference), despite immunisation with the powerful immunogen gD, that on its own induce antibodies and protects from initial infection in this model [In contrast to CCL5, the VIT molecule could target CCR5 as well as CCR4, CCR6 and CCR8, which are on multiple T regulatory, T-Reg, subsets ,63. Theris model . The actThe ability to use defined genes to deconstruct the virus, and to transiently stimulate the immune response by VIT action via chemokine inhibitor pathway as shown here using a DNA modality, offers a highly effective and potentially safer approach. Applications to RNA could also be employed, but the attractions of thermostability that DNA offers combined with defined and efficient single fermentation manufacturing for scaling offers advantages for utility in many settings.Immunisation of the VLM + VIT DNA formulation protected against acute infection, latency establishment, and recurrent disease, with reduction in virus shedding, while in comparison the adjuvanted gD protein vaccine reduced recurrence but had no effect on virus shedding. In fact, adjuvanted gD protein immunisation appeared to increase recurrent HSV-2 virus secretion, trends seen in other animal models, but not for HSV-1 ,39,65. EVirothera Ltd. has filed for patent protection for the aspects of the VLM, VIT and CIR vaccines described and their applications.


We aim to provide real-world evidence on the use of ocrelizumab for treating multiple sclerosis (MS), with specific regard to prescription pattern, adherence, persistence, healthcare resource utilization and related costs, also in relation to other disease-modifying treatments (DMTs).We included 2495 people with MS from the Campania Region  who received first or switch DMT prescription from Jan 2018 to Dec 2020, and with at least 6-month follow-up. We collected hospital discharge records, drug prescriptions, and related costs, and calculated persistence (time from first prescription to discontinuation or switch to other DMT), adherence (proportion of days covered (PDC)), annualized hospitalization rate (AHR) for MS-related hospital admissions, and DMT costs.n = 399; age = 45.74 ± 10.98 years; females = 224), after dimethyl fumarate (n = 588) and fingolimod (n = 401); 26% patients treated with ocrelizumab were naïve. When compared with ocrelizumab, the risk of discontinuation was higher for other highly active DMTs , and low/medium efficacy DMTs . When compared with ocrelizumab, PDC was similar to other highly active DMTs , but higher for low/medium efficacy DMTs . When compared with ocrelizumab, AHR was similar to other highly active DMTs , and low/medium efficacy DMTs . When compared with ocrelizumab, DMT monthly costs were higher for other highly active DMTs , but lower for low/medium efficacy DMTs .Ocrelizumab was the most commonly prescribed DMT . Ocrelizumab was associated with the highest persistence, confirming its favorable benefit-risk profile. Costs for ocrelizumab were lower than those associated to similarly effective DMTs, in absence of changes in healthcare resource utilization. Ocrelizumab is approved for the use in both relapsing–remitting and primary progressive multiple sclerosis (MS) , 2. OcreHereby, we aim to provide real-world evidence on the use of ocrelizumab, with specific regard to prescription pattern, persistence, adherence, healthcare resource utilization and related costs, and also to compare ocrelizumab to other DMTs, based on administration  and activity .This is a population-based study, based on the retrospective analysis of routinely collected healthcare data, prospectively recorded from 2018 to 2020, on individuals with a diagnosis of MS living in the Campania Region of Italy. The original dataset has been fully described elsewhere . For theThe study was approved by the Federico II Ethics Committee (355/19). All patients signed informed consent authorizing the use of anonymized data collected routinely as part of the clinical practice, in line with data protection regulation (GDPR EU2016/679). The study was performed in accordance with good clinical practice and Declaration of Helsinki.The dataset was created by merging different data sources of the Campania Region . We specInclusion criteria were: (1) new DMT prescriptions from Jan 1, 2018, to Dec 31, 2020 ; (2) DMT prescription maintained for at least 6 months .Exclusion criteria were: (1) individual treatment periods already including a DMT at baseline ; (2) incomplete records; (3) lack of written consent to participate in the study; (4) residence outside of the Campania Region.DMT administration route: infusion , oral , and injection , using ocrelizumab as reference for comparison ;DMT treatment line: low/medium efficacy  and highly active treatments , using ocrelizumab as reference for comparison , 14.DMT prescriptions were collected, and following DMT groups were defined based on:Based on DMT prescriptions in the previous 12 months, ITPs were classified into treatment naïve (no treatment records in the previous 12 months) and switcher patients (presence of previous treatment records).DMT discontinuation was defined as a switch to another DMT or complete discontinuation  , 13.Adherence was calculated as the proportion of days covered (PDC) over 1-year time ; PDC ≥ 0.8 was considered adherent . ConsideHealthcare resource utilization included MS-related and non-MS-related hospital admissions, that were classified based on the main discharge diagnosis. The number of hospital admissions was then reported on annual basis ) , 15.Direct healthcare costs were derived from regional datasets, referred to corresponding healthcare resource utilization, and inflated to the most recent values (2020), to avoid variations in price per unit of service through different years , 15.We further collected age, sex, and, for patients with Hospital Discharge Records, Charlson Comorbidity Index , 16.Study variables are presented as mean (± standard deviation), number (percent) or median (range), as appropriate.Differences between DMT groups  were explored using Cox regression models , and linear regression models , as appropriate. Covariates were age, sex, year of treatment start , treatment duration, and adherence; statistical models were then run for the subgroup of patients with hospital discharge records, also including Charlson comorbidity index among covariates.p values, as appropriate. Statistical analyses were performed using Stata 15.0. Results were considered statistically significant for p < 0.05.Results were reported as adjusted coefficient (Coeff), adjusted hazard ratio (HR), 95% confidence intervals (95%CI), and n = 7080), we included 2495 individuals who were commenced on a DMT from 2018 to 2020, corresponding to 2918 ITPs . Reasons for exclusion are reported in Fig. From the population of people with MS in the Campania Region from 2015 to 2020 , fingolimod  and ocrelizumab , with ocrelizumab being the most frequently prescribed DMT in 2019. Also, we observed an overall drop in new DMT prescriptions in 2020 , 1325 with oral DMTs , and 901 with injectable DMTs . Looking at efficacy line, we included 724 ITPs with other highly active DMTs , and 1795 with low/medium efficacy DMTs . Most frequently prescribed DMTs were dimethyl fumarate (n = 104), followed by patients previously treated with fingolimod (n = 76), dimethyl fumarate (n = 54), teriflunomide (n = 51), glatiramer-acetate (n = 37), natalizumab (n = 34), interferon beta1a (n = 16), interferon beta1b (n = 13), alemtuzumab (n = 12), and peg-interferon beta1a (n = 2).Most patients treated with ocrelizumab were newly diagnosed and drug naïve (p = 0.02), oral  and injectable DMTs  , and low/medium efficacy DMTs  , but similar to other infusion , and injectable DMTs . When compared with ocrelizumab, adherence was lower for other highly active DMTs , and low/medium efficacy DMTs . Results were confirmed also after adjusting by Charlson Comorbidity index.Adherence to treatment is reported in Table p = 0.03), and similar to oral  and injectable DMTs . When compared with ocrelizumab, AHR was similar to other highly active , and low/medium efficacy DMTs . Results were confirmed also after adjusting by Charlson Comorbidity index.Healthcare resource utilization and costs are reported in Table p = 0.46), but lower for oral  and injectable DMTs . When compared with ocrelizumab, monthly costs for MS hospital admissions were similar to other highly active , but lower for low/medium efficacy DMTs . Results were confirmed also after adjusting by Charlson Comorbidity index.When compared with ocrelizumab, monthly costs for MS hospital admissions were similar to other infusion DMTs , but lower for oral  and injectable DMTs . When compared with ocrelizumab, monthly costs were higher for other highly active DMTs , but lower for low/medium efficacy DMTs . Results were confirmed also after adjusting by Charlson Comorbidity index.When compared with ocrelizumab, monthly costs were similar to other infusion DMTs , and related healthcare resource utilization and costs.When compared with other high-efficacy DMTs, ocrelizumab was used on much more complex populations , as already described by some previous studies –19. NotwWe also found high rates of adherence to ocrelizumab compared with lower and similar efficacy class. While we have to acknowledge that adherence analyses were run on the subset of patients with at least 12 months of follow-up, our rate of adherence is in line with previous similar studies , and oveThe main novelty of our study is the inclusion of healthcare resource utilization and costs. In particular, ocrelizumab was associated with lower direct treatment costs, but was associated with similar probability of MS-related hospital admissions and costs, when compared with other DMTs similar in administration route  and efficacy class . Similarly, in a previous US claims’ study including 189 patients treated with ocrelizumab, alemtuzumab or natalizumab for 1 year, authors showed reduced costs for ocrelizumab treatment and related procedures . OverallLimitations of our study include the generalizability of our results, since we only included patients from a specific Italian region. However, our cohort had similar distribution , when compared to other international studies , 18, 27,In conclusion, we confirmed previous results on high persistence and adherence rates of ocrelizumab, when compared with DMTs of similar efficacy and mode of administration. We also showed that ocrelizumab is less expensive than other high-efficacy DMTs, while possibly equally effective based on indirect measures on routinely collected healthcare data .


The use of facial protection has been adopted globally due to the COVID-19 pandemic. We dissected four respirators and one surgical mask into layers to determine whether or not the virus adheres to them. These individual layers were contaminated with the SARS-CoV-2 Delta variant, and its release by vortexing was performed. Samples were used to infect Vero cells, and a plaque assay was used to evaluate the adherence of the virus to the layers. Results showed that a cumulative log reduction of the layers reduced the load of the virus by at least sixfold. Our study confirms the effectiveness of facial protection in reducing the transmission and/or infection of SARS-CoV-2. The use of facial protection, including masks and respirators, has been adopted globally due to the COVID-19 pandemic. These products have been demonstrated to be effective in reducing the transmission of the virus. This study tested whether the virus can adhere to the different layers of respirators and a surgical mask. These individual layers were contaminated with the COVID-19 Delta variant, and we determined the degree of release after applying a mechanical force. Samples were used to infect cells, and the adherence of the virus to the layers was assessed. Results showed that a significant reduction in the number of viruses was observed. Our study confirms the effectiveness of facial protection in reducing the transmission and or infection of SARS-CoV-2. The pandemic of the novel coronavirus SARS-CoV-2, the causative agent of COVID-19, was initially identified in Wuhan, China, and has spread worldwide. According to the WHO, as of 15 January 2022, there have been 5,518,343 deaths and 318,648,834 confirmed cases of COVID-19 [COVID-19 is spreading through airborne transmission via aerosol. This occurs mainly when breathing, speaking, sneezing, coughing or talking . These tThe terms respirators and medical masks are used as synonyms, but there are differences according to their use . In the According to the US National Institute for Occupational Health and Safety (NIOSH), there are nine categories of air respirators. These categories include N , P  and R . The values 95, 99 and 100 indicate the minimum filtering efficiency (in percentages) [There are different types of masks or respirators with various levels of protection, which can be reusable or disposable. Generally, masks do not fit as tightly as respirators. For example, N95 respirators are classified by NIOSH as air filtration, filtering at least 95% of airborne particles. N95 respirators and surgical masks are the most common disposable masks, while reusable masks are mainly used in industrial setups with half or full facepiece respirators .According to the CDC and the NIOSH, surgical masks are approved by the US FDA and intended to use as protection for the wearer against multiple hazardous environments, such as those created by sprays, dangerous fluids, splashes and droplets. However, masks would not always confer respiratory protection because of their lower reliable level of filtration against smaller particles that could be inhaled, such as airborne particles.Surgical masks contain mainly three-layer structures with different functions. For example, the outer layer repels water, the middle layer functions as a trap of particles and prevents the penetration of microorganisms, and the inner layer absorbs the moisture ,10.On the other hand, N95 respirators are defined by NIOSH as a filtration system that retains at least 95% of different airborne sizes particles (from aerosols to large droplets), reducing the exposure to the user . RespiraThe penetration or filtration mechanism of aerosols containing SARS-CoV-2 in masks has been studied and reported ,10,12,132. All the work performed in this study was conducted in level 3 at the Facility for Infectious Disease and Epidemic Research .SARS-CoV-2 Delta variant (B.1.617.2) was provided by the British Columbian Centre for Disease Control Public Health Laboratory, BC, Canada. Vero E6 cells (ATCC CCL-81) were used to replicate the virus and perform the infections. Cells were grown in minimum essential media (MEM)  supplemented with 5% fetal calf serum (Invitrogen), pyruvate and nonessential amino acids. All the incubations were performed in an incubator at 37°C supplemented with 5% CO5 PFU/ml concentration. After 72 h, the supernatant was collected and cleared by centrifugation at 3200 × g for 10 min. The supernatant was aliquoted and kept at -80°C until further use. The titer of the virus was determined to be 5.9 × 105 PFU/ml by plaque assay.Viral stocks were prepared by infecting 80% confluent Vero E6 cells with 1 ml of the virus at a 5x105 Vero cells into 12-well plates. The next day, after confirmation of 80–100% confluency, the cells were infected with the virus for 1 h. Then, the virus was washed away, and the monolayer was overlayed with 1.5 ml of 2 × MEM supplemented with 2% cellulose  (v/v 1:1).A plaque assay was performed overnight by dispensing 1.5 × 102 were cut, separated into individual layers, decontaminated by submerging the pieces into 70% ethanol for 10 min, and dry overnight at 37°C inside a decontaminated biosafety containment level 2. The decontaminated layers were stored in empty sterile 1.5-ml tubes until further use.Five different masks or respirators  were use3 viruses (in 10 μl MEM). After 10 min (time to dry the drop), the layers were submerged in 300 μl of OptiMEM (Invitrogen) in a 1.5-ml screw-top tube and vortexed vigorously for 1 min. The supernatants of the samples were subjected to tenfold dilutions using OptiMEM in 1.5-ml tubes.The layers were exposed to 2 × 10Vero E6 cells were grown as detailed above. Cells were then checked for confluency, and the medium was removed from the wells. The serial dilutions (300 μl) were added to the corresponded wells, and the plates were incubated as explained above. The plates were placed back in the incubator for 1 h to allow infection of the cells. The plates were gently rocked every 15 min. Then, the virus and the medium were removed from the wells, and an overlay of 2% cellulose and 2x complete MEM (as detailed above) was gently added to each well. Plates were incubated for 72 h, fixed for 30 min with 4% p-formaldehyde  in phosphate-buffered saline. After removing the PFA, the cells were stained with 250 μl of 1% crystal violet (Sigma), dissolved in 20% methanol  for 30 min, washed three-times with water, dried and the number of plaques was counted and recorded.Statistical analysis was performed using the GraphPad Prism version 9.3.1 (350) . A p-value < 0.05 was considered significant.The adherence of SARS-CoV-2 to different types of buccal protection showed that all of the layers could retain the virus to different extents. In addition, adherent viruses were not released upon vortexing the samples, suggesting that they are tightly bound to the layers once they are dried.Analysis of the results showed that an accumulation of the log reductions of the layers ranged between 6.15–8.45 . In addiPeople use medical masks as protection from sprays and splashes of fluids by sick people. Although extended usage or reuse of masks has become common, especially during the present pandemics, this could lead to an infection of the person wearing the mask due to the possible presence of pathogens in different layers of the masks .Masks materials  are mainOf the different layers in a mask or respirator, the most important one is the filter layer, which is mainly produced through a melt-blown process, where a polymer (such as polyethylene) is exposed to blows of high-velocity air, forming a web shape of filaments. This filter layer (commonly used in N95 respirators) has an electrostatic charge; therefore, the polymer filtration capability for small particles increases because of electrostatic adsorption ,18. In oA model of viral retention based on the electrostatic charges on the surface of the virus and the modified polypropylene, termed electret, might explain one of the reasons for the log reduction of the virus.SARS-CoV-2 is decorated by the spike protein, which is crucial for the infection of the host. Assuming that other components of the virus are not contributing to the surface charge of the virus (lipids and nucleocapsids are part of the core of the virus ), we canThe isoelectric point of the spike protein is 6.2 . AlthougOn the other hand, studies reported negative surface charges on the electrets, varying between -200 and -1350 (V) ,26. ThusIn conclusion, we report that the tested respirators and the medical mask could reduce at least six logs of the viral load of the SARS-CoV-2 Delta variant. This reduction was probably related to an electrostatic adherence of the virus on the tested materials. Therefore, we conclude that these results support the fact that wearing masks or respirators reduces the load of the virus either dispersed in the environment from an infected person or reducing the viral load in healthy people.This study presents the findings that facial protection reduces the number of viruses by adherence. Future studies should focus on the facial safety of other materials used to fabric masks and respirators.The use of facial protection has been established to reduce the transmissibility of SARS-CoV-2.The viruses effectively adhered to the different layers of the mask or respirator.Facial protection may decrease the transmissibility and infection caused by SARS-CoV-2.


Flavivirus genus (family Flaviviridae). The virus is maintained in nature in a rural cycle between mosquito vectors, mainly Culex species, and avian hosts. Spillover from this cycle occasionally results in outbreaks in horses and humans where, in severe cases, the infection can induce neurological signs such as meningitis and encephalitis, and in some cases can lead to death. The virus has spread in the world in this century, and an increase in the number of outbreaks as well as their severity has been observed in Europe in recent years. Considering all of these aspects, a multidisciplinary approach including public, animal, and environmental health, is the best option to increase the knowledge on this problem, making this disease an excellent example of a “One Health” issue.West Nile virus (WNV) is a mosquito-borne pathogen that belongs to the Pathogens, readers can find contributions covering topics on different aspects of the virus, including its pathogenesis, vaccines, diagnosis and epidemiology, in both humans and animals. We have eight contributions in the form of original research and a review.In this Special Issue of Six of the research papers published in this Special Issue are focused on epidemiological studies on different areas of the world including different continents where the epidemiological history of WNV has been completely different. In Africa, the virus has been present since its first description in 1937 in the West Nile district in Uganda and can now be considered as endemic in the continent. In Europe, WNV emerged in the 1960s, but only sporadic cases occurred in the continent up to the 1990s. Since then, the frequency and relevance of outbreaks have increased, reaching the highest number of cases in 2018 (2083 reported cases in the European Union). The presence of the virus in the Western Hemisphere is much more recent. The virus was detected for the first time in New York in 1999 and quickly spread across North America causing an epidemic with a high number of human cases and bird deaths. Between 2001 and 2004 it was present in the Caribbean, and in South America viral genome was detected in Argentina in 2006, Colombia in 2012 and Brazil in 2018.Epidemiology in South Africa is the focus of the article from Bertram et al.  which deCulex pipiens population. Additionally, this study also describes the presence of the virus in areas without previous WNV circulation with infections in humans, horses and wild birds. Additionally, the isolation of WNV lineage 2 from a raptor in 2018 demonstrated the emergence of lineage 2. A second original paper that focus in Europe describes a serosurveillance [Three other contributions reflect the epidemiological situation in recent years in different European regions. Beck et al.  report oeillance  in humaneillance . ClinicaAn example of the epidemiological situation in a South American country is presented by Costa et al. , showingAlectoris rufa) to two Mediterranean WNV strains from Morocco and Spain via experimental infection, showing higher mortality and morbidity in the birds infected with the Moroccan isolate. In this study, differences in pathogenesis have been analysed to explain the different courses of infection and mortality. Although the virus was present in brain and showed a similar viral load, a more acute inflammatory reaction and necrosis were observed for the Moroccan strain. These data suggest that differences in neurovirulence between strains can be more significant than neuroinvasiveness in birds mortality, and that a higher virulence can be caused by a more acute and severe encephalitis.In relation to WNV pathogenesis Gamino et al.  delved iIn the area of diagnosis, Pérez-Ramírez et al.  present In the last manuscript in this collection, Saiz  discusseI hope that this Special Issue will contribute to further knowledge on WNV infection. As the Collection Editor, I would like to thank all of the authors, reviewers, and editorial personnel who have made this Issue a reality.


This novel human pathogenic tickborne thogotovirus is found in the eastern and central regions of the country. Thogotovirus genus; these negative-strand RNA viruses appear to be transmitted by ticks, and much of their biology remains unknown. In this review, we describe the emergence, virology, geographic range and ecology, and human disease caused by BRBV and discuss potential treatments for active BRBV infections. This virus and other emerging viral pathogens remain key public health concerns and require continued surveillance and study to mitigate human exposure and disease.The emergence of SARS-CoV-2 and the worldwide COVID-19 pandemic triggered considerable attention to the emergence and evolution of novel human pathogens. Bourbon virus (BRBV) was first discovered in 2014 in Bourbon County, Kansas, USA. Since its initial discovery, several cases of BRBV infection in humans have been identified in Kansas, Oklahoma, and Missouri. BRBV is classified within the  The first case of Bourbon virus (BRBV) was identified in June 2014 in Bourbon County, Kansas, USA, after severe febrile illness developed in a previously healthy middle-aged (>50 years of age) man  viral plaques in plaque reduction neutralization tests performed by using serum from the deceased patient and including a control HRTV strain. Subsequent electron microscopy revealed pleiomorphic viral particles consistent with the family Orthomyxoviridae . PThogotovirus contains several other emerging viruses: Araguari, Aransas Bay, Dhori (including the subtype Batken), Jos, Oz, Thogoto, and Upolu viruses  revealed that BRBV has genetic identity with Dhori and Oz viruses ranging from ≈59% in the most divergent GP gene to ≈82% in the most conserved PB1 gene (BRBV forms a pleomorphic (filamentous or round), ≈100–130-nm enveloped virion that is consistent with virions of other orthomyxoviruses  . EAll thogotoviruses use a single attachment GP to mediate virus attachment to and fusion with the host cell   , 4; theyA. americanum ticks); numerous mammals were seropositive for BRBV, including domestic dogs, eastern cottontail rabbits, horses, raccoons, and white-tailed deer  through a surveillance study performed in Virginia, USA (29). This recent finding provides evidence of possible transmission of Bourbon virus in 2 separate species of ticks, A. americanum and H. longicornis. In addition, that study expands the known region of Bourbon virus to include Virginia.As of 2022, Bourbon virus has now been detected in the Asian longhorned tick (


Existing studies have illustrated how the onset of physical disability or dementia negatively impacts economic wellbeing and increases out of pocket costs. However, little is known about this relationship in older individuals. Consequently, this study aimed to identify how the onset of physical disability or dementia in older adults affects economic wellbeing and out of pocket costs, and to explore the impact of gender in the context of Australia.The data was collected from a large, randomized clinical study, ASPirin in Reducing Events in the Elderly (ASPREE). Two generalized linear models (with and without interaction effects) of total out of pocket costs for those who did and did not develop physical disability or dementia were generated, with adjustment for sociodemographic characteristics at baseline.We included 8,568 older Australian individuals with a mean age of 74.8 years and 53.2% being females. After adjustment for the baseline sociodemographic characteristics, the onset of physical disability did statistically significantly raise out of pocket costs (cost ratio = 1.25) and costs among females were 13.1% higher than males.This study highlights that classifying different types of health conditions to identify the drivers of out of pocket costs and to explore the gender differences in a long-term follow-up is of importance to examine the financial impact on the older population. These negative financial impacts and gender disparities of physical disability and dementia must be considered by policymakers. Developments in health care over the last century have extended life expectancy . This haThe rapid growth of disability and dementia among older adults is strongly associated with a heavy financial burden on governments, communities, families, and individuals, with the global costs of dementia estimated to reach US$2 trillion by 2030 . For exaWhile disability has a positive association with disadvantage, there are also gender inequalities compounding these disadvantages , 14–17. We therefore performed analyses using the large and high-quality ASPirin in Reducing Events in the Elderly (ASPREE) dataset, which was conducted as a part of the international clinical trial on older adults to: (i) identify the association between the onset of physical disability or dementia in older adults and individual out of pocket costs for health care and economic wellbeing; and (ii) explore the differential impact of gender.The ASPREE was a double-blind, placebo-controlled randomized clinical trial of 100 mg aspirin daily on the primary endpoint of disability free survival, defined as the first occurrence of dementia, persistent physical disability, or death. ASPREE recruited 19,114 community-dwelling, healthy individuals aged 70 years or above in Australia and the U.S. (> 65 years for the U.S. minorities) between March 2010 to December 2014. Study medication for ASPREE ceased on June 12, 2017, and the median follow-up time was 4.7 years. The detailed primary results and design of the ASPREE study were reported previously , 23. OveAustralian participants in ASPREE were also asked to consent to a sub-study ASPREE Longitudinal Study of Older Persons (ALSOP) . This inThe first author and second last author designed the data selection methods, including missing data treatment. We chose a three-year follow-up period for this study as the recruitment of participants ended in December 2014, and the MBS and PBS datasets contain out of pocket information up to Sep 2016. Explanatory variables were selected based upon commonly used sociodemographic confounders.The primary endpoint of this analysis was the first occurrence of physical disability or dementia during the three-year follow-up from the study entry. Participants without the documented outcome were censored at three years or at the last data they were known to be event free. Physical disability was assessed every six months by the participant’s self-reported ability to perform the 6 Katz Activities of Daily Living (ADLs), which includes walking, bathing, dressing, transferring from a bed or chair, using the toilet, and eating . PhysicaOut of pocket fees for all services and medications covered under the MBS and PBS were identified directly from claim records. Out of pocket fees are the amount patients paid towards the MBS and PBS subsidized medicines and health services, and the Australian Government pays the remaining costs. Out of pocket payments can be incurred for services outside of public hospitals, and for services in private hospitals, both of which are funded through the MBS; and for pharmaceuticals, which are funded through the PBS. The Federal Government will pay a proportion of the health service and pharmaceutical fees, with patients also paying an out of pocket component in some cases. Private health insurers may reimburse the patients for their out of pocket costs for services provided in a private hospital. It should be noted that out of pocket fees paid through the MBS and PBS are only part of the full range of costs paid by participants. Additional out of pocket fees can include other costs such as insurance premium costs, in-home care services, mobility aids and equipment, transportation costs, as well as home modification , 12, 29.In addition, out of pocket fees can vary for the same service depending on a patient’s concession status, but will not necessarily be zero as is the case in other countries. Pharmaceutical access under the PBS has a concessional rate for eligible patients (such as people with low income or seniors) and an annual PBS ‘Safety Net’, where patients pay less for medicines once they reach a threshold amount of out of pocket costs in a year. For services covered under the MBS, out of pocket charges does not have a concession rate for people with low income and are subject to capped benefits  for some health services and there is also an annual Medicare ‘Safety Net’ (patients pay less for services once they reach a threshold amount of out of pocket costs in a year) , 31.For this study, total out of pocket fees were summed from Year one to Year three after the year when participants developed physical disability or dementia. For those who did not develop physical disability or dementia during the three-year follow-up, total out of pocket costs were added up over three years from the study entry. All costs were not adjusted for inflation due to the short time period.Economic wellbeing was reported based on participants’ responses to their ‘present level of income’ at the three-year follow-up ALSOP social questionnaire, with the options being: ‘very adequate’, ‘adequate’, or ‘inadequate’.Descriptive analysis was initially undertaken to identify the baseline demographic and socioeconomic characteristics of those who did and did not develop physical disability or dementia during the three-year follow-up. Means, standard deviations, proportions (MBS and PBS), and components (MBS) for out of pocket costs from Year one to Year three were presented, stratified by sex for those who did and did not develop physical disability or dementia. Then two generalized linear models were performed due to the skewed nature of the cost data . One genA flow diagram of included participants was constructed Fig. . All theTable  Table Table Table Table Table Table Out of pocket costs of older adults were positively impacted by the onset of physical disability or dementia, and the effect of physical disability was significant after adjusting for socioeconomic and demographic factors at baseline. The findings from the current study are aligned with previous research that has shown having a disability, poor mental health, or poor physical function increased out of pocket costs, and suggested that the higher costs were positively associated with the prevalence of financial stress and poverty, compared to people without such health conditions –7, 35. TIn addition, a larger share of the total out of pocket costs was derived from prescription medication use for both genders, rather than health services such as clinician consultations or investigations. This suggests that despite government subsidization of prescription pharmaceuticals, there are still substantial co-payments from PBS-listed medications for the older population. The result is consistent with earlier studies that illustrated that older adults with chronic health conditions had considerable out of pocket spending on prescription drugs . PreviouIn line with our findings on out of pocket expenses across health conditions, there are also distinct gender differences in total out of pocket costs. Women paid substantially higher costs on average than men even after accounting for differences in baseline characteristics. The results are consistent with prior studies in that the financial impact of dementia was likely to be greater for women than men of the same age –47, and Despite the universal MBS and PBS coverage system in Australia, the onset of physical disability did significantly raise out of pocket costs after adjusting for confounders. The out of pocket cost burden fell more heavily on older women with physical disability than older men, mostly driven by prescription pharmaceutical costs. These negative financial impacts and gender disparities must be considered by policymakers. Also, the different shares and components of MBS/PBS costs have implications for policymakers in identifying the types of services on which out of pocket fees are being incurred. In order to improve these gender disparities and make health services more affordable to older adults, policymakers could lower the co-payments by removing coverage caps or providing additional support to older women with physical disability.The strength of our analyses is that they drew on data from a contemporary and high-quality dataset of healthy older individuals. Nonetheless, there are a number of limitations. Although our sample size is relatively large, the number of participants who were identified with physical disability or dementia and who reported inadequate income is quite low, which may limit the generalizability of our results. The incidence numbers are also lower compared to the Australian population for physical disability (0.9% vs.8.8%) and dementia (0.4% vs. 3%) in this age group in 2016 , 51. OneIn conclusion, the onset of physical disability was associated with statistically significantly higher out of pocket costs, and costs among females were higher than males, after adjustment for baseline sociodemographic characteristics. This study provides a better understanding of how the onset of physical disability or dementia affects economic wellbeing and out of pocket expenditure in older adults by stratifying types of health conditions and differentiating by gender. It also highlights that classifying different types of health conditions to identify the drivers of out of pocket medical spending and to explore the gender differences in a long-term follow-up is of importance to examine the financial impact on the older population. Future research is recommended to include a larger sample size with the full amount of patient costs (if possible), more detailed data on current income (rather than wide income group), longer follow-up period to further explore the gender differences and financial impacts of poor health conditions on older adults.


West Nile virus (WNV), a single-stranded RNA virus belonging to the family of Flaviviridae, is an arbovirus transmitted to humans predominantly by mosquito bites. It exhibits a wide range of clinical findings ranging from asymptomatic presentation to severe several neurological disorders. WNV has afflicted several countries around the globe including Pakistan. News of yet another outbreak in the country by WNV is circulating again. Concerned authorities should act vigilantly before the endemic takes over completely and bring down the already bereaving healthcare of Pakistan. Dear Editor,Discovered and named after the West Nile district of Uganda, West Nile virus (WNV) is a positive-strand RNA virus replicating via RNA-dependent RNA polymerase cycle . It beloThe clinical manifestation following a WNV infection spans a wide range, with about 80% population remaining asymptomatic. Mild symptomatology includes systemic febrile illness, also known as West Nile Fever (WNF). The jeopardizing symptoms are seen in less than 1% population and develop when the virus invades the central nervous system (CNS), thereby leading to meningitis, encephalitis, or acute poliomyelitis-like syndrome . The sevThe virus has been endemic throughout the world including Israel, Africa, Asia, the Caribbean, Greece, Romania, Russia, and the United States of America (USA) . Among tTo ensure that the country does not undergo another healthcare hazard, all precautionary measurements must be employed. Government must allocate more funds to the healthcare department. Currently, Pakistan lacks testing services for WNV except for a few institutes . IncreasPublic awareness programs should be run throughout the country, educating masses on personal hygiene, use of mosquito repellants, and electrical devices. Database systems for proper reporting of any case must be well-developed and the cases should be followed-up. Lastly, research programs should be devised that will work on actively controlling the transmissions via investigating every possible route. As a lower-middle-income country, still recovering from coronavirus disease 2019 (COVID-19) blackout and dengue outbreaks, a potential threat of WNV can lead to a disastrous setback, thereby further depriving the healthcare economy of Pakistan. Hence it is imperative that the concerned authorities take immediate actions or WNV can be the next gigantic public health threat for the country.Not required.None.S·W and T.G.S conceived the idea, S·W, T.G.S, S·H.A, and R.M.U retrieved the data, did write up of letter, and finally, M.J.T and M.S.A reviewed and provided inputs. All authors approved the final version of the manuscript.1Name of the registry: Not required.2.Unique Identifying number or registration ID: N/A3.Hyperlink to your specific registration (must be publicly accessible and will be checked):Muhammad Sohaib Asghar.No financial support was provided relevant to his article.All authors report no conflict of interest relevant to his article.


Green stem disorder (GSD) of soybean is characterized by delayed leaf and stem maturation despite normal pod maturation. Previous studies have suggested that GSD occurrence is promoted by a high source–sink ratio, which is produced by thinning or shade removal at the R5 growth stage (the beginning of seed filling). Here the effects of different times and durations of shade removal after the R5 stage on GSD severity were analyzed. First, shade removal for more than 28 days after R5 increased GSD severity by more than 0.4 point in GSD score. Thinning treatment at R5 increased specific leaf weight by 23%, suppressed stem dry weight reduction, and upregulated 19 genes including those encoding vegetative storage proteins at R5 + 28d, indicating excess source ability relative to sink size. On the contrary, shade removal for 14 days after R5 decreased GSD severity by 0.5 point in GSD score. In this treatment, seed size was smaller, while seed number was significantly larger than control, suggesting that shortage of source ability relative to sink size. These results implied that soybean plants regulate GSD occurrences either positively or negatively according to a source-sink ratio during the R5 to R5 + 28d growth stages. Glycine max (L.) Merr.) is a disorder wherein stems and leaves remain green and retain moisture, regardless of mature pods and seeds, at the time of harvesting2  of soybean (12. Depodding resulted in an upregulated gene expression and accumulation of vegetative storage proteins (VSPs) in the stem and leaves15. Furthermore, other biotic or abiotic factors that possibly promote GSD, such as high soil moisture content and diseases during the reproductive period17 and pest attack18, result in sink limitation at the pod setting and filling stages. According to these studies, a high source–sink ratio induced by sink limitation could result in GSD occurrence14. The same logic was applied in the GSD occurrence promoted by source surplus without sink limitation. Islam et al.19 reported that high-N treatments suppressed leaf senescence from the stages R6 (the beginning of full seed stage) to R7 (the beginning of pod maturity) in soybean, resulting in GSD. The number of seeds and pods per plant, representing sink size, was not affected by the N treatments, but seed size was increased. These results are consistent with the hypothesis that the high ratio of source affects GSD.Previous studies have shown that removing pods (depodding) at the pod setting and filling stages promotes GSD occurrence21. The plants in the thinning treatment at the R5 stage showed more severe GSD symptoms than those in the thinning treatment at the R1 stage (the beginning of flowering)21. The sink size (seed and pod number) was not significantly affected by thinning or shade removal at the R5 stage, while the sink size significantly increased after these treatments at the R1 stage20, suggesting that the R5 stage is critical for determining the source–sink ratio and then GSD.Furthermore, our previous studies reported that increasing light availability by thinning (after dense cultivation) or shade removal (after side-shading cultivation) at the R5 stage (the beginning of seed filling) increased GSD occurrence in soybean compared to the control plants, which were grown at a dense plant population or were exposed to shading treatments23, decrease in photosynthetic activity24, and the redistribution of assimilates from the stem and leaves to seeds26. Thus, it is necessary to narrow down the critical or effective growth period after the R5 stage for GSD occurrence under improved light conditions. The objective of this study was to identify the important time and duration of improved light conditions for increased GSD severity and to reveal the effects of light availability on growth parameters and gene expression. First, we employed a side-shade equipment that reversibly altered the assimilation ability of the source by attaching or detaching a shade-sheet to identify the critical growth period for GSD occurrence. Second, plant growth traits and transcriptomes of the stems at the critical growth stage were analyzed under the field conditions, wherein the plant growth habits were different from those in a growth chamber27.The period after the R5 stage includes developmentally important events, such as the determination of sink size (seed and pod number)Growth traits  in the no shade showed significantly higher scores Table  than theShading in entire growth periods (full shade) significantly increased GSD severity, with higher GSD score and number of leaves per node at the R8 stage (full pod maturation), and higher percentage of GSD ratio (= 5), and lower percentage of GSD ratio (≤ 3), compared to the treatment 6 (no shade) Table . As an iThe treatment 1  showed significantly lower GSD score, fewer number of leaves per node at the R8 stage, and significantly higher GSD ratio (≤ 3) 47.2%) than the control (full shade)  and 3  showed significantly higher GSD score and significantly higher number of leaves per node at the R8 stage than the control  were mainly associated with photosynthesis (44.8% in the R5 + 14 d and 56.1% in the R5 + 28 d datasets)  subfamily29. ANAC047 is a senescence-associated gene that promotes senescence and programmed cell death in leaves30. The genes encoding endo-1,4-β-mannanase and xyloglucan endotransglucosylase/hydrolase were also downregulated under thinning treatment (Table 31.A NAC transcription factor Glyma.16G043200) gene was downregulated in the R5 + 14 d and R5 + 28 d datasets  increased GSD severity as well Table . This ph5% Table . There w5% Table . It suggAn intermediate GSD score of full shade enabled to analyze whether the light availability treatment in experiment 2 promotes or suppresses GSD occurrence. Thus, these characteristics coupled with the reversibility of the shading treatment helped in the identification of the critical period for GSD occurrence between 14 and 28 d after the R5 stage.13 and stem16, suggesting an alteration in the leaf function from an assimilation organ to a storage organ for excess assimilates. In experiment 3, suppressed leaf abscission, increased leaf specific weight, and reduced loss of stem dry weight (redistribution of assimilates from the stem to seeds) from R5 to R5 + 28 d in the thinning treatment indicated that improved light conditions from R5 to R5 + 28 d resulted in excess assimilation products relative to the sink demand , VspB (Glyma.08G200100) and two lipoxygenases (Glyma.15G026400 and Glyma.15G026500). These results are consistent with those of Brown and Hudson27.Brown and Hudson27. In soybean, NAC transcription factors GmNAC81 and GmNAC30 promote leaf senescence38. As D gene in sorghum induces programmed cell death and determines stem water content39, Glyma.16G043200 may also promote senescence and programmed cell death in soybean stem and may be related to GSD occurrence.The gene encoding NAC transcription factor Glyma.16G043200), which was significantly decreased upon thinning in the present study  of soybean was promoted by a high source–sink ratio, that was increased by either sink limitation or source ability enhancement. Source ability was upregulated by light availability improvement such as thinning and shade removal. The increase in sink and source was, however, possible only by around R5 + 14 d and R5 + 28 d growth stages, respectively. Source and sink abilities could be represented by the sum of seed weight, dry weights of stem and pod and by seed number at maturity, respectively.42 in the experimental fields of National Agriculture and Food Research Organization (NARO), Western Region Agricultural Research Center, Hiroshima, Japan . The groundwater level in all the experimental fields was maintained at 30 cm below the surface using the farm-oriented enhancing aquatic system FOEAS43, and 3 g m−2 N, 10 g m−2 P2O5, and 10 g m−2 K2O were supplemented to the soil one day before sowing. To check biotic stress, insecticides and fungicides were regularly sprayed in the fields.Experiments 1, 2 and 3 were conducted using the soybean cultivar ‘Sachiyutaka’ (Maturity group 6)20. The shade-sheets used in the equipment were black-colored polypropylene sheets, with a shade strength of 82% 20 and were raised when one of the uppermost leaves in the plots grew 3 cm taller than the uppermost side of the shade equipment. Thus, the height of the equipment paralleled plant heights throughout the experiment. The shading treatment was applied from sowing to R8  or never applied . There were two replicates in 2017 and four replicates in 2018. The main stem of each plant was supported using a gardening pole to prevent lodging , without any shade-sheet on the upper part (width: 0.1 m) of the equipment, which mimicked a canopy where some of the uppermost leaves are not shaded by the neighboring plants under field conditions  as well as the control treatment were conducted with shading from sowing. The control treatment was common with Experiment 1. In the treatments 1, 2, and 3, shade-sheets were removed at the R5 stage. Then, shading treatment was resumed 14 d and 28 d after the initiation of the R5 stage in the treatments 1 and 2, respectively. The shade-sheets were not resumed after R5 in the treatment 3. In the treatments 4 and 5, shade sheets were removed after 28 d of the R5 stage (R5 + 28 d) and 42 d of the R5 stage (R5 + 42 d), respectively.−2, with a plant spacing of 0.15 m in 0.3 m rows, throughout the cultivation period. In contrast, the plant populations in the thinning plots were changed from dense to sparse (5.56 plants m−2 in 0.6 m rows and 0.3 m plant spacing) at the R5 stage. In the thinning plots, the aboveground parts of the plants in every alternate row and those of every alternate plant in the remaining rows were excised. To prevent lodging, plants were supported by poles standing on both ends of the rows and greenhouse polyethylene bands, which were stretched out between the poles at 40 cm above the ground  and 3.0 m × 2.1 m plots on June 25, 2018 (four replicates). There were two types of plots with different plant population size: dense  or nine (2018) plants from each dense and thinning treatment plot, except the plants at the plot borders, were randomly selected after the R8 and 28 d of the R5 stage (R5 + 28 d) for the analysis of growth parameters. Moreover, six (2016) or nine (2018) thinned plants, except the plants at the plot borders, were randomly sampled at the R5 stage to analyze the growth parameters of the plants at the start of the thinning treatment.Growth parameters, including leaf area, leaf number, dry weight of leaflets, and stem dry weight, were measured for each sampled plant of experiment 3. The leaf area was measured using an area meter LI-3100C , and the dry weights of each plant part were measured after drying them at 80 °C for 3 d before measurements.44. The severity of GSD was determined using the three indexes reported in the previous study, namely GSD score, leaf number per node at the R8 stage, and GSD ratio20. GSD score was calculated using a method modified from that of Furuya and Umezaki45, and each plant was assigned a GSD score (1–5) based on the number of remaining leaves per node and the color of the stem at the R8 stage, where a high GSD score represented more severe GSD symptoms. In addition, the ratio of the number of plants with no leaves (GSD score: < 3) and the number of plants with one-third of the leaves remaining at the R8 stage (GSD score: 5) to the number of sampled plants were calculated as GSD ratio (≤ 3) and GSD ratio (= 5), respectively for each plot. After the R8 stage, seed weight of 100 seeds and seed number were measured for each sampled plant. Dry weights of the stems and pods were measured after drying them at 80 °C for 3 d.The dates of the R5 and R8 stages for each plant were determined using the method of Fehr and Caviness46.For transcriptome analysis as part of experiment 3, two replicates with three main plant stems, each from dense and thinning treatments after 14 and 28 d of treatment (R5 + 14 d and R5 + 28 d), were collected and freeze-stored in liquid nitrogen at − 80 °C in 2016. Total RNA was extracted using phenol/SDS and treated with DNase I, followed by column purification. The quality and quantity of RNA were ensured using Nanodrop and Agilent 2100 Bioanalyzer, respectively, and RNA-Seq libraries were constructed using Illumina’s Truseq Stranded mRNA LT Sample Prep Kit (as per the manufacturer's instructions) and sequenced using Illumina HiSeq 4000 paired-end sequencing technology, with a read length of 100 and sample size of 7.46 giga base. The sequences were then aligned with the soybean reference genome (Gmax_275_v2.0) using TopHat2 2.0.9http://www.bioinformatics.bbsrc.ac.uk/projects/seand) and Java (1.8.0_151) (https://www.oracle.com/java/). Gene expressions were quantified using the RNA-Seq pipeline quantitation based on merged transcripts, counting reads over exons and correcting for feature length, using log-transformed data. A total of 54,878 transcriptionally active regions (probes) were generated for analysis. However, for the R5 + 14 d dataset, only the first field replicate was used for analysis because of the low RNA quality of the second replicate, whereas both the field replicates of the R5 + 28 d dataset were grouped for analysis. The probes were filtered using the intensity difference test (p < 0.05) and then further screened for a logarithmic value (to the base 10) above -2.0. The filtered and screened probes were then analyzed using gene ontology (GO) enrichment analysis of the web service of SoyBase (https://www.soybase.org/) with annotations from the Wm82.a2. v1 (Glyma 2.0).For the data analysis, filtering and analysis were performed using the software SeqMonk v1.38.2 (p < 0.05 or p < 0.01) was tested using the analysis of variance (ANOVA) or t-test. Dunnett and Tukey’s tests were used (p < 0.05 or p < 0.01) to compare the mean values of the control and other treatments and the mean values of plant growth parameters between the treatments, respectively. GSD scores and GSD ratio were analyzed after the Box-Cox and angular transformations, respectively, to meet the assumption that the data be approximately normally distributed. The analyses were performed using the statistical software BellCurve for Excel version 3.21 .A completely randomized design was used for each experiment. Statistical significance of differences (https://www.gene.affrc.go.jp/index_en.php). This research was approved by the Western Region Agricultural Research Center, NARO as the research number of 1,040,101, 2016–2018. All the experiments carried out on plants in this study were in compliance with relevant institutional, national, and international guidelines and legislation.The soybean cultivar ‘Sachiyutaka’ was provided by National Agriculture and Food Research Organization (NARO). The cultivar, which was developed by NARO and registered as the registration number of 11,367, was used in accordance with the plant variety protection and seed act of Japan. The plant resource can be accessed via NARO Genebank project (Supplementary Information.


Aspergillus fumigatus common reference strain Af293 biofilms in intermicrobial competition with Pseudomonas aeruginosa. We compared the sensitivity of two infected and one virus-free Af293 strains to antifungal drugs. All three were comparably sensitive to drugs affecting fungal membranes  or cell wall glucan synthesis . In contrast, forming biofilms of virus-free Af293 were much more resistant than AfuPmV-1-infected Af293 to nikkomycin Z (NikZ), a drug inhibiting chitin synthase. The IC50 for NikZ on biofilms was between 3.8 and 7.5 µg/mL for virus-free Af293 and 0.94–1.88 µg/mL for infected strains. The IC50 for the virus-free A. fumigatus strain 10AF was ~2 µg/mL in most experiments. NikZ also modestly affected the planktonic growth of infected Af293 more than the virus-free strain . Virus-free Af293 biofilm showed increased metabolism, and fungus growing as biofilm or planktonically showed increased growth compared to infected; these differences do not explain the resistance of the virus-free fungus to NikZ. In summary, AfuPmV-1 infection sensitized A. fumigatus to NikZ, but did not affect response to drugs commonly used against A. fumigatus infection. Virus infection had a greater effect on NikZ inhibition of biofilm than planktonic growth.Infection with Aspergillus fumigatus polymycovirus 1 (AfuPmV-1) weakens resistance of  Aspergillus wild-type strains have been naturally infected [Aspergillus, including effects on fungal physiology and virulence, have been recently reviewed and detailed [Aspergillus is the Polymycoviridae family, which was initially reported in 2015 [Polymycoviridae family currently accommodates one genus, Polymycovirus, with 10 species, including Aspergillus fumigatus polymycovirus. Polymycoviridae and related viruses usually have four to eleven [Aspergillus physiology or virulence. Where effects on fungal physiology have been delineated, the results have shown either enhancing or deleterious effects [Virus infections are widespread in the fungal kingdom, and since the 1970s, many detailed . Among t in 2015 , and rec in 2015 . The Polo eleven  dsRNA geo eleven ,5, althoo eleven ,7,8. LitA. fumigatus laboratory strain [P. aeruginosa inhibited infected Af293 to a greater extent than virus-free Af293, dominantly shown in the face of iron stress in competition with P. aeruginosa [P. aeruginosa volatiles [P. aeruginosa [Af293 is a very common and fully sequenced ruginosa . The virruginosa . The samolatiles , where truginosa . Recentlruginosa .Here, we compared the effects of the conventional antifungal drugs amphotericin B (AmB), voriconazole (VCZ), micafungin (MICA), and caspofungin (CASPO), as well as the chitin synthase inhibitor nikkomycin Z (NikZ) on isogenic-infected or virus-free fungus.Materials: RPMI 1640 medium, 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide inner salt (XTT), and menadione were purchased from Sigma-Aldrich . Nikkomycin Z (NikZ) powder was donated by Valley Fever Solutions, Tucson. Voriconazole  was prepared in DMSO. Further dilutions were prepared in RPMI. Final DMSO concentrations were below 1% , which does not affect A. fumigatus biofilm metabolism, allowing omission of separate DMSO controls. Amphotericin B (AmB) was obtained from Gilead Sciences , caspofungin (CASPO) was obtained from Merck , and micafungin (MICA) was obtained from Fujisawa Pharmaceutical Co., Ltd. . Large batches of all drugs were frozen as aliquots. For each experiment, a fresh aliquot was used.Strains, isolates, and institutional review: A. fumigatus isolates used in this study. The use of all strains mentioned in Aspergillus 18–42 by protoplast transfection [The naturally infected Af293 strains 10–53 and 18–95 were kept in separate laboratories  for over 10 years. Strains 18–95 were cured from AfuPmV-1 using the protein synthesis inhibitor cycloheximide [Assays used to quantify fungal growth of biofilm or planktonic cultures:Biofilm: We placed 100 µL/well of a 105 conidia/mL suspension in RPMI per strain and assay into a 96-well plate with five replicates. Plates were incubated overnight at 35 °C before determining optical density at 562 nm using a plate reader . Background  was subtracted. Absorbance of the virus-free 18–42 strains was regarded as 100%, and absorbance of infected strains was put in relation to that.Planktonic growth: One milliliter of a 105 conidia/mL suspension in RPMI per strain and assay was placed into 4 mL tubes. Tubes were incubated for 48 h at 35 °C before determining transmission at 530 nm, using a spectrophotometer . The background was subtracted by normalizing to RPMI (transmission of 100%). Fungal growth was determined by the formula: growth = 100 − T530. Growth of the virus-free strains 18–42 was regarded as 100%, and growth of infected strains was put in relation to that.Aspergillus forming biofilm metabolism assay: A. fumigatus conidia  were distributed into the wells of 96-well plates in a volume of 50 µL/well. Drug dilutions were added in 50 µL/well to the final concentrations indicated. Final volumes in wells during assays were 100 µL. Effect of RPMI 1640 medium on fungus served as the negative control. The assay plates were incubated at 37 °C overnight, and hyphal growth was verified by optical microscopy before performing XTT assays.All experiments were evaluated by XTT metabolic assay as detailed previously ,17. BrieDetermination of 50% antifungal activity (IC50): Figures and Figure Legends indicate drug dilutions in RPMI, in 1:2 dilution steps, that encompassed a concentration that was closest to inhibiting 50% of fungal metabolism and is referred to as the IC50 of a drug on a certain isolate.Minimal inhibitory concentration (MIC), minimal fungicidal concentration (MFC): One hundred microliters of NikZ were distributed into tubes. Then, 900 µL of standardized inoculum was added to the tubes to achieve final concentrations of NikZ 0.5–128 µg/mL. Tubes were incubated for 48 h at 35 °C before reading MICs. Planktonic growth and inhibition in an MIC assay were evaluated as previously described [escribed ,19. MIC Statistics: Results were analyzed using Student’s t-test if two groups were compared, and 1-way ANOVA combined with a Tukey’s post-test for multiple comparisons. All data in this study are expressed as a mean ± SD. Data are also reported as the percent of control. Each assay was performed with three to four biological and four to twelve technical replicates. Representative experiments are shown.NikZ significantly interfered with the metabolism of forming biofilms of 10AF at concentrations > 0.47 µg/mL, with the IC50 between 0.94 and 1.88 µg/mL in most experiments A. MicrosA. fumigatus strain 10AF and the naturally AfuPmV-1-infected A. fumigatus strain Af293 (10–53), we found 10–53 to be more sensitive to NikZ than 10AF (A. fumigatus strains (When NikZ (2 µg/mL) effects were compared between the virus-free strain han 10AF A. Micros strains B; the fr strains C.We next compared three Af293-based strains for their sensitivity to NikZ, of which two strains (18–95 and 19–40) were infected, and a third strain, 18–42, was virus-free see A. A doseA. fumigatus infections. Virus infection of A. fumigatus did not increase its sensitivity to MICA  were weaker than on infected strains . With reWe observed that virus-free Af293, growing planktonically A, as welSimilarly, biofilm assays showed that virus-free fungus had significantly more metabolism than infected fungus A. We conWe measured biofilm metabolism in 18–42 cultures that started with the same amount of conidia as used in This study showed that even when we compensated for the differences in basal metabolism between virus-free and infected, NikZ effects on infected strains were still greater. Thus, the increased basal metabolism of the virus-free strain does not explain the decreased NikZ effect on it.Aspergillus is not a rare event. There are multiple virus families with members that are known to infect Aspergillus, encompassing Chrysoviridae, Narnaviridae, Partitiviridae, Totiviridae, and Polymycoviridae [Aspergillus species has been described to be between 10 and 80% [A. fumigatus isolates are infected [Virus infection of oviridae . The inf and 80% ,21, whilinfected ,23. Infeinfected ,24.Aspergillus virus infection is controversial. There are reports showing hypovirulence in a mouse model [The clinical relevance of se model , increasse model ,26,27, ose model ,27,28. Ise model ,28.A. fumigatus strains, in the form of planktonically growing cultures, but also in the form of biofilms, were comparably sensitive to conventional drugs affecting fungal membranes , or cell wall glucan synthesis . In contrast, forming biofilms as well as planktonic cultures of virus-free Af293 were much more resistant than AfuPmV-1-infected Af293 to nikkomycin Z (NikZ), a drug inhibiting chitin synthase. The IC50 for NikZ on biofilms was between 3.8 and 7.5 µg/mL for virus-free Af293 and between 0.94 and 1.88 µg/mL for infected isogenic Af293 strains. The IC50 for the virus-free A. fumigatus strain 10AF was approximately 2 µg/mL in most experiments. NikZ also modestly affected the planktonic growth of AfuPmV-1-infected Af293 more than it affected the virus-free strain . Virus-free Af293 biofilm showed increased metabolism, and when growing in the form of biofilm, or planktonically, showed increased growth compared to infected Af293.We also found that virus-free and mycovirus-infected Aspergillus renders the fungus more susceptible to stresses affecting cell wall formation, especially towards NikZ, affecting chitin synthase. It would be of interest to compare the effects of other chitin synthase inhibitors, e.g., [Our results indicate that virus infection of s, e.g., , to NikZWe observed that virus-free fungus grew better compared to infected fungus, suggesting that viral infection might impair cell wall synthesis, particularly at specific points in the synthesis of the wall. Although we could show that resistance of virus-free biofilm to NikZ was not caused by its better growth, we indicate that virus-altered cell wall synthesis is more prone to detrimental effects caused by inhibition of chitin synthesis. This inhibition might result in osmotic stress for the fungal cell, resulting from impaired cell wall synthesis, and either lead to reduced growth or cell injury.Penicillium digitatum, with a polymycovirus resulted in increased susceptibility to an azole drug [Penicillium and Aspergillus are both filamentous fungi, the higher susceptibility of infected Penicillium to azole drugs might be explained by differences in cell wall stress response [In contrast to our data, infection of a plant pathogenic fungus, ole drug . Althougresponse .A. fumigatus alters responses to a variety of stresses, such as iron deficiency [Pseudomonas volatiles [A. fumigatus more susceptible to other stresses is currently under investigation. Higher susceptibility of infected strains to a variety of stresses points to the virus affecting molecules that have been proposed as master regulators of stress responses or cell wall stress responses [We previously showed that virus infection of ficiency ,11, Pseuolatiles , or higholatiles . Whetheresponses ,32,33. I


Background: High-efficiency particulate air (HEPA) filters are currently recommended when using recirculated air to eliminate the risk of pathogen transmission such as SARS-CoV-2 from one patient care area to the next. We tested the efficacy of lower-grade air filters in eliminating airborne virus transmission. Methods: We conducted an experiment in 2 adjacent exam rooms in an unoccupied hospital emergency unit. The HVAC system contained a 15,000-cubic-feet-per-minute rooftop air handler. All outside air and exhaust dampers were closed during the trial (full air recirculation). We conducted experiments in 3 tests arms with varying grades of MERV filters : (1) control without filters, (2) MERV8+14 filters, and (3) MERV8+16 filters. We repeated 20-minute virus challenge runs 3 times per test arm. Live attenuated influenza vaccine , was aerosolized into the HVAC system via a commercial nebulizer. Air was sampled using 3 six-stage Andersen air samplers placed in the center of the adjacent room. Environmental particle counts were collected using a particle counter . Results: Concentrations of viral RNA were determined by qPCR, and viral concentrations (vg/mL) in each stage of each arm were compared directly. Pairwise comparisons of the virus and particle burdens across each stage of each test arm were made using a general linear model. LAIV was detected in the control arm at a virus burden of 2,277 vg/mL, indicating a >6.5 log reduction of the virus released in the HVAC system . In the second arm, the MERV8+MERV14 filters demonstrated in a 13-fold decrease in viral burden compared to the control arm (mean virus burden: 169 vg/mL, p Our study demonstrates that viral containing particles can be transported via a hospital HVAC system from one patient room to the next. Considering the decrease in detectable virus within the HVAC system, the combination of MERV8+MERV16 filters reduced the virus burden reaching an adjacent room to levels well below the human infectious dosages for influenza and other highly infective viruses. Conclusions: Our findings indicate that MERV8+MERV16 filters provide protection against virus transmission through HVAC systems and are a cost-conscious alternative to HEPA filters.Funding: NoneDisclosures: None


Monkeypox is caused by a sylvatic, double-stranded DNA zoonotic virus. Since 1 January 2022, monkeypox cases have been reported to WHO from 106 Member States across six WHO regions, and as of 2 October 2022, a total of 68,900 confirmed cases, including 25 deaths, occurred. Here, by using a whole genome approach, we perform a genetic and phylodynamic survey of the monkeypox virus Clade IIb B.1, which is the lineage causing the current multi-country outbreak. Results suggest that outbreaks seem to be isolated and localized in several epidemic clusters with geographic consistency. Currently, monkeypox appears to be a virus with a flattened genetic variability in terms of evolutionary path, with a very slow rate of growth in the population size. This scenario confirms that the monkeypox virus lacks the evolutionary advantage, given by the high level of mutation rate, which is very strong in RNA viruses. Of course, constant genome-based monitoring must be performed over time in order to detect the change in its genetic composition, if any. On 13 May 2022, WHO was notified of two confirmed cases and one not-confirmed case of monkeypox (from the same household) in the United Kingdom (https://www.who.int/emergencies/disease-outbreak-news/item/2022-DON383 (accessed on 10 October 2022)). As of 2 October 2022, 68,900 laboratory-confirmed cases of monkeypox have been reported to WHO from 106 countries in all WHO Regions ) ) see . Indeed,In such a context, here, we performed a genetic and phylodynamic survey aimed at providing some key points on the genetic variability of the monkeypox virus Clade IIb lineage B.1 updated to all genomes publicly available on 10 October 2022. The research aimed to verify the occurrence of epidemic cluster in the Clade IIb B.1 (which is the most common lineage currently widespread) and the variation in terms of genetic variability from the first reported cases to current days. This kind of information is very important because it allows us to understand if the viral population size is growing or stationary during times.https://gisaid.org/ (accessed on 2 October 2022)) ) see . The updIn order to avoid bias linked to the occurrence of deletions, insertions, and recombination events, the test Φ  was applied, following Bruen et al. .−5 [4 × 10−5–6 × 10−5], proposed by Firth et al. [Population dynamics, based on genomes of monkeypox virus Clade IIb lineage B.1 (collection date range: 4 May 2022–14 September 2022), was reconstructed by implementing the evolutionary of 5 × 10h et al.  for varih et al.  followinh et al. .In order to verify the temporal signal of molecular phylogenies, a tip-to-root regression was performed by using the software TempEst 1.5.3 . The trehttps://gisaid.org/hmpxv-phylogeny/, last updated 2 October 2022.In addition, the genomic epidemiology of monkeypox virus Clade IIb lineage B.1 has been reconstructed by using the next strain available at The Φ statistic test suggested the lack of events attributable to deletions, insertions, and recombination. The Φ test, which is based on the principle of compatibility , indicatPopulation dynamic of monkeypox virus Clade IIb B.1  suggestsThis scenario is consistent with a very slow rate of growth in the population size. This point of view is also confirmed by the phylogenomic reconstruction , which i−2). The causes of this result can be multiple [This condition has also been confirmed by the test for the temporal signal, performed with a tip-to-root regression, which indicates a lack of a positive association between sequence divergence and sampling dates ).As far as human health is concerned, the biological condition of the monkeypox virus is a plus because it does not allow the virus to have a rapid spread, such as SARS-CoV-2 or many other RNA viruses . Indeed,However, the monkeypox multi-country outbreak should not be underestimated, and molecular monitoring at the genome level must be constantly undertaken to acquire a more wholesome and well-rounded viewpoint. The importance of unstopped surveillance is also required because of the occurrence of asymptomatic infections, which may be unknown, and thus the total number of infections can be underestimated . The virIn conclusion, the data herein suggest that the multi-country outbreak caused by the monkeypox virus seems to be localized in several epidemic clusters with geographic consistency. However, the current low capability in the differentiation of the monkeypox virus must not be understood as a reason to let down the guard against the outbreak. On the contrary, these findings further suggest the importance of a constant genome-based survey.