Caenorhabditis hosts and Orsay virus, a positive-sense single-stranded RNA virus that naturally infects C. elegans. We assayed species across the Caenorhabditis tree and found Orsay virus susceptibility in 21 of 84 wild strains belonging to 14 of 44 species. Confirming patterns documented in other systems, we detected effects of host phylogeny on susceptibility. We then tested whether susceptible strains were capable of transmitting Orsay virus by transplanting exposed hosts and determining whether they transmitted infection to conspecifics during serial passage. We found no evidence of transmission in 10 strains , evidence of low-level transmission in 5 strains (virus lost between passage 1 and 5 in at least one replicate) and evidence of sustained transmission in 6 strains  in at least one replicate. Transmission was strongly associated with viral amplification in exposed populations. Variation in Orsay virus susceptibility and transmission among Caenorhabditis strains suggests that the system could be powerful for studying spillover and emergence.A lack of tractable experimental systems in which to test hypotheses about the ecological and evolutionary drivers of disease spillover and emergence has limited our understanding of these processes. Here we introduce a promising system:  For example, SARS-CoV-2 spilled over into human populations  and becaSpillover requires that pathogens have the opportunity and the ability to exploit a new host; emergence requires that this opportunity and ability persist through time ,7. OpporEcological factors  can promote or hinder spillover by modulating host exposure risk or host susceptibility ,7. LikewMus musculus [Drosophila melanogaster [Daphnia species [Arabadopsis thaliana [Caenorhabditis elegans [Experimental model systems have been essential for testing hypotheses about infectious disease biology –23. Indemusculus , Drosophnogaster , Daphnia species , Arabadothaliana  and Caen elegans . HoweverCaenorhabditis nematode species are appealing model host candidates. Indeed, C. elegans and various bacterial and microsporidian parasites are staples of evolutionary disease ecology [C. elegans. That changed with the discovery of Orsay virus [ ecology ,28. Specay virus .C. elegans, is a bipartite, positive-sense, single-stranded RNA (+ssRNA) virus that transmits readily in laboratory C. elegans populations through the fecal-oral route [Caenorhabditis hosts and Orsay virus as a system for studying spillover and emergence, it is necessary to know the extent to which the virus can infect and transmit in non-elegans Caenorhabditis species. So far, such exploration has been limited to one other species, C. briggsae, which was determined to be refractory to infection [C. briggsae has been found to be susceptible to three related viruses [Orsay virus, a natural gut pathogen of al route . This vial route  and typial route . Moreoveal route  that cannfection . Notably viruses ,32–34.Caenorhabditis–Orsay virus system for studies of disease spillover and emergence, we first test a suite of Caenorhabditis species for susceptibility to Orsay virus, and then we test the extent to which susceptible host species can transmit the virus. We establish lower bounds for both susceptibility and transmission ability, and we test for effects of host phylogeny on these traits. Although host ranges of various pathogens have been studied by infection assays (e.g. [To explore the suitability of the ys e.g. –38) or b or bCaenys e.g. ,39), the, theCaen. 2 (a)Caenorhabditis species to Orsay virus by measuring virus RNA in virus-exposed host populations using quantitative PCR (qPCR). We obtained 84 wild isolate strains belonging to 44 Caenorhabditis species (one to three strains per species) from the Caenorhabditis Genetics Center (CGC) and from Marie-Anne Félix (IBENS). We tested each strain for Orsay virus susceptibility using eight experimental blocks  in 60 mm diameter plates with a lawn of bacterial food (lawns were seeded with 200 µl of Escherichia coli strain OP50 in Luria-Bertani (LB) broth and allowed to grow at room temperature for approximately 24 h [g for 1 min between each wash. After the five washes, we lysed the nematodes by transferring the nematode pellet along with 500 µl of water to 2 ml round-bottom snap cap tubes, adding approximately 100 µl of 0.5 mm silica beads and shaking in a TissueLyser II (Qiagen) for 2 min at a frequency of 30 shakes per second. We then removed debris with two centrifugation steps of 17 000 × g for 5 min, each time keeping the supernatant and discarding the pellet. Samples were stored at −80°C.We assayed the susceptibility of ely 24 h ). We expely 24 h , onto thely 24 h . We mainWe used qPCR to measure viral RNA in these samples. Primers and probe were Forward: GTG GCT GTG CAT GAG TGA ATT T, Reverse: CGA TTT GCA GTG GCT TGC T, Probe: 6-FAM-ACT TGC TCA GTG GTC C-MGB. We performed 10 µl reactions composed of 1.12X qScript XLT One-Step RT-qPCR ToughMix (Quantabio), 200 nM each of forward and reverse primers and probe, and 2 µl of sample. Reaction conditions were 50°C (10 min), 95°C (1 min), followed by 40 cycles of 95°C (3 s), 60°C (30 s). Assays were run on a 7500 Fast Real-Time qPCR System . Cycle threshold (Ct) values were determined using the auto-baseline and auto-threshold functions of the 7500 Fast Real-Time software .C. elegans laboratory strain N2 was exposed to water instead of virus. Controls 2 and 3 were positive controls where C. elegans strains known to have moderate (N2) and high (JU1580) susceptibility were exposed  = 15.7, s.d.(Ct) = 2.0; control 3, strain JU1580: mean(Ct) = 12.7, s.d.(Ct) = 2.2). Benchmark 4 was used to determine a Ct threshold for overt infection (i.e. susceptibility); we added virus to OP50-seeded NGM plates without nematodes and treated them identically to our plates with exposed nematodes during extractions. Therefore, these plates were used to quantify the amount of exposure virus that remains after the washing and extraction procedure (benchmark 4: mean(Ct) = 38.4, s.d.(Ct) = 2.6). Benchmark 5 was used to quantify the maximum amount of virus that could be present without replication and thus to generate a highly conservative Ct threshold for infection; it was determined by diluting 3 µl of exposure virus into 497 µl water, which corresponds to the final volume of our extractions. Samples with more virus than benchmark 5 therefore give unequivocal evidence of virus amplification (benchmark 5: mean(Ct) = 22.0, s.d.(Ct) = 0.6). In practice, benchmark 5 is overly conservative as a threshold for determining infection because virus is expected to be washed away during the wash steps, extractions are likely to be less than 100% efficient, and the virus may degrade between exposure and extraction. We therefore used benchmark 4 and the within-strain standard deviation in Ct among plates to set a threshold for determining infection status based on Ct. We calculated variance in the Cts for each strain (with undetectable virus assigned a Ct of 40), found the mean variance and took the square root; the result (sqrt(var(Ct)) = 4.1) is equivalent to the standard deviation in Ct values within a strain. We set a threshold of one standard deviation more virus than the maximum amount of virus detected in benchmark 4 plates (Ct = 33.6), yielding a threshold of Ct < 29.5. Strains were considered susceptible if at least one replicate population had more virus than this threshold. Note that had we used benchmark 5 rather than benchmark 4 to determine infection status, only 4 of 21 strains would have changed susceptibility designation . To confirm that virus was replicating within novel hosts deemed to be susceptible, we measured virus levels over time in three of our susceptible, novel host strains .Each experimental block also contained five sets of controls and benchmarks . Control (b)C. elegans laboratory strain N2 exposed to 3 µl of virus filtrate and three replicate negative control populations of strain N2 exposed to 3 µl of water. When populations were recently starved, 20 adult nematodes  were chosen at random and passaged to virus-free plates with fresh food (E. coli strain OP50 lawns prepared as above). Remaining animals were washed from the starved plates, virus was extracted and viral RNA quantified via qPCR as above . We passaged each replicate line five times, or until there was no detectable viral RNA by qPCR. Controls were passaged five times regardless of virus detection.We conducted transmission assays for all strains where at least one replicate population was determined to be infected in our susceptibility assay. First, three replicate populations were initiated as above and exposed to 3 µl of virus filtrate. At the same time, we initiated three replicate positive control populations of We assigned each passage line a transmission score of 0, 1, 2 or 3 based on detection of viral RNA through the passages. A value of 0 was assigned when viral RNA was not detected in the exposure population; a value of 1 was assigned when viral RNA was detected in the exposure population but not in the first passage population; a value of 2 was assigned when viral RNA was detected in the first passage population but became undetectable on or before the fifth passage population and a value of 3 was assigned when viral RNA was still detectable in the fifth passage population. (c)Caenorhabditis [Diploscapter pachys as the outgroup and constrained the tree to be ultrametric  using the ‘chronos’ function in the ‘ape’ package [We quantified phylogenetic relationships among nematode species using data from the most recent published phylogeny of habditis . We root package . We sele package .We then fit suites of Bayesian phylogenetic mixed effects models to the susceptibility and transmission data using the ‘MCMCglmm’ package ,45,46 inC. elegans  and in ‘ape’ ,49). TheThree model components were included or excluded to generate our suite of models for the transmission data . Our mos8) and parameter expanded priors for random effects that result in scaled multi-variate F distributions with V = 1, nu = 1, alpha.mu = 0, alpha.V = 1000 [We used the MCMCglmm default priors for fixed effects  for allribed in . We calcribed in . The DIC. 3 (a)Caenorhabditis strains of the 84 experimental strains tested  and C. elegans was significant . The importance of phylogenetic distance from C. elegans was also supported by the observation that susceptible strains were less well distributed across the phylogenetic tree than random . We also used R2 values from the best model and DIC weights calculated from the suite of models to further explore the importance of phylogenetic effects. Phylogenetic distance from C. elegans explained 89.0%  of the variance in susceptibility (figure 2b) and had a DIC weight of 0.773. The random effect of pairwise phylogenetic distance explained 5.15%  of the variance in susceptibility (figure 2b) and had a DIC weight of 0.710. Importantly, both phylogenetic effects together explained 94.1%  of the variance (figure 2b), and models that included at least one of these phylogenetic effects had a weight of 0.939. Further, the model lacking either phylogenetic effect had a low DIC weight of 0.061, demonstrating additional support for the importance of phylogenetic effects [figure 2b); we were not able to compute a DIC weight for this component since it was included in all the susceptibility models.Our statistical analysis uncovered the importance of host phylogeny in explaining differences in susceptibility. Our best model included both phylogenetic effects . In this (b)C. elegans strains as well as positive control replicates (C. elegans strain N2) maintained high levels of virus through five passages  for up to 12 passages, which yielded similar results to those in The primary exposure populations (passage 0) in our transmission assay were treated nearly identically to populations in our susceptibility assay. As an internal control, we thus note high concordance between Ct measures in both assays (correlation coefficient = 0.85). In a separate experiment, we completed passages for additional replicates of two susceptible strains  and a random effect of phylogenetic distance between pairwise sets of species. Notably, the fixed effects were moderately correlated (correlation coefficient = 0.477).As with the susceptibility data, we again identified factors associated with differences in transmission through model analysis. Our best model included a significant effect of viral amplification (Ct) in primary exposure populations , a non-significant effect of phylogenetic distance from b,c) of the variation in transmission ability and had a DIC weight of 0.862. Phylogenetic distance from C. elegans explained 46.6%  of the variation in transmission ability and had a DIC weight of 0.558, and pairwise phylogenetic distance between sets of species explained 4.3%  of the variation in transmission and had a DIC weight of 0.546. Combined, the phylogenetic effects explained 50.9%  of the variation in transmission ability, and models including at least one of the phylogenetic effects had a weight of 0.792. The R2 values and DIC weights indicate strong support for an effect of viral amplification in primary exposure populations and at least some support for each phylogenetic effect in explaining transmission ability despite the non-significant effect of phylogenetic distance from C. elegans in the best model. Interestingly, in the second-best model . Little of the variation in transmission ability was explained by species  or strain .Viral amplification in primary exposure populations explained 44.8%  out of 84 tested belonging to 44 species. When susceptible strains were assayed for transmission ability, 10 strains were dead-end hosts in all replicates and 6 strains  showed virus persistence for five passages in at least one replicate. The remaining five susceptible strains showed stuttering chains of transmission in at least one replicate. Our findings constitute lower bounds for the number of species and strains that are susceptible to Orsay virus and can transmit it; increased sampling of strains or increased replication could very well have identified more instances of susceptibility or transmission especially since these phenomena may be the result of stochastic ecological and evolutionary processes. Furthermore, we note that susceptibility and transmission findings are likely dependent on experimental conditions as we expect aspects of ecology such as dose and food quantity to impact spillover and emergence. Here, we found that susceptibility was associated with two phylogenetic effects: distance from C. elegans and phylogenetic distance between pairwise sets of species. Transmission ability was weakly associated with these phylogenetic effects according to analysis of DIC weights but strongly positively associated with viral amplification in primary exposure populations. Overall, we argue that the variation we observed among Caenorhabditis species and strains in susceptibility and transmission ability primes the Caenorhabditis–Orsay virus system to be valuable for experimental studies on the ecology and evolution of pathogen spillover and emergence.In our study examining the host range of Orsay virus, we determined that at least 13 C. elegans were more likely to be susceptible to infection, and closely related hosts had more similar susceptibilities regardless of their relationship to the native host. We expect that the importance of phylogenetic effects would only become more readily detectable if our unplaced Caenorhabditis species were placed on the phylogeny, since their lack of placement cost us statistical power. Importantly, we recovered an effect of phylogenetic distance from C. elegans even though few species are closely related to C. elegans .Caenorhabditis hosts. It is important to note, however, that the patterns we see with our susceptibility and transmission assays may not fully predict spillover and emergence patterns among Caenorhabditis hosts in the wild. Exposure risk is a key determinant of spillover and emergence [Caenorhabditis species in nature is unknown since we know little about the distributions of Caenorhabditis species and their viruses [C. elegans and C. briggsae, do have overlapping distributions [C. briggsae [C. briggsae and C. elegans [Here we have documented spillover and transmission of Orsay virus in mergence , but in  viruses . The twoibutions , but appibutions . Howeverbriggsae ,32–34 subriggsae  than C.  elegans .Caenorhabditis–Orsay virus system joins a small set of empirical systems suitable for studying spillover and emergence. Prior studies using other systems have yielded useful insights into these processes. For example, bacteria-phage systems have been used to show that the probability of virus emergence is highest when host populations contain intermediate combinations of native and novel hosts [Drosophila–virus systems have been used to show that viruses evolve in similar ways when passaged through closely related hosts [The el hosts , that pael hosts ,81 and tel hosts , to deteel hosts , and to el hosts ,81. Drosed hosts  and to sed hosts .Caenorhabditis–Orsay virus model can be uniquely useful for studying how ecology impacts spillover and emergence in animal systems since population characteristics like density, genetic variation and immunity can be readily manipulated and virus transmission occurs without intervention by a researcher. Caenorhabditis hosts have complex animal physiology, immune systems and behaviour, meaning that this system can be useful for revealing the importance of variation in these traits. In this study, we identified multiple susceptible spillover hosts that have variation in transmission ability. In the future, these hosts can be used not only to probe how ecology impacts spillover and emergence, but also to better understand how and why spillover and emergence patterns may differ across hosts.The


Monkeypox virus is a double-stranded DNA virus species that causes disease in humans and mammals. It is a zoonotic virus belongs the genus Orthopoxviral, the family of Poxviridae, associated with the smallpox virus in many aspects. The first human case of monkeypox was reported throughout the Democratic Republic of Congo in 1970. In April 2022, several cases were recorded in widespread regions of Africa, the Northern and western hemispheres. The current review spotlights taxonomic classification, clinical presentations during infection, and the pathogenicity of the monkeypox virus in humans. Furthermore, the current review also highlights different diagnostics used for virus detection. The zoonotic virus monkeypox virus (MPXV) was discovered in 1958 after an outbreak of a smallpox-like disease in macaques at a Danish research facility—hence the name monkeypox . The firMonkeypox virus has evolved into two separate clades: West Africa and Congo Basin . DiffereIn addition, monkeypox from Central Africa inhibits T-cell receptor-mediated activation, suppressing proinflammatory cytokine production within human cells from earlier infected monkeypox patients . The MPXOrthopoxvirus genus of the Poxviridae family includes numerous zoonotic infections, particularly monkeypox [Poxviridae family is divided into two taxonomic groups: Entomopoxvirinae and Chorodopoxvirinae. The subfamily classification depends upon whether the virus infects insects, as in Entemopovirinae, or vertebrates, as in Chorodopoxvirinae [The onkeypox . To replonkeypox . Poxviruonkeypox . The Poxxvirinae .chorodopoxviruses [Yatapoxvirus, Orthopoxvirus, Caprioxviru, Molluscipoxvirus, Suipoxvirus, Capripoxvirus, Avpoxvirus, and Parapoxvirus  infected by oral or pharyngeal (papular stomatitis) or skin contact (cellular dermatitis), typically with a moderate granulomatous reaction, causing a variety of clinical manifestations in animals.Poxviruses contain the human smallpox virus, declared eradicated in 1980 by the World Health Organization. Other poxviruses that can infect humans include the cowpox virus, smallpox virus, and Akhmeta virus, as well as viruses from different animal species that can cause a pandemic when combined with the monkeypox virus. The monkeypox virus is the most prevalent of these in humans. Bovine pox is another zoonotic disease with a large population in the animal kingdom, a clear relationship to livestock exposure in humans with variable prevalence and severity and a progression of several weeks. Paraboxvirus results in the formation of large nodular or vascular lesions. The species generates less immunity than Orthopoxviruses, increasing the likelihood of reinfection. Molluscum contagiosum is a common disease in humans, children, and adults caused by species of the genus Molluscipoxvirus, with specific human swellings and erythematous papules mimicking soft tumors, usually with few and variable numbers of immunizations. In the planting area, it is mainly distributed in roots, trunk, and limbs. Immunity is self-limited. The spreading of Molluscum contagiosum disease via direct contact with the genital lesions is facilitated by sexual contact and is also spread by Candida.Moreover, In contrast to other viruses, smallpox viruses have a much lower mutation ability than RNA viruses, such as influenza viruses. However, these viruses have extensive genetic ability, which explains their ability to attack and evade the virus and the immune system ,35.Human monkeypox outbreaks started in the USA in 2003. The first rats were imported from Ghana and sold as exotic pets, which started the trend. These rodents are believed to spread the infection to prairie dogs living in close areas that were also being sold as pets ,37.There are multiple routes of transmission for the monkeypox virus , all of In endemic areas of Africa with limited infrastructure and resources, exposure to the feces of infected animals can be a significant risk factor. Many people also choose to sleep outdoors, on the ground, or live close to or visit forests where infected animals are significantly more abundant . In thosPoxvirus mature structure  has a diThe pathogenicity and pathophysiology of the monkeypox virus begin with virus transmission, which starts with close contact of animal to human or human-to-human. Smallpox and monkeypox infect through the host’s respiratory or oropharyngeal mucosa. The virus enters through the inoculation site and begins replication in the respiratory and oropharyngeal mucosa. Viruses spread to the local lymph nodes in primary viremia. However, these viruses enter lymph nodes and organs via blood circulation in secondary viremia. This process represents the incubation period of 7 to 14 or 21 days .External virion proteins, cellular glycosaminoglycans on the target cell’s surface, and extracellular matrix components are most likely involved in monkeypox virion attachment. Poxviruses enter host cells via a low-pH endosomal pathway or direct fusion with the plasma membrane at neutral pH, releasing the viral core in the cytoplasm. A complex of 12 non-glycosylated viral membrane proteins is required for intracellular mature virions and enveloped extracellular virions to fuse with the cell . FollowiIn non-human primates, MPVX typically causes a short-lived rash. Plague and 1–4 mm epidermal papules that develop into pustules and crust over are the first clinical signs. A typical lesion has a red, necrotic, and depressed center surrounded by epidermal hyperplasia. These “smallpox pustules” can appear anywhere on the body, but the face and extremities are the most common. Most infected animals recover quickly; however, fatal cases occur, particularly in neonatal monkeys. Following vaccination, MPVX-caused illness was discovered in various rodents, including prairie dogs, dormice, and squirrels. Clinical symptoms in each of these cases  vary, buThe clinical features of traditional smallpox with a high mortality rate are classified as typical, altered, variola sine eruption, hemorrhage, and flat type . The hemEnzyme-linked immunosorbent assay (ELISA) can detect specific IgM and IgG antibodies in the serum of monkeypox-infected patients after 5 and 8 days of infection, respectively, for serological testing. A four-fold increase in serum antibodies can be used to diagnose monkeypox virus infection at both the acute and convalescent stages. Specificity is insufficient due to antigenic cross-reactions between MPXV and other poxviruses. Consequently, this method is inefficient in identifying monkeypox virions and is frequently employed in epidemiologic studies. Similarly, identification by an electronic microscope is not entirely reliable because virions cannot be distinguished morphologically .Real-time polymer chain reaction (RT-PCR) is a genetic method for detecting monkeypox. The conserved regions of extracellular envelope protein gene (B6R), DNA polymerase gene E9L, DNA-dependent RNA polymerase subunit 18 (RPO18) gene, and complement binding protein C3L, F3L, and N3R genes are usually selected as targets for PCR amplification. Furthermore, for MPXV DNA detection, recombinase polymerase amplification (RPA), loop-mediated isothermal amplification (LAMP), and restriction length fragment polymorphism (RFLP) have also been developed for detection purposes .The monkeypox virus (MPXV) produces many viral proteins that help it to hide from the body’s immune system . These c4+ and CD8+ T cells, thereby interfering with adaptive immune responses [Monkeypox infection results in the accumulation of less dsRNA than vaccinia virus and inhibits the phosphorylation of the pattern recognition immune receptor protein kinase R (PKR) and Eukaryotic Initiation Factor 2 alpha (eIF2), thereby inhibiting the activation of antiviral responses . It has esponses .According to data, the vaccines used for smallpox are the most effective against monkeypox and reduce the severity of clinical symptoms . There aJYNNEOSTM, made from modified vaccinia Ankara Bavarian Nordic strain, is a non-replicating and live attenuated Orthopoxviral and was approved by the FDA in September 2019; it is currently used to treat monkeypox and smallpox . Data in® also has a live vaccinia virus. The FDA approved it in August 2017 to replace the Orthopoxviral vaccine Dryvax, which was withdrawn by the manufacturer [® for non-variola Orthopoxviral infections for the duration of the pandemic [ACAM2000facturer . ACAM200pandemic .Neutralizing antibodies appear to be an essential component of the immunological mechanisms underlying cross-protection through vaccination against the vaccinia virus. Vaccinating monkeys with a human smallpox vaccine provides protection against monkeypox, which is consistent with the potential of the smallpox vaccine to provide cross-protection for humans against monkeypox ,18. SincOrthopoxvirus infection, cidofovir was used to treat resistant atopic dermatitis in a 28-month-old boy who had developed severe eczema after contact with his father. The boy survived with no long-term consequences [The FDA approved Cidofovir for the first time in 1996 to prevent cytomegalovirus eye diseases in patients with acquired immunodeficiency syndrome. Cidofovir has broad antiviral efficacy against many viruses, particularly herpes and adenovirus. Regarding its use in equences .Cytomegalovirus, Orthopoxvirus, and adenovirus infections [Bricidofovir was approved by the FDA for the first time in June 2021 to treat smallpox injections . It has fections ,69. Brinfections . Followifections . In May fections .The FDA recognized Tecovirimat in 2018 to cure the smallpox virus . The EurThe FDA approved vaccinia-resistant globulin intravenous in 2005 to treat the immunization difficulties caused by vaccinia infection . The vacFollowing the discovery of isolated cases in the western hemisphere, the monkeypox virus, previously restricted to African regions, poses a threat to humans worldwide. Because human-to-human transmission is common via respiratory globules and direct contact with an infected person’s mucocutaneous sores, social isolation and contact tracking are essential. MPX has been confirmed in adults in their twenties. This is due to smallpox vaccine pass-through immunity, which is lost in the elderly and the young population (aged < 45 years) not vaccinated in numerous countries, especially in Europe. This virus develops and replicates in the cytoplasm before entering the main viremia and infecting surrounding lymph nodes. Monkeypox infection can result in encephalitis, dehydration, respiratory distress, and bronchopneumonia. Corneal scarring is the first and most concerning issue, which can lead to vision loss. It is critical to provide the necessary maintenance treatment to reduce the risk of these diseases as much as possible. Supportive therapy, such as moist occlusive bandages, may be used in areas where the rash is concentrated. Organizations are focusing on understanding how these infections emerge intermittently across Europe and the Western hemisphere as new cases of monkeypox are confirmed worldwide. It is critical to investigate any potential treatments as well as understand the full spectrum of each monkeypox symptom as well as the long-term consequences of the virus and symptoms.


Globally, foodborne diseases result in a significant disease burden with low- and middle-income countries disproportionately affected. Estimates of healthcare costs related to foodborne disease can aid decision makers to take action to mitigate risks and prevent illness. However, only limited data on the African continent are available, especially related to more severe sequelae. We provide estimates of direct and indirect (non)-medical costs of patients with diarrhoea, Guillain-Barré syndrome (GBS), and invasive non-typhoidal salmonellosis (iNTS) in three healthcare facilities in Gondar, Ethiopia.Campylobacter spp., enterotoxigenic Escherichia coli, non-typhoidal Salmonella enterica) were analysed with ANOVA and chi squared tests. Contribution of healthcare facility, dehydration severity, sex, age and living area to transformed costs was identified with linear regression. Results are in 2020 USD per patient. To extrapolate to national level, 2017 national incidence estimates were used.We used healthcare data from patient records, interviews with family caregivers and 2020 healthcare resource unit costs. Descriptive statistical analysis was performed. For diarrhoea, differences in mean and median transformed costs between healthcare facilities and etiologies , 267.70 USD for GBS, and 47.79 USD for iNTS. Differences in costs between diarrhoea patients were mainly associated with healthcare facility. Most costs did not differ between etiologies. Total costs of a diarrhoea patient in the specialised hospital were 67 USD, or 8% of gross national income per capita. For direct medical plus transport costs of a GBS and iNTS patient in the specialised hospital, this was 33% and 8%, respectively. Of the 83.9 million USD estimated national non-typhoidal Direct medical costs per patient due to GBS and iNTS were 30 respectively five times those due to diarrhoea. Costs of a patient with diarrhoea, GBS or iNTS can be a substantial part of a household’s income. More severe sequalae can add substantially to cost-of-illness of foodborne hazards causing diarrheal disease.The online version contains supplementary material available at 10.1186/s12889-022-14539-1. Globally, foodborne disease (FBD) result in a significant public health impact, with low- and middle-income countries (LMICs), and especially in Africa, affected relatively more than high income countries . In 2010There are several studies available about the economic burden due to foodborne or diarrheal disease on the African continent. For The Gambia, Kenya, and Mali in 2011, direct medical costs of hospitalization due to diarrhoea due to any etiology in children up to 5 years were estimated between 0.70 and 2.20 USD per patient depending on the country, direct non-medical transport costs between 0.19 and 0.55 USD, and indirect non-medical costs due to lost income of caregivers between 1.55 and 4.99 USD . For Thehttps://www.ilri.org/research/projects/urban-food-markets-africa-incentivizing-food-safety-using-pull-push-approach): Campylobacter spp., enterotoxigenic Escherichia coli (ETEC) and non-typhoidal Salmonella enterica. To estimate the disease burden related to foodborne pathogens, the World Health Organization (WHO) Foodborne Disease Burden Epidemiology Reference Group (FERG) used a hazard-based approach based on health states [Campylobacter spp., we also included Guillain-Barré syndrome (GBS), an autoimmune disease that can be triggered by different infectious agents, including Campylobacter jejuni, when specific surface antigens mimic gangliosides in the host nervous system [Campylobacter infection [Salmonella enterica may also lead to invasive non-typhoidal salmonellosis (iNTS) with the highest incidence reported in Africa [This study aims to help fill this gap by gathering data about the illness costs related to sequalae associated with foodborne diarrheal disease agents selected by the project “Urban Food Markets in Africa – incentivizing food safety (Pull-Push Project)” in Ethiopia , about the illness history before coming to the healthcare facility , and about healthcare resources used at the facility related to direct medical costs that the patient was responsible for (co-)paying . Data for direct non-medical costs included ambulance service, because an ambulance is often used as a transport mode to bring a patient to the healthcare facility rather than a vehicle to provide emergency care. An ambulance is preferred, because most people do not own a car, public transport is often unsuitable to bring a patient to the hospital, and a taxi is often more expensive.Data for direct medical costs from family caregiver interviews included direct healthcare costs made prior to and during the visit to the hospital and data for indirect non-medical costs lost income. After a physician notified the data collector when a patient with diarrhoea was being treated, the data collector contacted the family caregiver while waiting during the treatment. Interviews were held in a private setting. Informed consent was obtained orally from all interviewed caregivers. The data collector noted the patient record number to later collect the date of discharge. Interviews were held in July 2020.The data collector gathered facility specific costs per unit of the healthcare resources in Ethiopian Birr (ETB) as used in the healthcare facilities in August 2020 , i.e., the majority of patients in our dataset, was performed for diarrhoea. For GBS and iNTS, data were only available from the specialised hospital. QQ-plot analysis showed costs were not normally distributed. Log10 transformation was used for total costs, stay costs, and drug costs. For diagnostic test costs and special services costs x0.5 and x0.75 were used, respectively, because several patients had no costs in these categories. ANOVA was used to compare mean transformed costs. Levene’s test was used to check for the homogeneity of the variance between groups. If variances were not equal, Tamhane’s T2 test was used as post-hoc test to compare the means between groups, otherwise a Fisher’s Least Significant Difference test was used. Medians of the transformed costs were compared using chi squared tests. A pairwise comparison with an adjusted p-value using the false discovery rate method was used as post-hoc test.Analysis of differences in mean and median of costs between type of healthcare facility and between etiology of interest in our study , age group , living area , and severity of dehydration . Therefore, linear regression was used to identify the contribution of each in addition to healthcare facility type to each category of transformed costs:The specialised hospital, severe dehydration, male, under 5 years, and urban living area were used as baseline in the regression analyses.All analyses were performed in IBM SPSS Statistics 25, except for the post-hoc median tests which were performed in R-package “rcompanion” .Campylobacter spp., ETEC and non-typhoidal Salmonella enterica in Ethiopia were estimated by multiplying estimated mean direct medical and (in)direct non-medical cost estimates per patient with diarrhoea, GBS or iNTS with 2017 national incidence estimates. Diarrhoea incidences were retrieved from Havelaar, et al. [Campylobacter spp. and non-typhoidal Salmonella enterica, respectively, which were extracted from the FERG database.National cost-of-illness due to FBD caused by , et al. . GBS andOf the 420 records of patients diagnosed with diarrhoea we collected, records of 406 patients were complete. Of these, roughly half were from the specialised hospital (205), and a quarter each from the private clinic (93) and the health centre (108)  and highest in the specialised hospital (1.5 days). Only in the specialised hospital, seven patients stayed more than three days. It should be noted that in our study admittance into the healthcare facility from two hours up to 24 h was recorded as stay of one day, because the fee was the same for all these patients. In all three healthcare facilities, most patients were treated in the paediatric ward or outpatient clinic.Supportive drugs and antibiotics were used in many patients in all three healthcare facilities, but the type differed. In the health centre, the only supportive drug used was oral rehydration solution (ORS), whereas in the private clinic glucose 40% and metoclopramide and in the specialised hospital Ringer’s solution were used in addition to ORS. Antibiotics were used for most patients in the health centre and private clinic, and for approximately half of the patients in the specialised hospital. In the health centre, the most used antibiotics were cotrimoxazole and metronidazole, in the private clinic ceftriaxone and ampicillin, and in the specialised hospital ceftriaxone and cotrimoxazole. Paracetamol was used more in the health centre than in the other two facilities. Both steroidal (dexamethasone) and non-steroidal (diclofenac) anti-inflammatory drugs were used in the private clinic, not in the other two facilities. Supportive drugs for the gastro-intestinal tract were used in the health centre and the private clinic. A few patients in the health centre and specialised hospital received anthelmintics.At least one stool test was used for more than 90% of the patients in all three healthcare facilities. At least one blood test was used for 70% of the patients in the private and specialised hospital, whereas in the health centre it was hardly used. A urinalysis was used for approximately 50% of the patients in the specialised hospital, but for less than 5% in the private clinic and the health centre. Blood culture was only used for 5% or less of the patients in all three facilities.Specialist consultation was used for 11% of patients in the private clinic, 21% of patients in the health centre, and 27% of patients in the specialised hospital. Intravenous fluids were provided to approximately 75% of patients in the health centre and private clinic, and to 88% in the specialised hospital. Special diet was only provided to 24% of patients in the health centre and to 16% of patients in the specialised hospital, but it was not provided in the private clinic.p < 0.001) than in the specialised hospital of 9.53 USD per patient or in the private clinic of 10.56 USD per patient. Median total direct medical costs were lowest in the health centre with 6.56 USD per patient (p < 0.001), followed by the specialised hospital with 9.16 USD per patient, and the private clinic with 10.02 USD per patient. This was caused by significant differences in special services costs, diagnostic test costs and stay costs between the healthcare facilities. Mean drug costs did not differ significantly between the healthcare facilities, but the median drug costs were significantly lower in the private clinic compared to the other two facilities (p = 0.001). In contrast, mean and median stay costs were higher in the private clinic compared to in the other two facilities (p < 0.001). Across all types of healthcare facilities, drug costs were highest, followed by special services costs, diagnostic testing costs and stay costs. The higher mean special services costs in the private clinic were caused by higher costs for intravenous fluids. Intravenous fluids made up approximately 80–90% of the total special services costs in all three healthcare facilities. Patients in the private clinic did not use a special diet. Distributions of the patient costs for special diet, specialist consultation, and intravenous fluids all differed significantly (p < 0.001) between the healthcare facility types.Mean total direct medical costs over the three healthcare facilities were 8.96 USD per diarrhoea patient Table . Mean toDifferences in total costs and in stay costs between patients with diarrhoea were associated with the healthcare facility Table . PatientCampylobacter spp., ETEC andnon-typhoidal Salmonella enterica  higher in the health centre (16.29 USD) than in the specialised hospital (5.70 USD). The private clinic did not have an ambulance service. Two third of the patients in the health centre and a quarter of the patients in the specialised hospital used ambulance services.Mean ambulance costs were 7.22 USD per patient  was approximately four times higher than the mean stay of a patient with diarrhoea (1.5 days). Specialist consultation was used for all patients with iNTS, whereas for less than a third of patients with diarrhoea. Special diet was provided to all patients with iNTS, whereas to only 16% of patients with diarrhoea. Intravenous fluids were used similarly for both patients with iNTS and with diarrhoea. More types of drugs and with higher administer frequency and more types of diagnostic tests were used for patients with iNTS than for patients with diarrhoea. The higher healthcare resource use resulted in mean direct medical costs of a patient with iNTS being approximately five times higher than those of a patient with diarrhoeaCampylobacter spp. and iNTS to diarrhoea by non-typhoidal Salmonella enterica were 0.00042 and 0.13, respectively. Total estimated cost-of-illness in Ethiopia due to FBD related to Campylobacter spp., ETEC and non-typhoidal Salmonella enterica were 343.4 million USD, of which 55% was related to Campylobacter spp., 24% to non-typhoidal Salmonella enterica and 21% to ETEC  was due to iNTS. Of the cost-of-illness related to Campylobacter spp. of 187.8 million USD, 0.4 million USD (0.2%) was due to GBS.The age-weighted probabilities of developing GBS as a sequela to diarrhoea by Our study estimated direct medical, direct non-medical and indirect non-medical costs for patients with diarrhoea and direct medical and direct non-medical costs for patients with GBS and iNTS in Gondar, Ethiopia in 2020. To our knowledge, this is the first study that collected such data about GBS and iNTS in Ethiopia. For diarrhoea, few studies on Ethiopia and other countries on the African continent are available. Per patient with diarrhoea in the specialised hospital, mean direct medical costs were 9.53 USD and in previously visited facilities 13.60 USD, direct non-medical transport costs approximately 15 USD and indirect non-medical costs (lost income) 29.12 USD. Thus, total costs per patient with diarrhoea in the specialised hospital were approximately 67 USD. Direct medical costs in the specialised hospital were thus approximately 15% of the total cost per patient. Total costs in our study are approximately three times higher than the median of direct medical, non-medical and indirect costs in the specialised hospital in the city of Gondar plus costs of treatment before the hospital visit of 22.25 USD per patient coming to the outpatient pharmacy in 2016 . In our For GBS and iNTS, we gathered only data about the direct medical and direct non-medical costs of healthcare resources available on patient records in the specialised hospital. Sum of direct medical costs (267.70 USD) and direct non-medical ambulance costs (26.17 USD) estimated in our study was 293.87 USD per GBS patient and sum of direct medical costs (47.79 USD) and direct non-medical ambulance costs (25.37 USD) was 73.16 USD per iNTS patient. These costs are only part of the total costs related to GBS and iNTS. We did not gather direct medical costs made in previously visited facilities. Direct non-medical costs included only ambulance costs, they did not include transport costs of bringing the patient to the hospital with other means of transport nor the costs of visiting the patient. Furthermore, we did not gather data about the indirect non-medical costs. These do not only include lost income of family caregivers, but potentially also of the patients themselves, because all patients with GBS and eight of the patients with iNTS were 18 years or older. Total costs for a patient with GBS or iNTS can be substantially higher than the costs gathered in our study.The World Bank estimated gross national income (GNI) per capita in Ethiopia in 2020 at 890 USD (database indicator code NY.GNP.PCAP.CD). Thus, mean total costs of a diarrhoea patient in the specialised hospital of 67 USD were 8% of the GNI per capita. The mean direct medical and direct non-medical ambulance costs per patient with GBS (293.87 USD) and per patient with iNTS (73.16 USD) were 33% and 8% of GNI per capita, respectively. Thus, healthcare costs of a patient with diarrhoea, iNTS or GBS in a specialised hospital can be a considerable part of family income.Campylobacter spp., ETEC and non-typhoidal Salmonella enterica were 343.4 million USD. This was 0.4% of the 2017 GNI of 78.379 million USD (The World Bank database indicator code NY.GNP.ATLS.CD), relatively significant in relation to the size of the national economy. Our estimate is lower than the cost-of-illness estimate of 0.6 billion USD related of Jaffee, et al. [Salmonella enterica, Campylobacter spp. and ETEC whereas they included all foodborne diseases. iNTS and GBS contributed 12.2 and 0.2% to the national cost-of-illness related to non-typhoidal Salmonella enterica and Campylobacter spp., respectively. This suggests that the cost-of-illness related to more severe sequelae need to be included in addition to those related to diarrhoea when assessing the costs related to foodborne hazards causing diarrheal disease in LMIC. The national estimates were based on a relatively small sample in three healthcare facilities in Gondar. Given the skewed distributions of costs, larger sample sizes in more locations across Ethiopia will provide more robust national estimations.On national level, estimated cost-of-illness due to FBD related to Campylobacter spp., ETEC and non-typhoidal Salmonella enterica. It could be that the mean costs of a diarrhoea patient in the cost categories and healthcare facilities for which we did not find significant differences between the pathogens are similar. However, for part of these costs, especially those for each specific healthcare facility, we might not have found significant differences because of small sample sizes. Furthermore, we don’t know the accuracy of the etiology data collected from patients’ records. For example, many red blood cells and pus cells were recorded as etiology for diarrhoea of several patients . Mean direct medical plus (in)direct non-medical costs per patient with diarrhoea, GBS and iNTS were 8%, 33% and 8% of the GNI per capita, respectively. These costs can be a substantial part of a household’s income. iNTS and GBS contributed 12.2 and 0.2% to the estimated national cost-of-illness related to non-typhoidal Salmonella enterica and Campylobacter spp., respectively. More severe sequalae can add substantially to cost-of-illness of foodborne hazards causing diarrheal disease. Larger sample sizes in more locations across Ethiopia will provide more robust estimates and further efforts to quantify e.g., costs borne by health care insurance and government will complement these estimates.Mean patient-related direct medical costs in a specialised hospital in Gondar of a patient with GBS (267.70 USD) and with iNTS (47.79 USD) were 30 respectively five times those of a diarrhoea patient (8.96 USD). Differences in mean total direct medical costs of diarrhoea patients were mainly associated with the healthcare facility and not with etiology (Additional file 1. Supplementary material S1. Data gathering tools.Additional file 2. Supplementary material S2. Unit costs of healthcare resource use.Additional file 3. Supplementary material S3. Healthcare resource use from patient records. Additional file 4. Supplementary material S4. Median direct medical costs per diarrhoeapatient by etiology and healthcare facility.


Bats host a large variety of viruses, including some that may infect other vertebrates and humans. Research on bat-borne viruses attracted significant attention in recent years mainly due to epizootics caused by viruses having bats as hosts. The characterization of the viral communities of bats was then prioritized, but despite increasing efforts, there are large disparities in the geographical ranges covered and the methodologies employed around the world. As a result, large gaps remain in our current understanding of bat viromes and their role in disease emergence. This is particularly true for megadiverse regions in Latin America. This review aims to summarize the current understanding about bat viruses that inhabit Brazilian biomes, one of the most bat species-rich and diverse regions of the globe. Taking into account all known bat-associated viral families studied in Brazilian biomes, we found that almost half of all bat species (86/181 species) were not investigated for viruses at all. Moreover, only a small fraction of viral lineages or families have been studied more in depth, usually employing targeted methods with limited power to characterize a broad virus diversity. Additionally, these studies relied on limited spatiotemporal sampling and small sample sizes. Therefore, our current understanding of bat viral communities in the Brazilian biomes is limited and biased at different levels, limiting zoonotic risk assessments of bat-borne viruses. Considering these limitations, we propose strategies to bridge the existing gaps in the near future.IMPORTANCE Bat-borne viruses have attracted much attention due to zoonotic outbreaks with large consequences to humans. Because of that, virus characterization in bats has been prioritized in tropical regions of the globe. However, bat-virus research in Latin America and particularly in Brazil, which are among the most bat species-rich regions of the world, are highly biased toward zoonotic viruses and known bat reservoir species. These results have direct implication for virus studies in general but also for new zoonotic virus and spillover events characterization. The limited knowledge we currently have about the virome of Brazilian bats drastically limits any broad assessment of zoonotic viruses they carry and calls for coordinated and large-scale studies to fill this crucial knowledge gap. Chiroptera) compose the second most diverse mammalian order with 1,456 known species from 21 families (https://batnames.org) that participate in diverse ecological processes such as plant pollination, seed dispersion, and soil renewal up to habitat modifications , and fewer studies were conducted in Oceania and North America , 18, 19.Bat virome characterization is also biased when it comes to molecular methodologies employed for viral detection, once the majority of virus detection methods were developed to target single viral lineages of zoonotic concern and not to capture the full diversity of bat viruses . These mhttps://www.sbeq.net/lista-de-especies). We highlight the currently limited and biased character of research on the Brazilian bat virome. Additionally, we summarize the methodologies used for characterization of bat viruses, showing that most studies used targeted low-throughput assays. Finally, we propose methodological approaches to address existing gaps.In this review, we addressed the current understanding of viruses detected in bats inhabiting Brazilian biomes , were likely motivated by concerns about fatal spillover events to humans in Brazil once different bat species were described as important RABV reservoirs .Studies of bat viruses in Brazilian biomes have been mostly performed from 1990 to the present . From the 81 published studies analyzed, there is a clear focus on servoirs to31. COf the 181 bat species known to exist in Brazilian biomes, 95 were included in at least one study, while 86 species (47.5%) have not been screened for viruses so far . The samDesmodus rotundus , Coronaviridae (21), Orthomyxoviridae (16), Paramyxoviridae (8), and Hantaviridae (9), while the remaining RNA virus families were detected in 5 or fewer bat species (Rhabdoviridae (43), Coronaviridae (23), Flaviviridae (13), and Paramyxoviridae (10), while the remaining virus families were detected in fewer than four bat species. Herpesviridae was the only DNA virus family detected in more than 6 bat species, with 17 species positive in the review database and 21 species positive in the ZOVER database , the ZOVER database is based only on virus genetic information available from other public databases. This means that we were able to detect both infection (presence of RNA/DNA and antigens) and exposure (antibodies) of bat hosts in the review database, while the ZOVER database only provides information about the infection status of the host. Moreover, there are several examples of virus sequences available in the ZOVER database tagged as “unpublished” , indicating that these data were publicly released with no peer review. Nevertheless, the majority of the bat species have not been comprehensively studied since most virus families were detected only in 1 to 3 bat species .Smacoviridae, Anelloviridae, Papillomaviridae, Poxviridae, Alloherpesviridae, Asfarviridae, Polydnaviridae, Papillomaviridae, and Parvoviridae, and RNA viruses: Retroviridae, Nairoviridae and Picornaviridae) were only characterized using HTS methods, screening a limited number of individuals and species , and such information cannot be recovered from the ZOVER database. Therefore, we used the detection of virus families per host species as a proxy of the number of studied species from each bat family. This likely leads to an underestimation of the total host species/specimens investigated contributing to the strong correlation between studied and positive species described in The temporal sampling and the number of individuals sampled per species are heterogeneous. Longitudinal sampling was conducted only in 46 of 81 cataloged studies. Moreover, 27 of the 46 studies with longitudinal sampling focused on RABV (Supplementary File 4). A recent meta-analysis of bat coronavirus surveillance identified longitudinal repeated sampling  as the most significant determinant for viral detection , suggestRousettus aegyptiacus . Four of 13 studies investigating coronaviruses employed oral/nasal/anal swabbing due to the upper respiratory and intestinal replication and known viral shedding route though oral/anal fluids (Supplementary File 4). It is important to note that three studies that screened coronaviruses in bats inhabiting Brazilian biomes used various tissues of internal organs from convenience samples obtained from RABV surveillance programs (Supplementary File 4). Convenience samples are known to be biased and generate misleading results for pathogen and disease prevalence  used only amplicon-based methodologies varying from strain-specific PCR (SS-PCR) to family/subfamily-level degenerated RT-PCR (FSD-PCR) (Various methodologies were employed for characterization of viruses in Brazilian bats. The large majority of the studies employed low-throughput (LT) methodologies  , while sFSD-PCR) . Twelve FSD-PCR) . These sE. bonariensis) was investigated using an HT method only, and 10 species were investigated using LT and HT   are also among the most studied ones  . Seven oied ones . All 12 ied ones , while 3ied ones . The negRABV was extensively characterized via VIIIM and DIAMA, and to a lesser extent these methods have been used for hantaviruses and paramyxoviruses. These methods provide precise information about symptoms induced by RABV infection in mice models and the possibility to isolate abundant viral material. However, they are laborious methodologies requiring euthanasia or sampling sick/dead bats. Therefore, these methodologies are not well suited for more broad virus discovery and virome community characterization.Only one reviewed study used ELISA to detect Henipa- and Filo-like viruses . AntibodAmplicon-based screenings have been applied for diagnosing several viral pathogens mainly because of low cost, high sensitivity, and speedy results, allowing high turnover rates and screening of a large set of samples. It is also possible to estimate the virus genome copy number and couple it with sequencing of amplified fragments.One key constraint of all these methodologies is the need for previous information about viral molecules , which is lacking for the large majority of poorly studied viruses infecting animals . TherefoDegenerated primers targeting conserved regions may allow a broader detection of divergent viruses; however, high genetic divergence precludes broad surveys. Nowadays, there are brute force methodologies that allow more unbiased detection and characterization of virus genetic material. For instance, high-throughput sequencing (HTS) approaches can be applied to characterize the complete genomes of DNA and/or RNA viruses . But dueThe viral diversity characterization predominantly performed using targeted low-throughput approaches imposes clear limits on our ability to understand the true diversity of viromes in Brazilian bats. Our findings are in close agreement with a recent review describing methodological biases involved in the attempts to broadly characterize coronaviruses worldwide .The results compiled in this review bring to light the limited understanding of viromes of Brazilian bats. Several biases shape the greater picture: (i) a complete lack of studies for almost half of known bat species occurring in the country, (ii) an extensive focus on few zoonotic viruses or virus families, (iii) sampling design failing to take into account host population size and spatiotemporal population and infection dynamics, (iv) a heterogeneous use of tissue and convenience sampling, and (v) the deployment of targeted LT methods. On the other hand, the number of studies using HTS for virome exploration is growing in Brazil and South America. However, they remain focused on a few bat species and small sample sizes due to high costs. Therefore, the biases and gaps identified point to our limited basic understanding about the virome of neotropical bats. Uncovering viromes of bats and new zoonotic viruses requires hypothesis-driven experimental design that will reveal not only their core virome, but will also uncover the biotic and abiotic factors modulating infection through time and space. Additionally, it can allow the identification of risk factors pertaining to cross-species transmission events.(i) Sampling strategy: the importance of bat and virus traits. Bat populations or metapopulations vary widely in size and geographical range, hence appropriate sampling of bat populations should be estimated taking into account these parameters. Infection rate through time is another key factor to take into account while planning sampling design. Prevalence and transmission may vary drastically in space and time, being correlated with the reproductive season and antibody waning in juveniles. Random longitudinal sampling may yield a more comprehensive picture of long-term infections and overall prevalence in a given population. However, this sampling strategy could miss short-lived acute infections in geographically isolated populations. A disease surveillance sampling strategy may be a useful alternative, where the sampling design is planned based on probability of sampling infected individuals . But it is highly dependent on previous knowledge of disease and the etiological agent, which is largely lacking for bats in Brazil.Comprehensive assessments of virus infections and diversity in bats should use multiple tissue/body fluid collections as much as possible, since mechanisms of infection, replication, and transmission route vary. Sampling strategies trying to answer questions about zoonotic potential can also use specific tissues/fluids that are more relevant in the transmission cycle. Coronaviruses, for example, infect respiratory and intestinal tracts in mammals , hence o(ii) Sampling strategy: methodologies for the characterization of viromes. Understanding the sensitivity and specificity of different methodologies is of utmost importance for assessing the virome diversity. Different molecular assays can be used for complementary testing, but viral enrichment protocols preceding deep HTS are the most powerful workflow for exploring virus communities. Such methodology will set the stage for in situ viral discovery and characterization of core viromes of bats and other animals in the next few years. These HTS technologies allow us to move from questions such as “What can we afford to do for virome characterization?” to “What should we do for comprehensive virome characterization?” Such approaches may, for instance, allow the sequencing of the virome of every individual sampled, not requiring sample pooling and providing a direct measurement of infection rate and coinfection of several viruses in the same individual. Challenges of HTS should be kept in mind: growing data sets of hundreds of samples are currently amenable only using powerful computational infrastructure and trained researchers .For each study we extracted title, year of publication, viral family/lineage studied , country, state, bat species, number of sampled individuals per species, number of positive individuals, methodology of virus detection, sampling strategies , and type of sample (tissue or body fluid). We extracted data to species level for viruses and bats, and used family-level taxonomy for visualization. The methodologies employed for virus detection and characterization were classified according to throughput: low throughput (LT), high throughput (HT), and studies using both methodologies (LT + HT). We detected the following methodologies used in the published articles: Virus Isolation through Intracerebral Inoculation of Mice (VIIIM), the Virus Isolation in Cell Culture (VICC), Electron Microscopy and Antigenic Profile (ELANT), Western Blot (WB), Direct Immunofluorescence Antigen Monoclonal Antibodies (DIAMA), Enzyme-Linked Immunosorbent Assay (ELISA), Strain-Specific PCR (SS-PCR), Family/Subfamily Degenerate PCR (FSD-PCR), Nested PCR (N-PCR), DNA Virome (DNAVIR), RNA Virome (RNAVIR), and Full Virome (FVIR) .A number of studies have sampled and characterized viruses from bat excrements (e.g. guano) where many virus genomes were likely derived from food sources and internal or external bat microbiota. Therefore, we only extracted information of viruses that were known to infect vertebrates, irrespective of the original sample .http://www.mgc.ac.cn/cgi-bin/ZOVER/main.cgi; last accessed May 2022) (https://www.sbeq.net/lista-de-especies). This database was chosen because it is the only database that compiles all available viral genetic information found in bats.In order to assess the available virus sequence information, we retrieved the number of entries per bat species and virus family from the ZOVER database (ay 2022)  using thhttps://www.r-project.org/) employing packages such as ggplot2 (Figures were rendered with R statistical language ( ggplot2 , webr 0. ggplot2 , dplyr 1 ggplot2 , tidyr 1 ggplot2 , ggpubr  ggplot2 , reshape ggplot2 , ggforce ggplot2 , ggridge ggplot2 , treemap ggplot2 , ggdist  ggplot2 , gghalve ggplot2 , packcir ggplot2 , ggrepel ggplot2 , patchwo ggplot2 , and ggv ggplot2 . Maps we ggplot2 .


The disease has also been spreading throughout other regions, such as Africa, the Americas, Eastern Mediterranean, and Western Pacific. According to OurWorldinData, as of July 25, there have been 16 313 confirmed cases of monkeypox worldwide in 75 countries. On July 23, WHO declared that monkeypox was a public health emergency. Monkeypox virus belongs to the Orthopoxvirus genus, the same genus as the virus that causes smallpox. Though there is no specific treatment for monkeypox, the genetic similarity between the viruses that cause the two diseases allows for the use of treatments and vaccines that are specific to smallpox.Until the beginning of 2022, human monkeypox was not a well-known disease among the general population. Sporadic cases of monkeypox occurred mainly in Central Africa and West Africa, where the virus is endemic. However, since May, 2022, cases of monkeypox have been confirmed outside of Central Africa and West Africa and the outbreak has been increasing at an alarming rate after the first case was confirmed in the UK on May 6conference report by Bunge and colleagues (2022) published in Vaccine showed that there has been an increase in cases since 1970, when the first human case of monkeypox was confirmed: from 48 confirmed cases in six African countries (between 1970 and 1979), increasing to 343 in 1980, and 520 in 1990. The first case outside the endemic region occurred in 2003 in the USA, with 47 cases caused by infected animals from Ghana that were sold as exotic pets. At that time, human-to-human transmission was not very common, with most cases related to animal reservoirs. The increase in cases of monkeypox could be related to, among other factors, climate change, armed conflicts in the region, rainforest exploitation, mobile populations, and waning herd immunity due to the eradication of smallpox.African countries have been dealing with the monkeypox virus for at least 50 years, and scientists have been warning about a potential spread to other regions. Smallpox is highly infectious and it is transmitted by variola virus. After the eradication of smallpox in 1980, an epidemiological niche was created generating an opportunity for the spread of monkeypox virus. A phylogenomic analysis by Luna and colleagues (2022) published in Travel Medicine and Infectious Disease evaluated 337 genomes and showed that the current monkeypox virus has a specific monophyletic lineage compared with the viruses from previous outbreaks, with a mutational signature that could possibly favour transmission and dispersion. Before the 2022 monkeypox outbreak, monkeypox virus was classified into two clades: the Congo Basin Clade (clade 1), which is associated with severe symptoms and higher mortality rates, and the Central Africa clade (clade 2) which is associated with milder cases For the 2022 outbreak, the increase in transmission could be related to the genomic divergence from these two clades and a stochastic evolutionary event, which could explain the successful transmission and spread of monkeypox virus throughout different countries. Luna and colleagues proposed a third clade that is derived from clade 2 and is comprised of the hMPXV-1A clade, as well as the following lineages: A1, A1.1, A.2, and B.1, the latter emerging in Europe. Clade 3 is believed to have derived from cases of monkeypox from between 2018 and 2019. The 2018 cases occurred in Israel (one case) and the UK (two cases). In 2019, one case of monkeypox in Singapore and another in the UK were reported, which were both related to previous exposure. Genomes from the 2022 outbreak are still under investigation to assess whether they all belong to clade 3 or if a new clade is emerging. Transmission is characterised by sexual encounters and direct contact with body fluids, sores and scabs, and exposure to fomites; it can also occur by placenta transfer from mother to foetus. Though considered a mild disease, the main symptoms of monkeypox include fever, rash, and lymphadenopathy, as well as complications such as pneumonitis, encephalitis, sight-threatening keratitis, and secondary bacterial infections.In the 2022 outbreak, transmission has been distinct from previous African cases and human-to-human transmission is common. A A study by Grosenbach published at the New England Journal of Medicine investigated the efficacy of the poxvirus antiviral Tecovirimat in a monkeypox model, which led to survival rates of over 90% in non-human primates. Though the authors could not expose humans to smallpox intentionally, they conducted a randomised double-blind trial with humans to try to mimic the efficacious dose observed in the non-human primate model. Previous model studies predicted that a dose of 600 mg twice a day for 14 days would provide exposure in excess compared with animal models. Multiple-day dosing in human patients resulted in steady-drug accumulation that were approximately 50% (AUC0-24h) and 40% (Cmax) higher than the first dose, with steady-state levels seen at day 6. Thus, the Center for Disease Control and Prevention recommends Tecovirimat for those diagnosed with monkeypox virus. According to WHO´s immunisation guidelines, mass vaccination is not required, and both pre-exposure prophylaxis (PrEP) and post-exposure prophylaxis (PEP) are recommended depending on the situation. PrEP is recommended for those who have a high risk of exposure, and PEP for contact cases within 4 days of first exposure. Vaccines used are from second or third generation smallpox vaccines, JYNNEOS  and ACAM2000 (IMVAMUNE). JYNNEOS has been recommended primarily because it is an attenuated, non-replicating live vaccine, from the Modified Vaccinia Ankara (MVA-BN) strain. Those who have immune deficiencies can thus benefit from this vaccine. ACAM2000 is not recommended for immunocompromised individuals as it can lead to serious disease. According to Fine and colleagues (1988) published at International Journal of Epidemiology, the vaccination efficacy is as high as 85%. However, these results date back when the first outbreaks occurred.Smallpox vaccination ceased 40 years ago with the eradication of the virus, thus immunity against poxvirus is limited and susceptibility to the virus has increased over the years. Though smallpox was declared eradicated in 1980, antivirals were created for poxvirus treatment. vaccine shortage is expected. A one-dose shot has been used as strategy in some countries, such as Germany and the UK. Earl and colleagues (2008) published at PNAS administered one dose of MVA in macaques to assess protective immunity for monkeypox virus, antibodies were detected by ELISA after 7 days for MVA, and found a dominant response for IFN-ƴ. The authors found that a single dose of MVA could induce a rapid antibody and CD8+ and IFN-ƴ+ response. For protection against monkeypox virus, all vaccinated animals survived, providing evidence to show that a single dose is effective against infection. Pittman and colleagues (2019) published at the New England Journal of Medicine compared the efficacy of MVA vaccine with ACAM2000 for smallpox. They assigned two groups: one group that was assigned two doses of MVA and a third dose of ACAM2000, and a second group that was assigned to only a single dose of ACAM2000. Neutralising antibodies were higher for MVA than ACAM2000 group, and results indicated that one dose of MVA induced neutralising antibody titers similar to ACAM2000. For now, a single dose strategy might be one solution to the problem of low vaccine supply.With the increase in cases of monkeypox around the world, eBioMedicine we welcome robust studies to address the 2022 outbreak of monkeypox virus.Vaccination for those at high risk of exposure to monkeypox should be a priority for policy makers, and low-income and middle-income countries should also vaccinate their high-risk populations. And though we have effective tools against monkeypox virus, real-world data from the 2022 outbreak is necessary to identify any shortcomings. Surveillance, epidemiology, detection, and control of the virus should be high priorities for the scientific community and public health officials. Communication with the population is also necessary to increase awareness and avoid the possibility of stigmatisation. At


Metapneumoviruses, members of the family Pneumoviridae, have been identified in birds  and humans . AMPV and HMPV are closely related viruses with a similar genomic organization and cause respiratory tract illnesses in birds and humans, respectively. AMPV can be classified into four subgroups, A–D, and is the etiological agent of turkey rhinotracheitis and swollen head syndrome in chickens. Epidemiological studies have indicated that AMPV also circulates in wild bird species which may act as reservoir hosts for novel subtypes. HMPV was first discovered in 2001, but retrospective studies have shown that HMPV has been circulating in humans for at least 50 years. AMPV subgroup C is more closely related to HMPV than to any other AMPV subgroup, suggesting that HMPV has evolved from AMPV-C following zoonotic transfer. In this review, we present a historical perspective on the discovery of metapneumoviruses and discuss the host tropism, pathogenicity, and molecular characteristics of the different AMPV and HMPV subgroups to provide increased focus on the necessity to better understand the evolutionary pathways through which HMPV emerged as a seasonal endemic human respiratory virus. The ongoing global health crisis caused by the SARS-CoV-2 pandemic has unequivocally reminded us of the impact that a newly emerging zoonotic respiratory virus can have on a naïve human population. Although the origin of SARS-CoV-2 remains to be established , an evolHuman metapneumovirus (HMPV) is also assumed to have evolved following a zoonotic virus infection from an unknown avian reservoir host species, as this virus shares a common ancestor with avian metapneumovirus subtype C (AMPV-C) . AnalysiMeleagris gallopavo) termed “acute respiratory disease syndrome”, or most commonly known as “turkey rhinotracheitis (TRT)” , bu, bu94], AMPV strains have also been shown to contain G proteins of varying lengths. The size of the G ORF of AMPV-A, -B, and -D are reported to be 1176 nt (391 aa), 1245 nt (414 aa), and 1170 nt (389 aa), respectively  55]. Th. Th55]. The metapneumovirus F protein is a trimeric type I membrane glycoprotein of 539 aa (HMPV) or 537–538 aa (AMPV)  84] and and84] aThe F proteins of metapneumoviruses are not dependent upon the attachment protein G to mediate fusion of the virus particle with the host cell membrane, meaning that the G protein is thus dispensable for virus growth in transformed cell lines ,92. MoreThe SH protein of metapneumoviruses is a type II integral membrane glycoprotein of 174–175 aa for APMV  and 177–Gorilla beringei beringei) and chimpanzees (Pan troglodytes) [S. pneumoniae and K. pneumonia. The source of infection was not determined, as the HMPV-infected gorilla died early in the outbreak and had not been in contact with park personnel or veterinarians [HMPV is a human respiratory virus that has no known animal reservoirs, but zooanthroponotic infections have been reported in endangered great apes such as mountain gorillas (lodytes) ,124,125.lodytes) ,126. Furlodytes) . Patholoinarians . Furtherinarians .Mus musculus) model has been largely predominant in HMPV studies due to the availability of reagents, easy handling, and lower costs [Sigmodon hispidus) has also been investigated as a small animal model of HMPV infection, possibly due to its successful use in the RSV field [Mesocricetus auratus) [Cavia porcellus), Syrian golden hamsters, and ferrets (Mustela furo) [In the past two decades of HMPV research, several animal models have been established which show varying degrees of permissiveness to virus replication. The mouse . The coSV field ,140. Sevauratus) , althougauratus) ,137,139.auratus) ). Other la furo) , which ela furo) .Macaca fascicularis), rhesus macaques (Macaca mulatta), African green monkeys , and chimpanzees have all been shown to be permissive to HMPV infection [Cynomolgus macaques , and Muscovy ducks have displayed seroconversion, but no clinical signs following infection with an American turkey lineage AMPV-C strain [AMPV-C strains can be divided into two lineages, namely the American turkey strains, known to be the etiological agent responsible for the outbreaks in turkey farms in the USA in the 1990s and early 2000s, and the Eurasian duck strains which mainly infect duck species . A recenC strain ,149. TheC strain . This reAMPV-D has been isolated twice from turkeys in France in 1985 and has not reemerged since then . Under eBranta Canadensis), house sparrows (Passer domesticus), barn swallows (Hirundo rustica), and European starlings (Sturnus vulgaris) [Larus delawarensis) in Minnesota and snow geese (Anser caerulescens) from Saskatchewan, Canada, were also found to be positive for AMPV-C [Spatula discors) [Columba livia) and a plethora of other wild birds in Brazil [Anas platyrhynchos), greylag geese, and common gulls via the use of a pan-paramyxo and pneumovirus RT-PCR assay [Mareca Penelope) (35261311). AMPV-C was also detected in 2017 in Ontario, Canada in wild waterfowl including the American black duck (Anas rubripes), American wigeon (Mareca americana), blue-winged teal (Spatula discors), mallard, northern shoveler (Spatula clypeata), wood duck (Aix sponsa), and ring-necked duck (Aythya collaris) [Although AMPV was first isolated from turkeys in South Africa in 1978, the true origins of AMPV are unknown. However, the rapid spread of AMPV within the UK and the close relationship of the early UK strains to the original South African strain suggested a role for wild and migratory birds in the transmission of AMPV ,151. Folulgaris) . Studiesulgaris) ,153. Samr AMPV-C . Anotherdiscors) . The knodiscors) . In 2011n Brazil . AMPV-C CR assay  and moreollaris) . AMPV-A ollaris) . CollectLittle research has been undertaken into the capacity of AMPV to infect mammals. A single study by Wei and colleagues has shown that AMPV-C can replicate and persist in the lungs of mice for at least 21 days in association with the development of histopathological lesions in lung tissues . These mIn recent years, the use of next generation sequencing has become omnipresent in the field of virus discovery and has enabled the characterization of an ever-increasing number of partial and complete genome sequences of novel viruses in wildlife species, domestic animals, and humans. This includes the discovery of a novel swine orthopneumovirus  which coMetapneumovirus research continues to encounter considerable technical challenges with respect to virus propagation in cell culture. Considering the data of numerous studies, metapneumoviruses may require several in vitro passages to produce notable CPEs, depending on virus strain or more generally, on the laboratory setup. Passaging of virus isolates in a particular cell line allows the virus to adapt to these artificial surroundings, producing mutant variants that grow more efficiently. Although these mutant laboratory-adapted isolates produce higher titers to use in future experiments, they do not necessarily reflect the phenotype of a circulating wild-type virus. For future metapneumovirus research studies, particular care should be taken that full genome consensus sequences of recombinant viruses are identical to that present in the original clinical swab material from birds or humans. Generally, it is crucial to sequence isolates pre- and post-experiments to define the molecular basis of adaptation to novel hosts. Metagenomics has also facilitated a rapid expansion in the last decade in the number of available metapneumovirus complete genome sequences which notably includes sequences generated de novo from clinical samples such as the novel highly divergent monk parakeet and gull AMPV strains. Collectively, this may necessitate updated molecular clock modeling to reassess the divergence date of AMPV-C and HMPV.In summary, obtaining a better understanding of the evolutionary origins of HMPV and the barriers to metapneumovirus cross-species infections will require a better understanding of the molecular and phenotypic differences between AMPV-C and HMPV. This will necessitate more rigorous investigations based on the use of virus isolates which retain the consensus sequence present in clinical samples in conjunction with in vitro and ex vivo primary cell models of avian and human respiratory epithelium. Illuminating the mechanisms through which an avian virus evolved to become an endemic human respiratory virus may hold valuable lessons for better understanding how zoonotic viruses are able to circumvent the barriers to cross-species viral infections.


Based on this knowledge, soil and other environmental samples could provide rapid and reliable information on the disease outbreak and serve as indicators of the risk posed by the affected locality.The survival of African swine fever virus (ASFV) on different matrices and its infectivity in wild as well as domestic swine is still a matter of interest. ASFV is resistant to environmental effects; this fact is enhanced by the presence of organic material. Therefore, the aim of this work was to determine the ability of laboratory ASFV to survive in soil at different temperatures (4 and 22 °C) and with and without the presence of blood using culture procedures. The suitability of the procedure for determining the viability and titre of the ASFV field strain by the hemadsorption method was also verified, when a higher decrease in virus infectivity in the case of clay compared with peat was demonstrated. The stability of the virus was clearly temperature-dependent, the infectious virus was detected after 112 days, and the viral DNA was still detected in the matrix 210 days after inoculation in a relatively high and stable concentration (between 10 Suidae, including both domestic pigs and wild boars (Sus scrofa) [Ornithodoros and common warthog (Phacochoerus africanus); (2) the pig–tick cycle, and (3) the anthropogenic cycle [Asfarviridae family and belongs to the group of large nucleocytoplasmic DNA viruses. The outstanding feature of the infectious ASF virion is its multilayer architecture. An ASFV genome-containing nucleoid is surrounded by two distinct icosahedral protein capsids and two lipoprotein membranes [Recently, the African swine fever virus (ASFV), an aetiological agent of African swine fever, has become a severe threat in pig industry worldwide. ASFV affects members of the family  scrofa) . The epiicanus);  the pig–cle, and  the anthicanus);  the pig–ic cycle ,5. Probsic cycle . Howeveric cycle . Moreoveic cycle . Neverthic cycle . The ASFic cycle . The cauic cycle . ASFV isembranes . Moreoveembranes .In this work, we aimed to obtain a more complete picture of the ability of ASFV to survive in soil under different conditions. In the first part of the experiment, a laboratory ASFV strain Ba71V that is able to grow and produce visible cytopathic effects on a VERO cell line was used. Furthermore, we developed a method suitable for the detection of infective ASFV particles in the soil. To verify the procedure, we used recent field isolate, which does not produce cytopathic effects (CPE) on established cell lines. Thus, we had to use primary porcine alveolar macrophages and quantify the virus by hemadsorption test.v/v) gamma-irradiated foetal bovine serum  was used for VERO cultivation. A confluent monolayer of cells (cultivated for a maximum of 24 h) was inoculated with virus suspension. After 1 h of virus adsorption, DMEM with FBS (2% v/v) was added, and infected cells were cultivated at 37 °C under 5% CO2. An extensive cytopathic effect (CPE) was observed in the cell culture at six days post infection (DPI). Next, the cells were frozen at −80 °C and thawed twice. Cell debris was removed by centrifugation . Then, viral particles were concentrated employing Pierce™ Protein Concentrator  following the manufacturer’s instructions to obtain virus stock for survival testing. The prepared virus suspension was stored at −80 °C until further use. The virus concentration was determined by titration assay on VERO cells, and the final concentration was 7.5 × 107 TCID50/mL.ASFV strain Ba71V stocks obtained from the European Union Reference Laboratory for African swine fever (EURL-ASF) were used in the study. The Ba71V strain was propagated on VERO cells. Dulbecco’s Modified Eagle Medium High Glucose  supplemented with 10% , supernatant was filtered through 0.45 µm filter , diluted 50× in DMEM supplemented with 10% (v/v) FBS and 1% (v/v) Antibiotic-Antimycotic 100× . Four wells  of PAMs (5 × 105 cells per well) were infected with 200 µL of prepared inoculum. After 1 h of absorption, 1 mL of DMEM  was added, and infected cells were cultivated at 37 °C under 5 % CO2. Infected cells were observed daily for any microscopical changes. After 5 days, CPE was observed in comparison with non-infected control cells, and the culture plates were freeze-thawed for further passaging. The second passage was performed under the same conditions as the first passage. The following passage was performed on cell culture flasks  (5 × 106 PAMs per flask with growth surface of 25 cm2). The cells were inoculated with 500 µL of virus harvest (4× diluted freeze-thawed second passage), and after 20 min of absorption, 10 mL of DMEM  was added. Infected cells were incubated at 37 °C in 5% CO2. CPE was observed after 6–7 days of cultivation. Thereafter, the cells were freeze-thawed, and the aliquots with virus harvest were frozen in −80 °C until use.Field isolates were subcultured successfully on primary porcine alveolar macrophages (PAMs). PAMs were prepared according to Kavanová et al. . Field sTo work with well-defined virus, the stock virus of the Czech ASFV isolates (samples No. 175576/17 and 103483/18) prepared for further experiments was characterized by next-generation sequencing. DNA from the virus aliquots was isolated with the use of a QIAamp DNA Mini Kit . The DNA library preparation, Illumina sequencing, and nucleotide sequence mapping to ASFV reference sequences were performed by a commercial provider . The numbers of 250 bp pair-end reads per sample were 1,386,592 and 1,309,234 reads, and the percentages of mapped reads 9.5% and 11% . Nucleotide sequences for each ASFV isolate were assembled into contigs using the high-performance graphical viewer Tablet .The sequences of genes coding main structural proteins p30, p54, and p70 for both ASFV field isolates were deposited in the GenBank. They received accession numbers MW889885–MW889890.7/mL): (1) in DMEM with FBS (2% v/v), which represents less biological soiling, and (2) in DMEM with FBS (2% v/v) and 10% defined commercial sheep erythrocytes  to simulate more biological soiling. Thereafter, inoculated samples were stored in two different conditions until use: (1) at room temperature protected from light (22 °C) and (2) at 4 °C. All samples were tested in duplicate.Ba71V strain is able to produce CPE on a commonly used VERO cell line, so it is easy to quantify it using titration. Thus, we used this method for testing the long-term survival of ASFV in soil under different conditions. To ensure defined identical conditions during all sampling times, sand-clay soil was sterilized by autoclaving at 121 °C for 15 min and divided into 1 g aliquots in 15-mL centrifuge tubes . The pH of soil samples was determined in water extracts using a soil water ratio of 1:2 with a glass electrode and a direct-reading pH meter. All samples of sterile soil were inoculated with 500 µL of Ba71V virus strain  and placed in a Pierce™ Protein Concentrator . The supernatants were concentrated to original inoculum volume (500 µL) by centrifugation at 5000× g for approximately 15 min. The virus concentration was determined by titration assay on the VERO cells. The limit of detection in this assay is 101.5 TCID50/mL.The virus (Ba71V strain) was isolated from the inoculated soil samples (described in paragraph 2.4) at 0, 1, 14, 28, 42, 56, 63, 70, 112, 126, 147, 176, and 210 days post inoculation (DPI). The soil inoculated with virus was removed from storage, 10 mL of DMEM  was added, and samples were shaken vigorously for 20 min. The samples were centrifuged at 5000× Viral DNA was extracted from 100 μL of every concentrated supernatant originating from soil (see paragraph 2.5) with the use of the QIAamp Viral RNA Mini Kit  and eluted into 60 μL of PCR-grade water, according to the manufacturer’s instructions. 3 copies of IAC DNA, and 5 µL template DNA. To avoid possible carry-over contamination, 0.2 U of Uracil DNA Glycosylase (Roche) was used in each qPCR reaction. The optimised duplex qPCR was run in a total volume of 20 µL. Amplification and fluorescence detection were performed on the LightCycler 480 (Roche) using 96-well PCR plates under the following conditions: initial denaturation at 95 °C for 4 min and 45 cycles of 95 °C for 10 s, then 58 °C for 30 s. Consequently, the DNA standards  were used to construct a gradient, which served for the quantitation of ASFV  and as positive controls. The analysis of results was carried out using the “Fit Points Method” option of the LightCycler 480 Software release 1.5.0 .Based on the standard operating procedure of the European Reference Laboratory for African swine fever, real-time PCR (qPCR) was introduced and optimized to detect and quantify the ASFV genome . An inteIn the next step, we decided to demonstrate the efficiency of isolation of infectious virus particles from different soil types using ASFV field isolate instead of laboratory Ba71V strain. In contrast to Ba71V, field ASFV isolate does not produce the cytopathic effect (CPE) on established cell lines, and conversely, Ba71V strain was not able to produce typical signs of hemadsorption, e.g., rosettes. Therefore, we had to use primary porcine cells  and field isolate 175576/17 and quantify the virus by hemadsorption test.7 HAD50/mL) was added into 1 g of matrix and incubated for 15 min at room temperature. The titre of the field virus isolate was assessed by hemadsorption test based on the EURL-ASF standard operating procedure [50/mL) were determined. Alveolar macrophages were seeded on a 384-well plate at a concentration of 3×104 per well in DMEM  and incubated overnight at 37 °C in 5% CO2. ASFV was diluted  using macrophage medium , and 25 µL of each dilution was transferred to five wells of the micro-titre plate. The amount of 25 µL of 0.1% porcine erythrocytes (isolated from porcine heparinized blood by Histopaque-1077 (Sigma-Aldrich) gradient) was added after 20 min of adsorption. Erythrocytes were diluted in DMEM . The presence of the virus was assessed by hemadsorption 7 days after inoculation, and virus titres were calculated by the Reed and Muench method [The infectivity of ASFV isolate was tested after incubation in two different matrices: (1) soil (pH 7.78) or (2) peat (pH 3.85). The amount of 500 µL of virus . Thus, further experiments with field ASFV were conducted only with 175576/17 isolate.7 TCID50/mL) per 1 g, and the isolation from the soil at day 0 (immediately after inoculation) showed a decrease in the virus load to 1.26 × 106 TCID50/mL. The best ASFV stability was reported in the samples of soil incubated at 4 °C with an addition of sheep erythrocytes (1.5 TCID50/mL) was still detected after 112 days of incubation in the samples with and without sheep erythrocytes at 4 °C (The virus (Ba71V) was isolated from soil stored under different conditions at regular intervals, and the virus concentration was determined by titration assay on VERO cells. The samples of soil were inoculated with 500 µL of virus   of 1.40 × 10ulation) B,D.4 and 1.2 × 105 with and without erythrocytes, respectively) and peat  when compared with samples isolated directly from virus suspension .The experiment verifies the possibility of field strain ASFV isolation from soil and peat. Mean titres of viable ASFV are presented in ASFV resistance and stability in the environment are the main prerequisites for its indirect transmission. It has been proved that ASFV is extremely resistant to environmental conditions and can survive many freeze-thaw cycles as well as a wide range of pH levels and temperatures . SeveralWe showed that the stability of the virus is clearly affected by the ambient temperature, similar to Davies et al. . The vir6 and 107 GE/mL) were detected until 210 days post inoculation (end of the experiment) of soil samples. Unexpectedly, this stability of ASFV DNA was also observed at 22 °C. Lee et al. successfully detected the ASFV genome from mud and water samples from areas where ASFV-positive wild boar carcasses were found using the polyethylene glycol-based precipitation of viral particles [During the study, the substantial stability of ASFV genetic material has been proven. Zani et al. demonstrated relatively low virus contamination of soil surrounding wild boar carcasses buried in different levels of decomposition . Howeverarticles . UnfortuBased on this knowledge, soil and other environmental samples could provide rapid and reliable information on the disease outbreak and trends in animal population and can serve either as indicators of the risk posed by affected locality or as confirmations of area safety.