A previously healthy male patient had detectable monkeypox virus DNA in saliva 76 days after laboratory confirmation of infection. A comprehensive characterization of viral kinetics and a detailed follow-up indicated a declining risk for transmission during the weeks after monkeypox symptoms appeared. Orthopoxvirus genus of the Poxviridae family. Monkeypox outbreaks have historically been described mainly in central and west Africa (Monkeypox is a zoonotic infection caused by monkeypox virus (MPXV), belonging to the In May 2022, a multinational monkeypox outbreak surfaced; cases were reported from Europe, the Americas, Israel, and Australia. Compared with those in previous outbreaks, these reported patients show a different clinical manifestation of localized rashes and mucosal lesions predominantly in the genital area. Common systemic symptoms included fever and lymphadenopathy. The cases clustered in men who have sex with men . The next day, he observed a small skin change on his foreskin, progressing over the next days to a deeper, well-circumscribed lesion with local lymphadenopathy. Fever developed on day 5 and 6, peaking at 39°C. One week after symptom onset, the patient sought care at an outpatient clinic. By then, the fever had subsided. No new lesions appeared. He reported a history of receiving oral sex from several male partners within the 3 weeks before symptom onset. At a follow-up visit on day 11, the lesion had increased in size to 2 cm in diameter. Microbiologic analyses for herpes simplex virus, syphilis, and The genital lesion slowly healed but with increasing local lymphadenopathy; on day 25, the patient had a ruptured local lymph node with discharge. At a follow-up visit on day 53, the patient was feeling well but still had enlarged lymph nodes. The original genital lesion had diminished to 5 mm in diameter and bled slightly when touched. The wound from the ruptured lymph node had healed.We took repeated samples from the patient during the 10-week follow-up period from the genital lesion, the ruptured local lymph node, urine, semen, blood and the respiratory tract. We detected MPXV DNA in most samples  Table 1.https://github.com/DerrickWood/kraken2), followed by assembly of the nanopore reads using Flye (https://github.com/fenderglass/Flye), resulting in a single contig representing MPXV. The contig was polished using medaka (https://github.com/nanoporetech/medaka) for the long reads and then ntEdit (https://github.com/bcgsc/ntedit) for the short reads, which produced a nearly complete genome sequence. We compared this genome sequence by whole-genome alignment and tree construction using publicly available sequences  showed a single-nucleotide polymorphism distance of 4 nt (We performed electron microscopy on skin lesion material and observed viral particles characteristic for orthopoxviruses  Figure. equences . The ana of 4 nt .As of August 2022, the multinational monkeypox outbreak is still unfolding; new cases are being reported in an increasing number of countries. Many aspects of monkeypox infection in the ongoing outbreak differ from previous endemic and imported monkeypox cases, including clinical manifestations and route of transmission (This report highlights several aspects of monkeypox as an emerging infectious disease. First, the case manifested as a single genital lesion accompanied by enlarged local lymph nodes, leading to lymph node rupture. The appearance of localized genital lesions was consistent with recent reports from other countries in Europe (Second, we presented viral kinetics in different sample materials over time and show that, despite the localized lesion in this patient, viral DNA could also be found in urine, blood, and the respiratory tract. So far, this type of data has been published for few cases (Third, phylogenetic analysis revealed that the virus belongs to the Western Africa clade of monkeypox, which has been associated with lower mortality rates than the Central Africa clade (<8 days (Within the context of the emerging outbreak of monkeypox, we present comprehensive clinical and microbiologic data with long follow-up times revealing persistent PCR positivity. Previous reports have provided PCR data from single timepoints or short follow-up periods of <8 days . This anAdditional information on monkeypox patient in Sweden, 2022.


ADP-ribosylation (ADPRylation) is a versatile posttranslational modification in eukaryotic cells which is involved in the regulation of a wide range of key biological processes, including DNA repair, cell signalling, programmed cell death, growth and development and responses to biotic and abiotic stresses. Members of the poly(ADP-ribosyl) polymerase (PARP) family play a central role in the process of ADPRylation. Protein targets can be modified by adding either a single ADP-ribose moiety (mono(ADP-ribosyl)ation; MARylation), which is catalysed by mono(ADP-ribosyl) transferases (MARTs or PARP “monoenzymes”), or targets may be decorated with chains of multiple ADP-ribose moieties (PARylation), via the activities of PARP “polyenzymes”. Studies have revealed crosstalk between PARylation  processes in plants and plant–virus interactions, suggesting that these tight links may represent a novel factor regulating plant antiviral immunity. From this perspective, we go through the literature linking PARylation-associated processes with other plant regulation pathways controlling virus resistance. Once unraveled, these links may serve as the basis of innovative strategies to improve crop resistance to viruses under challenging environmental conditions which could mitigate yield losses. ADP-ribosylation (ADPRylation) is a well-known highly conserved reversible posttranslational protein modification which is involved in regulation of a wide range of key biological processes, such as DNA repair, cell signalling, programmed cell death, responses to biotic and abiotic stresses, and many others. Members of the poly(ADP-ribosyl) polymerase (PARP) family play a central role in the process of ADPRylation. During ADPRylation, ADP-ribose moieties are typically covalently  added to target proteins, which results in either decoration of the proteins with single ADP-ribose moiety (MARylation) or with poly(ADP-ribose) (PAR) chains of different lengths and branching morphologies (PARylation) ,2. MARylThe PARP family contains 17 mammalian proteins, of which only four are capable of generating PAR chains, with PARP1 being the best studied and most abundant member of this group . NumerouInterestingly, despite its potentially misleading name, most other PARP proteins possess MARylation activity. In mammalian cells, MARylation may play a role in immunity, transcription, regulation of the cytoskeleton and various stress responses including those occurring in the endoplasmic reticulum, which have recently been reviewed in detail by Challa et al. .With regard to the substrate specificity of ADPRylation, it is worth noting that protein targets can be modified via PARylation or MARylation at a wide variety of amino acid residues such as Ser, Asp, Glu, and Cys, constituting a diverse repertoire of ADPRylation targets ,9,10.Pieris rapae and pierisin-like proteins and scabins from Streptomyces bacteria act as cellular toxins and possess ADPRylation activity that mainly target DNA. Such bacterial DNA-modifying ADP-ribosyltransferases (toxins) are typically counteracted by ADP-ribosyl hydrolases (antitoxins or immunoproteins) residing in the same operon [In addition to proteins as a substrate for ADPRylation, it has recently become evident that some PARPs are capable of modifying nucleic acids. Mammalian PARPs can transfer ADP-ribose onto the 5′- or 3′-ends of both DNA and RNA nucleic acid substrates, if they possess either a 5′- or a 3′-phosphate ,10,11. Se operon . This sue operon .ADPRylation events (both MARylation and PARylation) are also known to be a significant factor involved in the development of responses to highly diverse group of RNA- and DNA-containing human and animal viruses, including Epstein–Barr virus (EBV), human immunodeficiency virus 1, the herpesviruses, human cytomegalovirus, hepatitis B virus, Ebola virus, Marburg virus, coxsackievirus B3, Japanese encephalitis virus, Sindbis virus, influenza A virus, poliovirus, dengue virus, Zika virus, herpes simplex virus 1, vesicular stomatitis virus, chikungunya virus and SARS-CoV-2 ,14,15. IAnother interesting link between ADPRylation and antiviral immunity may come from findings demonstrating that both DNAs and RNAs can be MARylated by various PARPs. It is, therefore, tempting to suggest that such a process could provide a very efficient way to suppress viral replication via the targeting of viral RNA or DNA. In this connection, the macrodomains present in certain viruses could operate as antagonists to prevent ADP-ribosylation of viral nucleic acids, thus representing a counterdefence strategy that facilitates virus replication. Similarly, as a reaction to coronaviral infection, the activity of human antiviral PARPs  is significantly upregulated and induces the proinflammatory defence response in the cell. Virus-encoded macrodomains can reverse this antiviral defence and enhance virus infection . The mecCollectively, these observations clearly show that the process of ADPRylation plays a critical role in mediating responses to biotic (virus attack) and possibly abiotic stresses.Arabidopsis  polymerases that mediate PARylation reactions ,17. The bidopsis . Howeverin planta they may fulfil analogous functions . The products of both these genes possess canonical PARG catalytic activity in vivo and in vitro  or cisplatin (an inhibitor of DNA replication) in Arabidopsis . In agreAt lower levels of DNA damage, PARP proteins recruit some DNA damage factors via PARylation processes to the DNA ,16,23. SIn addition to its role in DNA repair, PARP proteins and PARylation are involved in regulation of plant development . For insApart from DNA repair, PCD, and development, an emerging body of evidence implicates PARP and PARylation in many different stages of RNA biogenesis, metabolism and protein biosynthesis such as transcriptional initiation, polymerase II elongation and termination, chromatin remodelling and decondensation, co-transcriptional alternative splicing, RNA polyadenylation and mRNA export, RNA stabilization, ribosome biogenesis and translation ,25. MechAnother intriguing finding is that the PARP proteins may be involved in the assembly and maintenance of some subnuclear compartments such as the nucleolus and Cajal bodies, which are responsible for processing, assembly and transport of ribonucleoprotein (RNP) complexes ,25,26 whPlants are constantly exposed to different types of environmental (abiotic) stresses that can adversely affect their growth and development. These negative influences are likely to be further aggravated as a consequence of future global climatic changes, which will increase the risk of environmental stresses. Plants perceive abiotic stress signals and produce an appropriate response with altered metabolism, growth and development. At the molecular level, abiotic stress tolerance can be achieved by altering the accumulation of osmoprotectants, production of chaperones, superoxide radical scavenging mechanisms, exclusion or compartmentalisation of ions by efficient transporter and symporter systems . Over thSilencing of PARP genes in Arabidopsis and oilseed rape using RNA interference increased the tolerance to drought, high light, heat and methyl viologen (paraquat) treatment, accompanied with a decreased PARP activity and PAR accumulation ,4. The mPathogen-associated molecular pattern  triggered immunity (PTI) constitutes the first layer of plant immunity that restricts pathogen invasion . The N-tPharmacological studies using the PARP inhibitor 3AB have been shown to block a distinct subset of late responses characteristic of the flg22 and elf18 linked PTI such as callose deposition, whereas early responses such as the burst of reactive oxygen species (ROS) and early responsive gene expression are not affected ,34. TranIn contrast to pharmacological studies, genetic knockout experiments showed that Arabidopsis parp mutants exhibit a variety of different responses to flg22 or elf18 treatments including reduced, enhanced or unaffected callose deposition depending on plant age or experimental conditions [The more convincing evidence showing direct interplay between PARylation and plant immunity (via ubiquitination) was recently published by Yao et al. . It was as Streptomyces scabies encode scabins, a mono-ADP-ribosyltransferase toxins which target DNA molecules [It is also worth noting that some plant bacterial pathogens such olecules . HoweverExperimental data obtained employing pharmacological PARP inhibition often contradict with genetic (knockout) results ,37. It iWhile much remains to be discovered, several lines of compelling evidence point toward the suggestion that PARylation plays important role in plant virus interactions. The first indication of the functional links between PARylation and plant virus infections came from the observation that PARG2 transcription was significantly upregulated in cucumber mosaic virus (CMV) infected Arabidopsis plants carrying an RCY1 gene which confers extreme resistance to CMV .In our work, we observed a consistent and concerted upregulation of various PAR-degrading enzymes including PARG, NUDIX and ADP-ribose pyrophosphatases (ADP-PPase) in response to infection of potato plants with potato virus Y (PVY). According to these changes, PAR accumulation was significantly increased upon PVY infection compared to non-infected plants. It is thus conceivable to suggest that the PVY–plant interactions are at least partially regulated by PARylation functions .In more recent work we showed that an exogeneous PVY-specific fragment of double stranded RNA (dsRNA) protects potato against PVY . UsuallyIn many cases, the nucleolus and CBs play a crucial role in the infection cycle of DNA and RNA-containing viruses ,44,45,46Thus, the data presented above strongly suggest that PARylation processes may be linked to how animal and plant hosts respond to virus infection, although such interplay in plants is less well studied.CBs and their signature protein, coilin, have been shown to affect a variety of interactions between host plants and viruses that have RNA or DNA genomes. Moreover, the effects of coilin on these interactions are manifested differently: coilin contributes to plant defence against some viruses, but in contrast may serve to increase virus pathogenicity in other viruses. These findings show that interactions with coilin (or CBs) may involve diverse mechanisms with different viruses, and that these mechanisms act at different phases of virus infection ,45,46. TIn nature, plants are usually exposed to diverse environmental stresses, which may modulate plant–virus interactions . It is sTo elucidate the mechanisms underlying the cross-talk between PARylation and virus infection outcomes, analysis looking at expression level (transcript levels and protein levels) and activity of PARylation-related enzymes  and PAR itself should be performed during the infection process. It would also be important to look at possible relocalisation of PARylation related proteins potentially induced by various viruses, as significantly altered distribution (including perhaps sequestration to defined cellular regions) may greatly affect PARylation efficiency. It is well known, for example, that the nucleolus can act as a sequestration centre which may capture and detain various cellular factors .In mammalian model systems, it appears that MARylation is required for host cells to respond to viral infection. It is, therefore, conceivable to examine the distinctive role of MARylation (compared with PARylation) in plant–virus interactions.It has recently been shown that nucleic acids (DNA and RNA) may be directly ADPRylated. Therefore, future studies should examine whether such modifications can occur with plant viral RNAs or DNAs and investigate the biological relevance of such process.It would also be critically important to identify PARylation targets among virus components  and host signalling molecules involved in plant responses to virus attack or to combined stresses. Application of systems biology approaches to analyse the data obtained will allow the identification of target genes and regulatory hubs which could be exploited to develop improved plant resistance to biotic and environmental stresses.On a practical level, genetic alteration of the PARylation (and possibly MARylation) process (by affecting PARP and PARG proteins) in crops may be achieved either by CRISPR-Cas technology   or by RNViruses represent one of the major plant pathogens (biotic stress factors), causing almost half 47%) of all emerging plant disease outbreaks (more than any other pathogen group; [% of all


To date, 40 Human Milk Banks (HMB) have been established in Italy; however, recent cost analysis data for operating an HMB in Italy are not available in the literature.This study was a cross-sectional study performed at “Bambino Gesù” Children’s Hospital in Rome, Italy in 2019. We assessed the one-year operational costs and, the per liter unit costs at our HMB.p = 0.016) comparing the cost for collected liters in the year 2019 and the cost for the maximum capacity of the bank for that year of activity. Analyzing each cost item that determines the charge of donor human milk (DHM), the highest costs are the salaries of medical and paramedical staff, and then the costs related to transporting. If the HMB works at maximum capacity and manages a greater number of liters of milk, this can represent an important saving. Conversely, the price of consumables is modest .During the 2019 year we collected 771 l of human milk supplied by 128 donors. The total cost was € 178,287.00 and the average cost was € 231.00 per liter. € 188,716.00 would have been spent had the maximum capacity for 904 l been reached. We found a significant difference (€ 231.00 vs € 209.00 per liter, The costs for a liter of DHM are quite high, but they must be related to the benefits, especially for preterm infants. Comparing the cost for collected liters in 2019 and the costs for the 2019 maximum capacity of the HMB, we calculated how much fixed costs of collection and distribution of DHM can be reduced, by increasing the volume of milk collected. To the best of our knowledge, this is the first complete cost analysis for an Italian Milk Bank. A thorough analysis could help to abate fixed costs and reduce the cost of a liter of DHM. The centralization of DHM can allow savings, rather than creating small HMBs scattered throughout the territory that would operate with lower milk volumes. Breast milk (BM) and donor human milk (DHM) from the Human Milk Bank (HMB) represent the optimal nutrition for all babies, from full-term babies to preterm and high-risk newborns, due to their anti-infectious properties, immune modulators, growth hormones, and the best nutrient composition available for infants for their physiological growth , 2. SeveHowever, this is not an inexpensive process. Limited data are available about the cost of DHM in Neonatal Intensive Care Units (NICUs) worldwide. Carroll and Herrmann found in the US in 2013 a wide variation in the mean cost of DHM per infant, ranging from US$27.04 to US$590.90 during hospitalization (about US$133.00 per liter) . Buckle Fengler et al. reported how providing preterm infants with DHM was significantly more expensive than using formula or MOM, but the cost of pasteurization was minimal (only € 3.51 per liter) .However, a recent meta-analysis  showed aNo recent data on costs of collection and management of DHM in Italy are available: the aim of our study was to estimate the one-year operational costs and the per liter unit costs of the Bambino Gesù Children’s Hospital Human Milk Bank.This study was a cross-sectional study performed at “Bambino Gesù” Children’s Hospital, Rome, Italy in 2019. The activity-based costing (ABC) method usually used for estimating the costs of blood banks was used to estimate the one-year operational costs and the per liter unit costs at our HMB .This study does not contain any studies with human participants performed by any of the authors. For this type of study informed consent is not required.For reporting our modelling study, we followed the Consolidated Health Economic Evaluation Reporting Standards (CHEERS) Statement .The “Bambino Gesù” Children’s Hospital HMB was established in 1989 and is the sole Human milk Bank in Rome and in the whole area of Lazio, connected to NICUs and part of the Italian Association of Donor Human Milk Banks. We follow and sustain breastfeeding specific advice providing support to mothers who are breastfeeding or who must maintain milk production during their baby’s stay in the NICU . The pro, 25. DHMProcessed DHM is provided in hospitals to preterm and low birth weight infants, children with congenital heart disease, and high-risk surgical infants, according to the provider’s prescription.Donors for our HMB are mothers whose babies are admitted to our hospital, and nursing mothers who have seen recruitment information. They are carefully selected within 12 months of lactation if they are in good general health  and have then tested negative for Human Immunodeficiency Virus, Hepatitis B, Hepatitis C, and syphilis. In addition, a bacteriological count is carried out on a small aliquot of breast milk once per human donation, and then randomly during the donation period . Donors The milk thus collected is then stored in a freezer at − 24 °C in the Lactarium Room. Inside the Lactarium, health workers prepare all the nutritional products of the hospital and, in particular, pasteurize the milk donated.In our study, all DHM donations and consumptions are expressed in the international unit of a liter. All costs used in the ABC program are expressed in euros (€).To facilitate calculation, we excluded costs incurred by donors using their mechanical pump and storage in their home freezer compartment, and we included only certified data collected from budgets, financial and expenditure reports of “Bambino Gesù” Children’s Hospital HMB in 2019.HMB and Lactarium direct costsClinical personnel ;Medical supply;Laboratory tests;Transport (including fuel and car park);b)HMB and Lactarium indirect costsIndirect supplies ;General costs ;Depreciation of infrastructure.We calculated direct and indirect costs of collection and management of a liter of milk to our bank’s DHM:The general costs, the staff costs and the depreciation of infrastructure allocation basis was the percentage of HMB’s personnel costs compared to the hospital’s total cost of personnel (0.04%).Then we allocated the assigned indirect costs down to each collected liter. For the sole doctor who worked not only in the HMB but also in the Neonatal Intensive Care Unit, charges were calculated according to the working hours spent at each cost center. In order to assess direct costs of collection and transport of milk, we analysed the driver’s compensation, insurance, tires, and gasoline costs.We estimated also direct and indirect costs of the Lactarium, analysing every single cost for the preparation and pasteurization of bottles of DHM, and then for the room cleaning.We also evaluated the charges for liters of our DHM delivered to four other hospitals in Rome.P-value was set at 0.05. Comparisons between groups were made using paired t-tests. Statistical analysis was carried out by using the Statistical Package for Social Sciences , version 13.0.Results are expressed as mean ± standard deviation (SD) if not differently indicated, and the We collected 771 l of human milk in 2019. Donors came from mothers whose sick babies are in our hospital  and volunteering at-home nursing mothers who have seen recruitment information . The total cost for 771 l of DHM supplied by 128 donors during the study period was € 178.287.00, and the average cost was € 231.00 per liter.p = 0.016) when comparing costs for collected liters in the year 2019 and costs for the maximum capacity of the bank as calculated for the year 2019. We spent € 178,287.00 to collect 771 l in 2019, whereas € 188,716.00 would have been spent if the maximum capacity for 904 l had been reached. All costs are reported in each particular, hereafter.Costs for collection and management of a liter of our bank’s DHM are summarised in Table The total cost of each type of direct cost during 2019 was divided by the number of the annual total donated liters, in order to have the cost / liter value. In Table In Table Direct and indirect costs of the Lactarium Room are reported in Table Starting in May 2019, our bank also started supplying DHM for free to other hospitals in our city, for a total cost of € 44,435.00: each charge is reported in Table Breast milk represents the best food choice for all babies, especially preterm and surgical infants . WheneveTo the best of our knowledge, our study was the first complete cost analysis for an Italian Human Milk Bank, whereas in Germany, Fengler et al. have reported an average cost of € 306.95 per liter of DHM . The aveWe demonstrated that the major item was the cost of staff. Therefore, the costs of DHM can be widely different in each country, according to the economic situation and to the cost of work.We believe that the microbiological safety outweighs the minimal costs of pasteurization, that Fengler et al. estimated to be 1.1% of the cost of a liter of DHM (€ 3.51 / € 306.95) .p = 0.016), underlining how much the fixed costs of collection and distribution of DHM can be reduced, by increasing the liters of milk collected. Therefore, this novel aspect should be carefully considered by local health organizations: the presence of several milk banks in the same region appears not justified, considering that these fixed costs would increase with more HMBs, while donor mothers from the same area can all donate milk to the same institution that manages larger volumes and distributes DHM to those who request it. Fengler’s and Daili’s groups had not addressed this aspect. Similarly, Hoodbhoy described that in the United Kingdom the estimated cost of 1 liter of donor breast milk has been estimated to be £150 – £290, but this has been not investigated [In comparing costs for collected liters in 2019 and costs for the maximum capacity of the bank, we found a significant difference . Jegier et al. previously reported that the mean cost of providing 100 mL of human milk varied from $2.60 to $6.18 for mothers who gave human milk for their very low-birthweight infants during the early NICU stay . ConversFurther multicentre studies are needed on the cost analysis of HMBs in Italy and other countries, and the economic benefits of using DHM from an HMB, as the best alternative to a MOM.The breastfeeding rate in high-risk newborns should be increased by specific steps, sustaining mothers during NICU hospitalization and after discharge . HMBs coFurthermore, we demonstrated that the cost of DHM can be reduced (−€ 22.00 per liter) by working at the HMB’s maximum capacity, because all fixed costs are distributed over the greater quantity of processed milk; existing staff have excess capacity to process the greater quantity without requiring additional staff; and the variable costs of consumables contribute the least to the cost per liter.In order to provide DHM to other hospitals in the same region, it would be conceivable to create an agreement with the additional centers to share the economic burden. The pooling of resources allows for the optimization of the use of fixed costs and could potentially limit variance in variable costs.To the best of our knowledge, this is the first cost complete analysis for an Italian Human Milk Bank. A thorough analysis of collection and management of DHM, processing milk at maximum capacity of the bank and thus reducing fixed costs, could reduce the cost of a liter of DHM, and provide benefits to a larger cohort of high-risk newborns.


After two years of extended lockdown, Kampala’s vast workforce of motorcycle taxi riders today looks a little different. Though the sector has long constituted a vital source of labour and income for many thousands of urban residents cut off from more decent opportunities elsewhere in the economy, a recent combination of lockdown pressures and digital transitions has created new forms of dependency upon the sector whilst simultaneously stripping some old ones away. In this article, we draw on in-depth qualitative data from interviews with riders, carried out at different stages of the pandemic, to show how the composition of labour within the sector has been reworked by a series of ‘selective exits’ and ‘substitution effects’ over the past two years. In exploring the nature and nuances of these parallel movements, our analysis not only reveals considerable socio-economic unevenness within the city’s motorcycle taxi sector itself but also sheds light on a new, broader configuration of urban inequality in the making. For close to two years during the COVID-19 pandemic, Uganda’s motorcycle taxi sector took a battering. What started out as a 14-day government-imposed suspension of public transport and passenger ferrying towards the end of March 2020, implemented alongside a wider raft of temporary measures designed to curb the virus’ spread in those early days of the pandemic, ended up as a shifting regime of lockdown and curfew that lasted 22 months. It was not until 7 February 2022, one month after schools reopened and two weeks after Uganda’s vibrant nighttime economy was resuscitated by the lifting of a 7pm curfew on the general population, that government restrictions on motorcycle taxis were finally dropped. This ubiquitous form of popular transport, known locally and regionally as boda bodas, was the last piece of the economy to be fully restored.Uganda’s boda boda sector is one of the country’s most significant labour markets. Though accurate statistics do not exist, it is generally understood that boda riding constitutes the single largest sector of male employment outside agriculture Amone, .1 Relati2For all the stigma attached to boda riding, particularly so in the congested and youthful contexts of large towns and cities, this is a sector of ‘vital’ economic activity in more ways than one  to an enduring ban on riding after dark?While Ugandan news outlets regularly featured coverage of these latest developments alongside a scattering of more analytically focused commentaries . Significantly, however, this does not appear to have been a like-for-like substitution. Many of those now turning to the motorcycle as a means of survival are arriving from elevated starting points relative to some of the industry’s most precarious and dependent longer-term riders, facilitated into the sector by an expanding array of possibilities for digital platform work and frequently bypassing pre-existing institutions of entrance and operation. Though the specific scale of this practice is difficult to determine, and while we must be careful about lumping all ‘exits’ and ‘entrants’ into discrete, cleanly bounded categories, there are both indications here of important social shifts within the city’s boda workforce itself alongside implications for how infrastructures of urban consumption and informal labour are being reworked and brought together in new ways  of their core livelihoods, whilst simultaneously manufacturing new dynamics and patterns of urban inequality. This story is developed over the course of the four following sections, starting with a brief overview of data and methods. We then, in separate sections, explore the two parallel processes outlined above—‘selective exits’ and ‘substitution effects’—in greater depth, drawing directly on the empirical data to illustrate how these changes have been unfolding and experienced by workers in Kampala’s boda economy. Finally, we conclude by drawing out some theoretical implications from the discussion.This article is based on qualitative data generated through in-depth semi-structured interviews, carried out over an extended period whilst Uganda’s lockdown measures were still in place. They form part of the lead author’s PhD project, which looks at livelihoods, politics and the changing nature of work within Kampala’s boda boda sector. Although additional methods have also been used as part of this study, for our purposes here we draw specifically on interview material collected at two different points in time.A first round of interviews was conducted by a small team of researchers at Makerere University’s Urban Action Lab towards the end of 2020, at a time when COVID restrictions prevented international travel and the completion of in-person fieldwork by the lead author. These initial interviews were organised through detailed online discussions between team members concerning the objectives of the exercise, the design of interview guides, sampling strategy, and plans for transcription and secure data storage. A total of 26 interviews with boda riders were carried out in November 2020, with researchers being careful to ensure variation across the sample of interviewees in order to capture diverse experiences and perspectives. To this end, rider selection was carried out with two key variables in mind: means of motorcycle access (i.e. those who own vs. those who either rent on a daily basis or are in the process of paying off a hire-purchase loan); and means of accessing jobs . This basic framework generated four categories of boda rider, which are fairly evenly represented within the final sample Table .Table 1COnce these categories had been established, we made further efforts to reduce sampling bias by designing a selection strategy that to some extent reflected the urban geography of Kampala’s boda boda sector. Rather than focus on a single location, potential interviewees were approached at a range of sites within the city, including: (i) busy commercial areas of high economic activity ; (ii) popular hangout spots where riders would often congregate to eat, rest or chat amongst themselves, including at times when they were not actively working; and (iii) quieter areas with higher levels of residential land use. Though these sites were chosen purposively, the strategic selection of multiple areas in this way helped to construct a more realistic picture of the sector’s internal composition than would have been possible using either a single-sited approach or one based on selection by administrative unit.A second round of interviews took place between October 2021 and February 2022, once the easing of COVID restrictions permitted international travel. Though an interval of this length was unplanned, it made preliminary analysis of the earlier interview material possible and proved useful in structuring the next steps. In particular, it allowed us to: develop a clearer sense of what changes in the city and sector seemed to be unfolding amid lockdown conditions; think through appropriate ways of probing these issues further; and more generally ground the design of subsequent interviews in analysis of the earlier material. During this five-month period, 51 additional interviews were carried out with boda riders. These typically lasted between one and two hours, drawing on life-work history approaches to open up a dialogue about people’s journeys into the sector as well as exploring their current experiences and perceptions of life in the industry Lewis, . By contIn the two sections that follow, we draw first on data from the initial set of November 2020 interviews to outline the beginnings of our story (‘selective exits’), before zeroing in on some selected accounts from our later interviews to develop a more detailed picture (‘substitution effects’). In particular, we use the in-depth recent experiences of one new entrant to the boda sector as a way of drawing out broader insights into the nuanced nature of change within the workforce during this time.When our initial interviews were carried out in November 2020, the Ugandan government’s restrictions on boda activity had been in place for just over seven months. During this time, their severity shifted in response to changing assessments of the public health threat posed by the virus, starting out as an outright ban throughout the earliest phases of the pandemic before easing to permit the carrying of cargo but not passengers for several months. By the end of July 2020, riders were once again allowed to take passengers but with both a series of ‘standard operating procedures’ and a 6pm curfew in place. These partial lockdown conditions remained relatively stable until Uganda’s second wave hit in the second quarter of 2021, which resulted in a return to total lockdown from June onwards and a ban on passenger ferrying. By August 2021, infection rates had slowed and restrictions once again began to ease. For the rest of the year, riders could take both passengers and cargo until nightfall, and this was largely how the situation remained until all measures were lifted on 7 February 2022—a fortnight after the rest of the economy had been fully reopened.Prior to the pandemic, the boda industry provided work and income to a colossal number of people, likely forming the country’s largest sector of urban employment for men. For at least the last two decades, it has kept numerous individuals afloat as an increasingly all-consuming politics of regime survival worked against sustained commitments to structural transformation and broad-based job creation  employment have often strategically incorporated bodas into their working lives further bears out this picture of internal heterogeneity, with teachers in particular having long used intermittent riding to supplement their wages or get through periods of delayed pay.kibaluwa system, riders usually pay the owner 10,000 UGX a day (just under 3 USD) in exchange for access, and are responsible for purchasing their own fuel as well as handling minor maintenance work. While it is often claimed that the owners or ‘bosses’ embedded in this system tend to be wealthy, politically connected (or involved) individuals, again the reality is much more diverse. Purchasing one or two motorcycles and renting them out through the kibaluwa system is a widespread money-making strategy among members of the middle class looking for easy investment opportunities  only as a last resort once other safety nets or financial avenues had been exhausted, selling it off to generate survival revenue or even becoming riders themselves. As one of our interviewees put it, it was ‘mostly those riding for [other] individuals [who] failed and left. Many bosses sold their bodas to sustain their families’.Another rider made similar remarks, suggesting that the worst effects of the pandemic were experienced by ‘colleagues [who] lost their boda bodas to the bosses and companies’.These reports of dropouts from the sector are reflected in observations from other sources, including from within the digital ride-hailing space. In a media interview just three months into the pandemic, one of the founders of Uganda’s largest ride-hailing company, SafeBoda, said that 30% of the platform’s registered riders had left Kampala and returned to the village  companies linked to the e-commerce platforms—in exchange for around a quarter of their earnings on each food / grocery delivery made.17Samuel’s reported earnings sit right at the upper end of what a boda rider in Kampala can expect to earn, even after several years of accumulating experience and contacts, and his testimony provides a few clues as to why he has been able to reach this status in such a short period of time. First, because of his relatively privileged starting point, Samuel managed to bypass both the Second, Samuel took advantage of almost any platform opportunity he deemed worthy, doing stints with different ride-hailing and e-commerce companies to figure out where the most lucrative spaces of boda work existed at the time—in much the same way as he had previously done as a young teacher, constantly seeking out the next best thing. Such an approach is by no means accessible to all in the sector, requiring as it does both a number of initial conditions or endowments to be in place—each platform has its own particular set of joining requirements, ranging from a good level of English to ownership of a smartphone meeting minimum standards—as well as the time and knowledge necessary to scope out all potential options.lubyanza), in the process attracting a great deal of resentment and even physical abuse from riders working the ‘right’ way, with the arrival of platform work it has become increasingly straightforward and indeed normal for individuals to enter the sector as independent, stage-less riders. This was precisely the route taken by Samuel, who started out as an ‘independent contractor’ for a European food courier company before frustrations with that work led him to full-time passenger-ferrying. To access customers, he initially registered with two digital ride-hailing companies but soon found himself regularly going offline to find and negotiate directly with passengers on the street, a method he quickly realised could be used to escape the low ‘piece-rate’ payments offered by the apps. At the time of our interview with him, Samuel had three ride-hailing apps installed on his phone, which he used normally between 11am and 4pm when ‘people are very few’ and logging on can help riders tap into the diminished pool of customers, and combined this with substantial shifts of offline, stage-less work during the much busier morning and evening periods—essentially taking the most lucrative strands of work in the sector and tying them together to form a ‘mobile and recombinant strategy of survival’  unregulated labour market. Riders typically attach themselves to a fixed location called a ‘stage’, which is usually positioned in a site that enhances exposure to potential customers: that might be outside malls or markets, at key junctions and intersections, or within one of the city’s many local neighbourhoods. According to the sector’s most influential yet informal rules, apart from in exceptional circumstances riders should only pick up passengers and cargo from their stage. After completing the journey, they are then expected to return to the stage and wait for the next job . While there have always been some who operate entirely outside this system in order to maximise their personal incomes  unemployed young men and school dropouts living in poverty Howe, , to its lubyanza figure—app companies are helping to instil a different set of norms around how work and incomes within the sector are distributed. In bypassing pre-existing institutions of entrance and operation, the shift towards platform work risks producing a digitally connected yet largely atomised class of labour within the workforce  workforce. The implications of this are mixed and ambiguous. On the one hand, opportunities provided by the platforms have helped some people weather the impacts of lockdown, creating new routes into the survival space of the boda sector and offering alternative methods of piecing together a livelihood strategy for those already in it. On the other hand, there are signs that these technologies are both segmenting and further fragmenting labour within the sector. By opening up new ways to operate independently of the traditional stage system—but this time with the backing and sheen of corporate power, somehow creating a more professionalised iteration of the much-maligned shimo in kiSwahili usage), dispersing workers across the city and ‘dis-locating’ connections among riders and customers. Given how Kampala’s digital riders often remark that the ‘app is their stage’ or that ‘the stage is on the phone’, it is possible that we have begun to see a partial manifestation of this threat on the streets of the Ugandan capital. On the other hand, current and ongoing efforts by parts of the state to formally regulate the industry continue to position the stage centrally in their plans, with repeated calls for a mapping and demarcation of all ‘official’ stages in the city alongside a renewed requirement for all boda riders to register with the municipal authority through one of them .As COVID restrictions ease more fully and opportunities in the wider economy return, some will cease to be riders and their economic relationship with the sector will come to an end. Others like Samuel, who have seen the financial gains that can be made from riding and realised how relatively straightforward it is to jump into the work on an independent basis, will continue to be intermittently engaged with the sector. As one interviewee told us, even though he was looking forward to the reopening of schools so he could get teaching again, ‘I can’t just dump it’.Whatever the lasting specifics of this social reworking—given the ongoing and unpredictable nature of these processes, it is too soon to tell whether the workforce’s new internal unevenness will develop into a durable rather than fleeting inequality Tilly, —the COVIBut although the boda economy provides livelihoods and incomes to a huge number of people, including in times of crisis, it does not work equally for everyone. Ultimately, Kampala’s boda sector is a site of layered, multidimensional inequalities, expressed not just in the uneven distribution of earnings and guarantees of work among an ever-diversifying workforce, but also in the way that particular kinds of boda labour are being incorporated into digitally mediated systems of consumption and provisioning for the city’s growing middle class. As the sector becomes increasingly central to the workings of a new configuration of urban inequality, its vitality remains—but the question of whose interests it is really serving, and how that might be changing in an era of lockdowns and ‘digital transformation’, remains an open one.


Chlorovirus genus of the Phycodnaviridae family includes large viruses with a double-stranded DNA genome. Chloroviruses are widely distributed in freshwater bodies around the world and have been isolated from freshwater sources in Europe, Asia, Australia, and North and South America. One representative of chloroviruses is Acanthocystis turfacea chlorella virus 1 (ATCV-1), which is hosted by Chlorella heliozoae. A few publications in the last ten years about the potential effects of ATCV-1 on the human brain sparked interest among specialists in the field of human infectious pathology. The goal of our viewpoint was to compile the scant research on the effects of ATCV-1 on the human body, to demonstrate the role of chloroviruses as new possible infectious agents for human health, and to indicate potential routes of virus transmission. We believe that ATCV-1 transmission routes remain unexplored. We also question whether chlorella-based nutritional supplements are dangerous for ATCV-1 infections. Further research will help to identify the routes of infection, the cell types in which ATCV-1 can persist, and the pathological mechanisms of the virus’s effect on the human body.The  Chlorovirus genus of the Phycodnaviridae family includes large viruses with a double-stranded DNA genome [Chlorella genus, with a particular virus species infecting a specific algae species. Chloroviruses are widely distributed in freshwater bodies around the world and have been isolated from freshwater sources in Europe, Asia, Australia, and North and South America [Chlorella heliozoae. A few publications in the last ten years about the potential effects of ATCV-1 on the human brain sparked interest among specialists in the field of human infectious pathology.The A genome . The nat America ,3. One rIt is known that in the human body, there are no sterile organs where there would be no microorganisms that belong to different kingdoms of viruses, bacteria, or fungi and together make up the human microbiome. Many viruses can colonize trans-barrier organs. For example, the Zika virus can pass through the blood–placental barrier and lead to neonatal microcephaly . The humThe goal of our viewpoint was to compile the scant research on the effects of ATCV-1 on the human body, to demonstrate the role of chloroviruses as new possible infectious agents for human health, and to indicate potential routes of virus transmission.z100l gene of the ATCV-1 virus. qPCR analysis detected ATCV-1 DNA in all 10 subjects whose samples contained two or more reads homologous to the viral genome and in 12 of 14 subjects with single reads homologous to the ATCV-1 genome. The results of the analysis showed negative results with human DNA, the buffer solution used, as well as ATCV-1 host DNA from Chlorella heliozoae, other PBCV-1 and CVM-1 chloroviruses, and their host organisms: Chlorella variabilis and Micractinium conductrix [In the metagenomic sequencing of DNA samples obtained from the oropharynx of 33 adults without known psychiatric disorders or diseases, Yolken et al. unexpectedly revealed a significant number of reads homologous to various regions of the genome of the ATCV-1 . Virus sp < 0.002), Delayed Memory (p < 0.039), and Attention (p < 0.011) from the Repeatable Battery for the Assessment of Neuropsychological Status (RBANS) as well as the total scores in this battery of tests (p < 0.014).The proposed qPCR method was applied to oropharyngeal samples from a larger group of 92 individuals, including 33 previously tested subjects. In 40 (43.5%) of 92 samples, ATCV-1 DNA was detected. At the same time, the DNA of the ATCV-1 virus was not detected in the blood in any of the tests. There were no statistically significant differences between subjects with and without viral DNA in demographics such as age, gender, ethnicity, education level, smoking, basal metabolic rate (BMR), travel history outside North America, and place of birth. In addition, the authors of the work evaluated the relationship between the detection of ATCV-1 DNA and the success of passing a battery of cognitive tests. A statistically significant association was found between the presence of ATCV-1 DNA and lower scores on the Visual Reaction Speed Test, Trail Making Test Part A  or Chlorella heliozoae infected with ATCV-1  to mice. During the experiments, the memory of spatial recognition was tested in a Y-shaped maze. Mice exposed to ATCV-1 were less likely to enter a new, previously closed maze arm (p = 0.015), while the levels of locomotor activity did not differ between groups. Testing of recognition memory showed a decrease in the time spent exploring a new object by mice from the experimental group (p < 0.001) and a familiar object in a new location (p = 0.008). At the same time, the passive avoidance test, which tests memory for negative stimuli, did not show statistical differences. In tests with an open field and a dark–light box on the level of anxiety, there were also no differences between the groups. In addition, mice exposed to ATCV-1 were found to have worse pre-pulse inhibition (p < 0.05), which may reflect the deterioration in sensorimotor filtration that is characteristic of many psychiatric and neurological diseases.In addition to the human study, Yolken et al. created a mouse model of ATCV-1 infection. They orally administered Despite fairly convincing evidence, the detection of the ATCV-1 virus was questioned in an article by Kjartansdóttir et al. . This coTo study the molecular processes behind the changes in the behavior and memory of mice exposed to the ATCV-1 virus, the authors of the work investigated gene expression in the hippocampus, a region of the limbic system of the brain that plays a key role in learning and memory . SignifiIn addition to the identified changes in the pathways associated with the activity of microglia and other immune cells, specific antibodies against ATCV-1 were detected in the experimental group of mice. These data may suggest that infection with ATCV-1 is followed by a pronounced immune response, which may lead to the activation of microglia and the release of pro-inflammatory cytokines that can affect memory and cause cognitive deficits through neuroinflammation mechanisms. In more recent work on the interaction of ATCV-1 with mammals, the virus was directly injected intracranially. There was a significant increase in neuroinflammation markers such as IL-6, iNOS, IFN-γ, and CD11b after administration . In addiAnother study also showed the ability of the ATCV-1 virus to activate macrophages in vitro in a cell culture of mouse RAW264.7 macrophages and a primary culture of inflammatory macrophages from C57BL/6 mice. An hour of incubation with the ATCV-1 virus increased the expression of IL-6 and inducible NO synthase genes, as well as the release of pro-inflammatory cytokines IL-6 and NO. An increase in lactate levels was observed under the influence of the virus, probably due to a shift in metabolism from oxidative phosphorylation towards glycolysis. Such metabolic changes characterize the transition of macrophages from a resting state to a pro-inflammatory M1 phenotype. A characteristic response of mammalian cells to viral infection is the production of interferons and interferon response factors such as IRF7. The expression of the IFN-β and IRF7 genes increased in macrophage cultures after incubation with the ATCV-1 virus, indicating the activation of innate antiviral immunity pathways .Chlorella heliozoae), and 24 hours later, the cell membrane was stained with CellMask dye to detect intracellular viruses. Confocal microscopy revealed stained viruses inside the cell and outside the cell membrane.To make sure that the virus is not only attached to the cell surface, but is also internalized, ATCV-1 stained with Sytox orange dye was added to the RAW264.7 cell culture . On average, for RAW264.7, this amount was 0.8 × 106 PFU/culture , and for the primary macrophage culture, 2.7 × 105 PFU/culture . However, after 24 hours, the amount of ATCV-1 PFU (isolated from the supernatant and macrophage lysates) was already 2.6 × 106 PFU/culture for RAW264.7 and 3.9 × 105 PFU/culture for the primary macrophage culture. After 72 hours, the amount of PFU was 19 × 105 PFU/culture for RAW264.7 and 5.0 × 105 PFU/culture for primary macrophage culture. Compared to the number of PFUs at the 1-hour time point, a significantly higher number of ATCV-1 PFUs were observed at the 24- and 72-hour time points, indicating the ability of ATCV-1 to persist and slowly replicate in macrophages.In addition, in a parallel experiment, the number of viruses bound to cells was measured after 1 hour of incubation and removal of the supernatant with unbound viruses. The initial dose was 1 × 10z280l encoding the main protein of the ATCV-1 capsid. The average amount of mRNA was 40 copies at the 24-hour time point, 139 copies at the 48-hour time point, and 100 copies at the 72-hour time point. However, to confirm viral replication, it was still necessary to detect the production of ATCV-1 proteins in the RAW264.7 cells. To do this, a rabbit antiserum against the purified ATCV-1 virus was obtained, and when conducting a Western blot with proteins from lysates of RAW264.7 macrophages incubated with ATCV-1, a 55 kDa protein was detected (mass similar to the main protein of the ATCV-1 capsid), the level of which remained constant from 16 h to 66 h after incubation with the virus, and another 17 kDa protein, determined at the point at 16 h and increasing in number from 48 h to 66 h. To determine whether these proteins are synthesized in macrophages or remain in the cells after phagocytosis of the initial amount of virus, a heat-inactivated ATCV-1 virus was used as a control. Despite the fact that at the initial time point 24 h after the addition of the virus, the intensity of the 55 kDa protein bands were equivalent in cultures infected with both the intact and inactivated virus, after 48 h and 72 h, the level of this protein in the cultures with the inactivated virus decreased. In addition, in the case of the inactivated virus, the 17 kDa protein was not detected. Thus, it can be argued that RAW264.7 macrophages, after infection with the intact ATCV-1 virus, do indeed synthesize some viral proteins. However, it is impossible to say for sure whether the synthesis of other proteins of the virus occurs in the cells, and they are not detected because their number is below the level of detection in this experiment, or the antiserum used in the experiment is not able to recognize them.To confirm the replication of the ATCV-1 virus in macrophages on a RAW264.7 culture, the qPCR method measured the expression of mRNA of the viral gene In addition to the above actions on the cells of macrophage cultures, the ATCV-1 virus has a cytopathic effect on RAW264.7 cells: 24 hours after the addition of the virus, plasma membrane blebbing, karyorrhexis, and apoptosis are observed in some cells (the heat-inactivated ATCV-1 virus did not cause such phenomena). However, the primary culture of inflammatory macrophages did not show a significant level of apoptosis even after 72 h, but the formation of cell membrane outgrowths characteristic of macrophage activation was observed. The RAW264.7 cells, after exposure to the virus, showed an increase in the level of activated caspase 3 and annexin V associated with cell membrane phosphatidylserine, which indicates the activation of programmed cell death through apoptosis, which is probably a protective antiviral mechanism that limits viral replication. However, it is possible that the phagocytosis of apoptotic macrophages by other macrophages promotes the spread of ATCV-1 to them and its persistence in the macrophage population. It was shown that the triggering of apoptosis mechanisms upon the infection of RAW264.7 macrophages with the ATCV-1 virus depends on the activation of the mitogen-activated protein kinases/extracellular signal-regulated kinases (MAPK/ERK) pathway. In Western blot with antibodies to activate phospho-ERK, its increase is observed as soon as 30 minutes after the introduction of the virus; it is detected after 60 minutes, but is absent after 3 hours. The pretreatment of cells with ERK kinase activation inhibitor U0126 prevents RAW264.7 apoptosis caused by the ATCV-1 virus .In this article, a BHK-21 fibroblast culture was also tested for the possibility of ATCV-1 infection, and the results were negative. The question of the ability of the ATCV-1 virus to infect various types of cells remains open: is the virus capable of penetrating the human CNS, infecting neurons or glial cells? Given the data obtained on macrophage cultures, is it possible for ATCV-1 to infect the microglia of the human nervous system, which act as resident macrophages of the CNS and have a common origin with macrophages of other body tissues? As shown, ATCV-1 is capable of persistence and replication in macrophages, which is accompanied by their activation with a pro-inflammatory phenotype, the release of various inflammatory factors, such as IL-6 and NO. In addition, activated macrophages are able to migrate, thus spreading the virus that persists in them. It is known that activated microglia with a pro-inflammatory phenotype can cause serious disturbances in the functioning of CNS cells: it can destroy synaptic connections, cause disturbances in the formation of new synapses, neurogenesis, synaptic plasticity, and cause disturbances in the normal energy and plastic metabolism of neurons and astrocytes, which can trigger the processes of programmed cell death. The activation of microglia and an increase in the level of pro-inflammatory cytokines and NO in the CNS characterizes, among other things, many neurodegenerative diseases.n = 18) had significantly higher levels of anti-ATCV-1 IgG1(p = 0.0182), but not other IgG subclasses, compared to the healthy control group (n = 13).A recent article  elucidatFurther, the authors of this article decided to determine whether ATCV-1 influences the development of motor neuron diseases (MNDs) using a transgenic mouse model of familial ALS, with mice expressing human genetically polymorphic Superoxide Dismutase 1 G93A. SOD1-G93A-transgenic mice or C57Bl/6 control mice were intracranially injected at day 35 of age with ATCV-1, heat-killed ATCV-1, PBS, or polyinosinic:polycytidylic acid (polyI:C), a stimulant of the antiviral immune response. The SOD1-G93A mice, injected with active ATCV-1, showed more rapid motor deterioration compared to the saline-treated transgenic mice. However, active ATCV-1 infection did not significantly affect mortality in this group. Inactivated ATCV-1 did not affect motor loss or survival, so only active ATCV-1 hastened the onset of MND in SOD1-G93A mice. An interesting finding in this study was that polyI:C injection significantly lengthened survival time, but had a minimal effect on motor loss onset. It is known that the ATCV-1 genome encodes a functional SOD-1 protein, which converges with findings that ATCV-1 increases motor deterioration in SOD1-G93A mice. To study the effects of ATCV-1 SOD1 on macrophage expression of immune factors, such as IL-6, IL-10, NO, and Interferon-Stimulated Genes (ISGs) promotor activity, RAW264.7 Lucia cells were transfected with a plasmid vector that expresses ATCV-1 SOD1. The results of assays with transfected cells indicate that ATCV-1 SOD1 augments the production of IL-6, IL-10, NO, and ISG expression, which are associated with the development of ALS in humans and MND in SODG93A transgenic mice.Chlorella sp. and Micractinium sp.), with a specific virus species infecting a specific algal species. The host for ATCV-1 is Chlorella heliozoae. Algae are able to establish endosymbiotic relationships with larger protists. Thus, the host for Chlorella heliozoae is Acanthocystis turfacea. One protist host can contain up to several hundred unicellular algae. Chlorella sp. in an endosymbiotic state is resistant to chlorovirus infection, whereas symbiont-free chlorella are susceptible to infection [Chlorella heliozoae. The consumption of protists hosting algae by small crustaceans can indirectly increase the titer of chloroviruses. In this case, a large number of single-celled green algae are released, which become vulnerable to viral infection due to the disruption of endosymbiosis. However, an increase in chlorovirus titers in the presence of predatory small crustaceans was only shown for viruses of Chlorella variabilis strains that are symbionts of Paramecium bursaria [Chlorella heliozoae and it is a symbiont of the Heliozoae species, it cannot be asserted that the concentration of Chlorella heliozoae viruses increases in the presence of predators. However, the study of this issue is extremely important in the context of ATCV-1 as a possible infectious agent for human health.Chloroviruses, to which ATCV-1 belongs, are widely distributed in nature. They are found in freshwater bodies throughout the world, and have been isolated from freshwater sources in Europe, Asia, Australia, and North and South America. The titer of chloroviruses in freshwater samples depends on the time of year and the location of the reservoir, but usually ranges from 1 to 100 PFU / ml; however, much higher values have been recorded, up to 100,000 PFU/ml. Chloroviruses infect chlorella-like algae (nfection . The titbursaria . Since tA distinctive feature of ATCV-1 is one of the lowest decay rates of viral particles among all algal viruses. The highest decay rate is observed in the summer period of the year, which is characterized by a high intensity of UV-B radiation, high daytime temperatures, increased activity of extracellular bacterial nucleases and other enzymes, and the consumption of viruses by nanoflagellates. However, ATCV-1 and some other chloroviruses are able to repair DNA damage from exposure to UV radiation using virus-encoded UV-dependent DNA glycosylase–pyrimidine lyase, which is able to eliminate thymidine dimers as well as other viral repair enzymes. The lowest rate of virus decay is observed in winter. Moreover, it has been shown that ATCV-1 can tolerate complete freezing for extended periods of time, allowing it to survive in season-related freezing waters ,18.Chlorella heliozoae appears obvious at first glance—swimming in a reservoir and swallowing water through the choanae into the nasal cavity, as well as passage of water through the nostrils—there are no reliable data on transmission routes. Other possible modes of human infection are not ruled out. When consuming water contaminated with infected Chlorella heliozoae, it can assume the alimentary route of ATCV-1 infection. Such water with ATCV-1 can enter the nasal cavity through the choanae, and then the virus can enter the brain through the olfactory nerves. Infection via Chlorella-based nutritional supplements is another possible alimentary route of ATCV-1 infection. Chlorella is commercially produced and distributed worldwide as a nutritional supplement and contains larger amounts of vitamins, including D, B9, and B12 [Chlorella sp. are used, for example, in the EU, where traditionally there are three species: Chlorella pyrenoidosa, Chlorella vulgaris, and Chlorella luteoviridis. But as Champenois et al.’s analysis showed, under real production conditions, control is not carried out properly, and Chlorella species are incorrectly identified [Chlorella can also become added into nutritional supplements, which is a potential threat of ATCV-1 infection both in the alimentary route and by the accidental inhalation of a dry suspension of Chlorella from a nutritional supplement when opening a food product package.Although the route of ATCV-1 transmission through contact with water containing infected algae  and B12 . For nutentified . This mePhycodnaviridae family in the cervico-vaginal secretion of women and their association with gynecological diseases [The discovery of the ATCV-1 virus as part of the human oropharyngeal virome prompts a new look at viruses from freshwater and other natural sources. There have been no previous studies describing the similar effect of viruses that infect algae on the human body, except one study that describes the isolation of viruses of the diseases . Topicaldiseases . The sci


The primary transmission route for foot-and-mouth disease (FMD), a contagious viral disease of cloven-hoofed animals, is by direct contact with infected animals. Yet indirect methods of transmission, such as via the airborne route, have been shown to play an important role in the spread of the disease. Airborne transmission of FMD is referred to as a low probability- high consequence event as a specific set of factors need to coincide to facilitate airborne spread. When conditions are favourable, airborne virus may spread rapidly and cause disease beyond the imposed quarantine zones, thus complicating control measures. Therefore, it is important to understand the nature of foot-and-mouth disease virus (FMDV) within aerosols; how aerosols are generated, viral load, how far aerosols could travel and survive under different conditions. Various studies have investigated emissions from infected animals under laboratory conditions, while others have incorporated experimental data in mathematical models to predict and trace outbreaks of FMD. However, much of the existing literature focussing on FMDV in aerosols describe work which was undertaken over 40 years ago. The aim of this review is to revisit existing knowledge and investigate how modern instrumentation and modelling approaches can improve our understanding of airborne transmission of FMD. Picornaviridae, genus Aphthovirus) causes a highly infectious and contagious disease of wild and domesticated cloven-hoofed animals, with outbreaks causing considerable economic consequences for the livestock industry worldwide. The disease is endemic in parts of Africa, Asia, the Middle East and South America, and sporadically causes outbreaks in previously free countries and regions [Foot-and-mouth disease virus (FMDV)  ,14. By cEmitted infectious aerosols can be spread via atmospheric transport causing long distance transmission events which pose a serious threat to the control of FMD outbreaks ,10,11. AAirborne transmission is generally assigned as the route of transmission where no other route is thought possible, for example if there are no animal movements from infected farms or shared equipment or personnel. Therefore, meteorological and epidemiological investigations are undertaken to explore the likelihood of airborne transmission, for example do the general wind directions match the sequence of disease spread among the infected premises. These investigations have indicated that airborne transmission was the most probable cause of several historic FMD outbreaks. For example, FMD infections in cattle occurring on farms in Jersey in 1974 and 1981 and the Isle of Wight in 1981, are thought to have originated from outbreaks in Brittany, France . Airborn50 is required to initiate an infection in susceptible ruminants [50 are required to cause an infection [The difference between species in susceptibility and the quantity of virus emitted is well documented in the available literature. Previous studies have shown as little as 10 TCIDuminants . Cattle uminants ,22. By cnfection .Generally, research which aims to assess the susceptibility of different species to airborne virus describe an indirect transmission experimental design. This consists of placing a needle-inoculated animal in a cabinet and exposing naïve animals to the excreted aerosols via a fitted mask which is connected to the cabinet through an exposure tunnel ,24,25. TStudies which aim to measure the efficiency of aerosols as a route of infection also describe an experimental set up designed to mimic a natural route of infection . These s8.6 TCID50 and 107.6 TCID50 in pigs [6.4 TCID50), O UKG 2001 (106.1 TCID50) and South Korea 2000 (105.8 TCID50) [Although swine have been shown to be less susceptible to airborne FMDV infection, infected swine are an important source of aerosolised FMDV as they are capable of excreting 100 to 1000 times more virus than infected sheep or cattle ,22,23. I in pigs ,32. By c TCID50) . However TCID50) .10 TCID50 higher than cattle, with the development of generalised lesions in all pigs and cattle, but not for all the sheep, despite comparable virus titres being emitted from cattle and sheep. Alexandersen and Donaldson [The studies used to measure emissions often differ from one another in regard to the experimental design and as such are not always directly comparable . The mosonaldson  used a s5 TCID50) and low (103 TCID50) doses of virus (C Noville and O UKG 2001). Emissions increased exponentially and then fell after three dpi and were undetectable after day 4 in the high dose group and day 5 in the low dose group [1 TCID50 and 102 TCID50 at one day post infection for cattle and pigs respectively. The titre decreased after one day in the breath of the cattle but remained consistent until day four in the breath of the pigs.Together with quantifying emissions from infected animals it is also important to understand the daily emission profiles generated by infected animals to improve estimations of the risk of airborne transmission events. Donaldson  reportedse group . Alexandse group  quantifi1 BFS 1860) had a better survival rate at relative humidity levels above 50% when using the same methodology. Barlow [1 BFS 1860 strain was more stable in aerosols when suspended in bovine saliva than in cell culture media, particularly when the relative humidity level was above 50%. More recently, Brown et al. [As well as the amount of virus emitted from infected animals, the stability of such strains in aerosolised FMDV is an important factor in assessing the risk of airborne spread. The stability of virus strains under experimental conditions has previously been investigated using a Collison nebuliser to generate aerosols into a Goldberg drum with the sampling of aerosols using an all-glass Porton impinger sampler . Using t. Barlow  also sho. Barlow ,49. Barl. Barlow  demonstrn et al.  showed r1.0–104.6 log10 PFU. In an experimental setting FMDV has also been detected in aerosols in rooms which had previously been contaminated by infected cattle [In addition to virus aerosols from the breath of infected animals, aerosols can also be generated from other sources such as skin and fomites  which can be re-aerosolised through daily husbandry practices . Gailiund cattle  showed t2 were required for successful airborne transmission and regions such as the Middle East, even though the scale of pig farming is much smaller, were still at risk of airborne spread. Klausner et al. [The risk of airborne transmission will vary between geographical areas depending on factors such as species present, local climate, animal density and management systems. In many FMD-free countries the climate is generally more temperate with conditions reaching the optimal levels of humidity, temperature and wind speed required to facilitate airborne transmission over long distances . In addir et al.  projecteMany studies have investigated the risk of airborne transmission of FMD into free areas using modelling approaches and as such have identified risk factors specific to airborne transmission. Garner et al.  investigBy comparison, the risk of airborne transmission of FMD in hot climates is relatively understudied, for example in areas of Africa and Asia where the disease is endemic. Instead, modelling has been more commonly used to assess the risk of disease incursions into free countries. There are publications which have modelled airborne transmission of FMD in hot climates but they have used temperate climate data which is not representative of the study area . It is lMany different types of instrumentation have been used to measure FMDV in aerosols. The advantages and disadvantages of the samplers most commonly used for detection of FMDV are outlined in To date, there has not been a sampler specifically designed for use with FMDV and most samplers used in FMDV studies were developed in the 1950s and 1960s. Many of the samplers were designed originally for the field of air quality and although samplers have since been developed for use with bioaerosols few have been tested with FMDV. Ideally, an air sampler suitable for collecting FMDV aerosols would be robust, lightweight, and compact for transportation. The sampler should also be easy to operate and to clean, without damaging sensitive parts by repeat disinfection. It is also preferable when sampling in close proximity to animals that the sampler is quiet when in operation. For use in field work, an aerosol sampler needs to be capable of operating and being stored in hot climates and have a long battery life for sampling in remote locations. Often the samplers used in the original FMDV studies were made of glass , making them easy to clean and disinfect but at the expense of robustness and portability ,37,41. SA large range of different types of samplers and their operation have been reported in the literature, but the most popular for use in FMDV studies are cyclones and liquid impingers. These samplers have the benefit of an increased chance of virus survival which is facilitated by elution into liquid . Doel etMore recently, there has been more options available for samplers specifically developed for bioaerosol research and diagnostic use, with a move towards portability, robustness and ease of use than previous samplers. The Coriolis micro sampler has recently been successfully employed to detect FMDV when in close proximity to infected animals both under laboratory conditions and in field settings ,7. RecenSimulating the airborne spread of FMD can be accomplished by using an atmospheric dispersion model. If implemented alongside other surveillance tools and methods, dispersion calculations can be used tactically to aid the decision-making processes directly following an outbreak ,66,67. I(i)Meteorological conditions: wind direction and speed (which will indicate where the plume will travel and how fast) . Data in(ii)Some models will be able to accommodate the impact of complex terrain and the presence of orographic features  at the source premise(s), pathways, and at the receptor sites, as these features may affect the general flow patterns impacting the virus plume . More ad(iii)Virus emission rates: the concentration of virus being emitted into the environment and the duration of virus shedding . Virus eEssential elements for the operation of a dispersion model are input data streams which can be collected over a suitable period of time from either direct measurements or inferences from empirical formula. More specifically, the input data needed include information on:Although dispersion models can be very useful, much will depend on the quality of the input data and the sophistication of the inherent science incorporated in the model. Some models will describe the general atmospheric motion well and others not so well. Gloster et al.  reportedMost of the previous studies centred on FMDV in aerosols have focused on quantifying emissions from infected animals and the survival of virus in aerosols. However, they have yet to fully characterise and quantify virus emissions from infected animals and contaminated environments, which provides a rich area for further study. Dust particles are generated constantly at varying levels within livestock farms and certain procedures  may lead to the resuspension of dust containing virus from surfaces. Aerosols from infected farms may carry varying levels of virus which will depend on the characteristics (particle size and composition) of the dust particles generated within a farm. A clearer understanding of how these processes generate aerosols would be useful to provide information on the distance of detection, as well as providing better predictions of farm-level emissions and, hence, the risk of airborne spread. Furthermore, it would be useful for modelling approaches to ascertain if changes in the prevailing weather (for example temperature and humidity) influence particle size, or if there are differences in emission profiles between healthy and infected animals. Therefore, in the future research instruments such as particle size spectrometers or wireless-sensor networks could be utilised to characterise the particle size of aerosolised dust from different farming systems. The application of such equipment could also be extended to the study of other livestock diseases that are prevalent and have large impact on farming economics, such as Porcine Reproductive and Respiratory Syndrome virus (PRRSV) or Avian and Swine influenza (SwIV and AIV).The survival of FMDV in aerosols and the difference in decay rates between serotypes and strains of FMDV has been investigated, but most of these studies were conducted in the 1980s using strains which are no longer circulating. There is a gap in knowledge regarding the survival rate of current, circulating strains and the trade–off, if any, between virulence and survival. Laboratory experiments to investigate the survival of FMDV strains when exposed to different environmental conditions, such as varying levels of humidity and temperature, could help bridge this gap in knowledge. Several approaches exist to suspend infectious viral pathogens in aerosols and expose them to different environmental conditions, such as using a Goldberg drum to hold aerosols in suspension, the microthreads technique designed to capture and suspend aerosols on spider webs and the capture and levitation of single or multiple bioaerosol droplets using the controlled electrodynamic levitation and extraction of bioaerosol onto a substrate (CELEBS) technique ,77,78,79Another important area that is relatively understudied in the field of FMD research is the use of air sampling as a surveillance tool in FMD control programmes. Colenutt et al.  showed tTransmission of FMDV via aerosols occurs less frequently than direct contact transmission. However, the importance of this indirect route of transmission has been demonstrated not only through experimental studies which aim to quantify emissions from infected animals but also in real-life outbreak situations. Both epidemiological studies and mathematical modeling have provided strong evidence that spread of disease over short and long distance was due to FMDV transported in aerosols. There is a large body of research on emissions from animals and how this varies over time and depends on host species and virus strain. This data has been useful in improving our understanding of the behaviour of FMDV in aerosols and the transmission risk they pose. However, the parameters often differ from study to study which can make it difficult to draw firm conclusions from the data and the data does not encompass current circulating strains of FMDV. Most of the published literature describes outdated methodology with samplers which are no longer available, while those currently available have not been designed for working with pathogens in high containment facilities or in rural, field locations. Further information regarding the survival of different FMDV strains and emission data from farms housing susceptible animals is needed to provide better and more up-to-date input data into mathematical models to make more accurate predictions of the risk of airborne spread.


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AS grade was confirmed to be a major determinant of LVET, irrespective of forward flow  and LVEF (above and below 50%). A regression equation was derived to index LVET (LVETI) to HR and SVI. By using this formula, LVETI detected severe AS more accurately  than LVET alone . Similar results were observed in patients with abnormal flow status. As an exploratory finding, we observed that the highest tertile of LVETI was associated with a higher rate of aortic valve interventions during follow-up. Conclusions: LVETI correlates with AS severity better than uncorrected LVET, independently from hemodynamic conditions, and may help to discriminate severe AS. This finding needs confirmation in larger prospective multicenter studies.Aims: The assessment of aortic stenosis (AS) severity is still challenging, especially in abnormal hemodynamic conditions. Left ventricular ejection time (LVET) has been historically related to AS severity, but it also depends on heart rate (HR) and systolic function. Our aim was to verify if correcting LVET  by its determinants is helpful for the assessment of AS severity, irrespective of hemodynamic conditions. Methods and results: We retrospectively studied 152 patients with AS and 378 patients with heart failure and no-AS. At multivariate analysis, LVET (assessed with pulsed-wave Doppler) showed a strong correlation with stroke volume index (SVI) (Beta 0.354;  Thus, oLV ejection time (LVET) is a potential measure of SA severity. It draws inspiration from the semeiological analysis of the arterial systolic pulse in patients with AS, characterized by prolongation of the upstroke phase with decreased peak (pulsus parvus et tardus) in association with an overall pulse prolongation. With the spreading of Doppler-echocardiography, LVET was measured on a transaortic CW Doppler as the time interval from AV opening to AV closure , althougLVET is known to be prolonged in AS and it is included in the Gorlin formula for AVA calculation . HoweverThe primary objective of this study was to verify the impact of LVETI on AS grading, even in different forward flow conditions. Then, as secondary and exploratory objectives, we sought to verify whether LVETI is associated with surgical or percutaneous aortic valve intervention at follow-up. Such information could be helpful to set up a large-scale validation study of LVETI as a predictor of the severity of AS.Echocardiograms from 233 consecutive adult patients with new or confirmed diagnosis of moderate-to-severe valvular AS (group A) and from 459 patients with heart failure (HF) without AS (group B) were retrospectively examined to provide an estimation of LVET values in patients with normal and reduced flow status, with and without AS.The time range of the study was set from February 2016 to September 2019. Exclusion criteria were: presence of mechanical or biological aortic prosthetic valve (52 patients in group A and 39 in group B), significant intraventricular peak pressure gradient ≥ 30 mmHg (2 patients in group A and 9 in group B), unicuspid valve (1 patient in group A), and poor image quality. Finally, group A included 152 patients and group B 378 patients. Demographic, clinical, laboratory and follow-up data were also collected from in-hospital records.Doppler-echocardiographic examinations were performed with a GE Vivid E9, E80 or S6  or Philips EPIQ 7c  ultrasound scanner. Echocardiographic images were stored in a digital format. All measurements were performed by 2 experienced investigators using the ComPACS software Rev. 10.10.20  according with the current European Association of Cardiovascular Imaging (EACVI)/American Society of Echocardiography (ASE) guidelines [2 and MG or Vmax < 40 mmHg and <4 m/s, respectively. In discordant cases in which an AVA < 1 cm2 and MG < 40 mmHg or Vmax < 4 m/s were found along with a low flow (LF) status, pseudosevere AS was differentiated from true LF low-gradient (LG) SAS by dobutamine stress echocardiography, cardiac catheterization (to obtain AVA) or a semi-quantitative assessment of calcium score (eCS) ≥3 [AS severity was defined according with current recommendations . Moderat(eCS) ≥3 , and re-(eCS) ≥3 .2) and used to define a LF status when ≤35 mL/m2. The flow rate (FR) (ml/s) was calculated by dividing the SV by the LVET and utilized to define a slow flow (SF) status when ≤210 mL/s [The LV stroke distance  was calculated as the TVI of the LVOT flow velocity recorded in the apical 5-chamber or long-axis view . The LVE210 mL/s .Patients with and without AS were classified according to their normal or abnormal flow status. The normal flow (NF) status was defined as the combination of normal SVI and FR, whereas the abnormal flow status was defined as the presence of LF or SF or both. Therefore, abnormal flow patients were those with LF but normal FR, SF but normal SVI, or concomitant LF and SF.Normal distribution was tested with the Kolmogorov–Smirnov test. Continuous variables were expressed as mean and standard deviation or median values with 25th and 75th percentiles if normally or non-normally distributed, respectively. Categorical variables were reported as counts and percentages. For continuous normal variables, Student’s t-test and ANOVA were used for comparisons between two or more than two unpaired groups, respectively. For continuous non-normal variables, the Mann–Whitney U test and the Kruskal–Wallis test were used for comparisons between two or more than two unpaired groups, respectively. Categorical variables were compared by the chi-square test. Pearson correlation was used for normally distributed variables, whereas Spearman correlation was used if at least one variable had nonparametric distribution.2) and/or FR (above and below 210 mL/s). LVETI was derived from linear regression analysis. LVET and LVETI mean values relative to different AS subgroups were compared. The receiver operating characteristic (ROC) curves for the detection of SAS by LVET and LVETI were calculated and compared with a z-test.The correlations between LVET, flow measures (SVI and FR), HR, AS grade , LVEF, SBP and mitral regurgitation (MR) were studied by univariate linear regression analysis. The covariates statistically correlated with LVET were included in the multivariate stepwise linear regression model to find the independent determinants of LVET. In the multivariate analysis, R-square for each step and changes in R-square were analyzed. Due to strong collinearity between SVI and FR (R > 0.5), 2 different multivariate models were performed, each one including either SVI or FR. Subgroups of interest for repeated analysis were identified by LVEF (above and below 50%), SVI   and MedCalc (version 11.2.1.0) . Differences were considered statistically significant at n = 23), dobutamine stress echocardiography (n = 3), and calcium assessment (n = 4). MAS was present in 73 patients, among which 11 patients had pseudosevere AS correctly diagnosed using cardiac catheterization (n = 5), dobutamine stress echocardiography (n = 1), and calcium score (n = 4). Patients with AS were older, with higher LVEF, SVI and LVET but similar FR to patients without AS , AS grade , LVEF  and FR  were directly associated with LVET, whereas HR  and MR  were inversely associated. Since SVI and FR were correlated in overall patients and subgroups , HR , AS grade  and LVEF  were independently associated with LVET. On the multivariate analysis including FR, only HR, AS grade and LVEF were associated with LVET . The medium and the highest tertiles of LVETI were associated with AV intervention at follow-up  compared to the lowest tertile (p < 0.001) (After a median follow-up of 2.6 years (interquartile range: 2.0–3.2 years), there were 72 AV interventions . The median time from echocardiography to AV intervention was 55 days (interquartile range: 15–173 days). Dividing the entire patients’ cohort in LVETI tertiles , there were 5 AV interventions in the lowest tertile, 18 in the medium tertile, and 49 in the highest tertile at follow-up  SVI, HR and AS grade are independent determinants of LVET regardless of forward flow conditions; (2) LVETI derived by indexing LVET for SV and HR discriminates AS grades better than uncorrected LVET, also in different hemodynamic conditions, i.e., in AS patients with normal and abnormal flow status; and (3) the highest tertile of LVETI is associated with the need for AV intervention during follow-up, regardless of flow status.As recognized by the current echocardiographic guidelines , AVA meaFrom a pathophysiological point of view, the duration of LVET is influenced by various factors. The shortening of LVET may be determined by heart failure, diminished preload, positive inotropic agents, mitral stenosis and regurgitation ,14. SpecThe relationship between LVET and AVA in AS is not linear. This is evidenced by the Gorlin and Gorlin equation for the calculation of AVA at cardiac catheterization :AVA=CO/. Even in the abnormal flow patient subgroup, the longest LVETI tertile showed a strong association with AV interventions at follow-up . Since t(1) This is a retrospective investigation. However, because our digital platform allows comprehensive storage of echocardiographic examinations, all necessary images were available for the required measurements;(2) LVET indexation requires an estimate of SV and LVOT diameter, which can be measured only in the presence of a parasternal long-axis view with sufficient image quality. However, if the LVOT diameter cannot be obtained, other methods to estimate SV may be considered ;(3) The LVETI formula derived from the entire patient cohort was applied also to the abnormal flow subgroups. We recognize that, in these subgroups, SVI and HR may account differently with respect to overall patients , and thu(4) The use of AV intervention as an outcome measure for SAS is limited because some SAS patients may not have undergone intervention, for example due to comorbidities. Moreover, it was the only outcome measure utilized in this study. We recognize that dedicated, prospective outcome studies are needed, possibly based on harder end-points ;2 in SF-LG AS (FR ≤ 210 mL/s) [(5) Namasivayam et al. showed the lack of prognostic value of AVA ≤ 1 cm10 mL/s) . Thus, t(6) In our data, LVET seemed not to be conditioned by SBP at admission  but this(7) Finally, this was a single-center investigation with a limited number of patients with SAS. A large number of these patients could be included in a multicenter study.In this study, LVET was corrected by its determinants to derive LVETI, a new index of AS severity, which may be helpful in recognizing SAS, especially in controversial cases. Future, large-scale validation studies of LVETI as a predictor of AS severity are needed.