Salmo gairdnery) fry and Atlantic salmon  post-smolt—frequently reach 80–90% of stocks. The virus causing the typical signs of the IPN disease in salmonids, named infectious pancreatic necrosis virus (IPNV), has also been isolated from other fish species either suffering related diseases (then named IPNV-like virus) or asymptomatic; the general term aquabirnavirus is used to encompass all these viruses. Aquabirnaviruses are non-enveloped, icosahedral bisegmented dsRNA viruses, whose genome codifies five viral proteins, three of which are structural, and one of them is an RNA-dependent RNA polymerase. Due to the great importance of the disease, there have been great efforts to find a way to predict the level of virulence of IPNV isolates. The viral genome and proteins have been the main focus of research. However, to date such a reliable magic marker has not been discovered. This review describes the processes followed for decades in the attempts to discover the viral determinants of virulence, and to help the reader understand how viral components can be involved in virulence modulation in vitro and in vivo. There is also a brief description of the disease, of host defenses, and of the molecular structure and function of the virus and its viral components.Infectious pancreatic necrosis (IPN) is a disease of great concern in aquaculture, mainly among salmonid farmers, since losses in salmonid fish—mostly very young rainbow trout ( Salvelinus fontinalis) suffering an infectious disease resembling a catarrhal enteritis. The virus causing the disease, the IPN virus (IPNV), was the first fish virus isolated in vitro  might influence its anti-apoptotic effect […] and partially affect […]” the result. Nevertheless, other authors have reached the same conclusions regarding the lack of influence of IPNV–VP5 on both viral replication [However, the same group soon reported a different statement. Santi et al. ,108 conslication  and infelication .Another point of discrepancy is the effect of the size of this protein on its action. Although for some authors VP5 activity does not depend on its size, and even though apoptosis is not dependent of that protein ,57,108, There are two possible explanations for all of these discrepancies. On the one hand, Ulrich et al.  suggesteJust considering that all viral proteins are involved in the activation (VP1) or blocking (the remaining) of IFN signaling, as demonstrated by Lauksund et al. , it is uAs described in In IBDV, Nouën et al.  discoverFinally, the VP4 function is not only linked to its protease activity , but it is also a potent IFN induction inhibitor (not associated to its protease activity), and in IBDV it has been demonstrated to trans-activate the synthesis of VP1 . Again, Reassortment: During coinfection of a cell with two variants of a viral strain, several recombination phenomena can occur which can provide diversity, adaptation and even the possibility to survive from lethal mutations. Among these recombination mechanisms, reassortment is the most common in segmented viruses and is known to be one of the most involved in the evolution and diversity of RNA viruses [ viruses ,113. In  viruses ,115, whi viruses , the fir viruses , and man viruses ,118. In  viruses  in the 1 viruses . Therefo viruses . The resPolyploidy. As already indicated in If with this review we expected to find out which marker, or set of well-defined markers, could be used to predict the level of risk of a new IPNV isolate from its molecular characteristics, by now we must be a little disappointed. In addition to the strong influence of the host (species susceptibility and host defense) and environment , the important influence of the virus itself is also well known, but there is no a single viral determinant of virulence. The viral genome and proteins, the interaction between them, a bunch of specific amino acid residues in VP2 , the efficiency or failure of viral proteins to target specific host proteins (affecting the efficacy of the virus to control certain cell processes), reassortment, and polyploidy: these are all parameters combined in a way that, with the information available to date, is too complex for us to understand and hence to be able to discover such a clear, definitive, and simple track to predict virulence levels with some reliability. Many attempts have been carried out to discover such a magic marker but, in most cases the final conclusion of the researchers has been that other factors must be involved. The closest we have been was with the aminoacidic residues at VP2 positions 217 and 221. We thought it could be the magic marker of IPNV virulence; however, too many exceptions were uncovered. Then, it was thought that it could be applied at least in the case of Sp-type strains; but there were some exceptions again. Therefore, we must consider how a group of genetic factors are combined to determine virulence. That is why research should be aimed at the analysis of all mutations—in both segments, and including the non-coding regions—associated with different (and well defined) levels of virulence in vitro  and in vivo .


Human herpesvirus 6 (HHV-6) is a β-herpesvirus that is highly prevalent in the human population. HHV-6 comprises two recognized species (HHV-6A and HHV-6B). Despite different cell tropism and disease association, HHV-6A/B show high genome homology and harbor the conserved U94 gene, which is limited to HHV-6 and absent in all the other human herpesviruses. U94 has key functions in the virus life cycle and associated diseases, having demonstrated or putative roles in virus replication, integration, and reactivation. During natural infection, U94 elicits an immune response, and the prevalence and extent of the anti-U94 response are associated with specific diseases. Notably, U94 can entirely reproduce some virus effects at the cell level, including inhibition of cell migration, induction of cytokines and HLA-G expression, and angiogenesis inhibition, supporting a direct U94 role in the development of HHV-6-associated diseases. Moreover, specific U94 properties, such as the ability to modulate angiogenesis pathways, have been exploited to counteract cancer development. Here, we review the information available on this key HHV-6 gene, highlighting its potential uses. Originally isolated in 1986 from blood lymphocytes of adult subjects affected by lymphoproliferative disorders, it was for that reason initially named human B-lymphotropic virus , and theLong considered a unique virus that is distinct in two variants, HHV-6A and 6B were recently recognized as different virus species  based onβ-Herpesvirinae subfamily of the Herpesviridae family, where it was included on the basis of its genomic similarities with human cytomegalovirus (HCMV) [Roseolovirus genus, and its infection is widespread worldwide [HHV-6 belongs to the s (HCMV) . Along worldwide . The twoorldwide ,8 as welorldwide ,10,11,12The two viral species use different cell receptors: HHV-6A uses CD46 , expressroseola infantum, a disease characterized by fever, diarrhea, and mild skin rash [Like all human herpesviruses, HHV-6 causes both productive and latent infections. The vast majority of documented primary infections and reactivation events are linked to HHV-6B, which usually infects children within the first two years of life, causing kin rash ,35. Compkin rash . Less iskin rash . Howeverkin rash . Pityriasis rosea and Kawasaki’s disease increased around ten-fold during the COVID-19 pandemic, compared to previous periods [After primary infection, HHV-6 establishes a life-long latent infection in the host, from which it may reactivate, especially during immune dysregulation . Several periods .L left, and DRR right), containing 9 ORFs, elements needed for viral DNA packaging (pac1 and pac2), and human telomere-like (TAACCC) simple repeats, including perfect and imperfect telomeric repeats (TMRs). These sequences are about 0.35 Kb long but imperfect TMRs are longer than the perfect TMR and both show length variation between strains. The lengths also appear to be different between free virus and iciHHV-6A/B [The HHV-6 genome consists of a double-stranded linear DNA segment around 160 Kb long, with slight differences between 6A (about 156 Kb in size for the GS strain) and 6B (about 161 Kb long for the Z29 strain) . Virus gHHV-6A/B ,66,67  and ribosome profiling revealed more complex transcriptomes , suggestThe most divergent regions are DRs and the right end of the U region, spanning ORFs U86 to U100 . In partMiniopterus schreibersii β-herpesvirus (MsHV) encode a U94/rep homolog [The U94 gene has no counterparts in any of the other known human herpesviruses, even in the closely related β-herpesviruses HHV-7 and HCMV, and is located in the antisense strand of the virus genome ,70. Amon homolog ,74, alth homolog . Despite homolog .rep gene product (RepAAV-2), which encodes four overlapping proteins required for AAV-2 DNA replication and modulation of the expression of homologous and heterologous genes [The HHV-6 U94 gene is a spliced gene encoding a 490-aminoacid protein that is highly conserved between species, as the difference consists of 10 amino acid residues . It was us genes ,78. The us genes ,80. It ius genes ,82, inhius genes , and repus genes . ras oncogene expression and HIV-1 transcription [U94 was shown to share 24% amino acid identity with RepAAV-2 and, consistent with this, to be able to restore the replication in Rep-deficient AAV-2 mutants , suggestcription . The HHVcription ,88. In acription . The expression of U94 is observed during the whole life cycle of the HHV-6 virus, both in the productive/lytic and latent phases of infection. During the productive infection, the U94 gene is expressed as an immediate-early (IE) α gene before the expression of early (β) and late (γ) genes . In in-vU94 transcripts are also found in-vivo in latently infected cells such as freshly isolated PBMCs from healthy donors, in the absence of other viral mRNAs ,93, and Most herpesviruses maintain their latent genome in an episomal circular form, but HHV-6A/B can integrate their genomes into the chromosomal telomeres and no episomes are observed ,78,96. TTarsius syrichta Roseolovirus 1 [The observation of a full-length integrated HHV-6 genome was first reported in the DNA of PBMCs ,97, and ovirus 1 . In the ovirus 1 ,103. It ovirus 1 ,105, andovirus 1 ,107,108;The mechanisms allowing HHV-6 integration are not yet elucidated, but it is likely that the TAACCC motifs present in the genome regions containing DR and packaging (pac) sequences are involved due to their homology with human telomeric repeat sequences ,110. ConInterestingly, U94 transcripts were detected in the majority (54.5%) of PBMC derived from 11 individuals with iciHHV-6 , supportHowever, the role of U94 in integration is yet unproven due to the lack of reverse-genetic systems and efficient integration assays able to quantify virus integration; more studies are needed to support the role of U94 in HHV-6A/B integration, especially in light of a recent report suggesting that U94 may be dispensable for virus integration . A mutanDespite its nonconstitutive function, during the natural infection by HHV-6, an immune response is elicited against the virus U94 gene product, both at the humoral and cellular levels. The onset of antibodies specifically directed against the U94 protein can be observed during the seroconversion in acutely infected subjects  and in aIn multiple sclerosis patients, >80% had anti-U94 IgG, with a mean titer of 1:515 (range 200–1200) . SimilarThese observations further suggest that immune-dysregulated individuals may not properly control the infection/reactivation of HHV-6A/B, allowing multiple virus reactivations and, hence, their possible pathological consequences. Moreover, the measurement of humoral and/or cellular response directed against the U94 antigen might be utilized as a risk marker for the development of HHV-6-associated diseases by contrast with anti-HHV-6 responses, which are almost unaltered in patients and controls due to the high prevalence of the virus in the human population.Interestingly, previous studies by our group also evidenced that U94 can also elicit a cellular immune response, showing a prominent reactivity of CD4+ and CD8+ T-cells toward the antigen, particularly the subset of CD4+ cells secreting both IFNγ and IL-2 , which iBased on different studies, at least some of the pathogenic effects induced by the virus may be directly correlated with the expression of the U94 product. For example, after many years of somehow controversial results on the association between HHV-6 infection and multiple sclerosis, it has been recently shown that U94 might have a role in the demyelination process since it inhibits the migration of oligodendrocyte progenitor cells (hOPCs) . This efOf note, the possible role of U94 in host cell function had been already postulated in previous studies following HHV-6 isolation from cardiovascular tissues in vivo . This firas [Of note, U94 not only inhibited capillary formation in vitro but also affected endothelial cell migration and blocked angiogenesis in rat aortic rings, suggesting possible useful applications where antiangiogenetic functions are needed, as, for example, in tumors. Toward this hypothesis, previous studies have shown that NIH3T3 cells stably expressing U94 suppressed transformation by the oncogene ras  and thatras .Src and inhibit cancer cell motility, invasion, and anchorage-independent growth [Src expression and its downstream signaling cascade, and induced a partial mesenchymal-to-epithelial transition in transduced cells, with a strong downregulation of TWIST (a recognized tumor-promoting invasion), N-cadherin, Snail1, and MMP2. In addition, U94 expression caused a dramatic decrease in tumor growth in vivo in NOD/SCID mice [Based on its potent antiangiogenic and antimigration activity in endothelial cells, the biological impact of U94 was recently tested in a human breast cancer cell line (MDA-MB-231), showing that it can downmodulate the expression of t growth . In detaCID mice . InteresCID mice , and supCID mice , downregCID mice , which aRecent advances have started to unravel the role of U94 and its possible effects at cell and tissue levels. In fact, originally investigated for its particular presence and role in HHV-6A/B, the U94 gene product has indeed revealed a plethora of actions that are even unrelated to the virus cycle. Reported findings on these aspects regarding the putative actions of HHV-6 U94 are summarized in As to the viral life cycle, reported U94 functions include virus latency, integration, and reactivation; however, more information would be needed to precisely define its role and mechanism of action. Large-scale population studies, as well as systemic monitoring of iciHHV-6, could provide conclusive answers on the biological mechanisms involved in U94 action. The virus reactivates frequently in latently infected adults, as shown by the isolation of the virus from saliva, and virus reactivation can also be observed in iciHHV-6 individuals. Multiple studies have associated virus reactivation with several clinical conditions, but the mechanisms of HHV-6 reactivation and the role of U94 in these processes are still poorly elucidated. Of note, overexpression of U94 inhibits β-herpesvirus replication, including HCMV, HHV-7 (strain CZ), HHV-6A (strain U1102), and HHV-6B (strain Z29) , thus hiAt the immune response level, the production of a purified recombinant U94 protein allowed the recognition of a specific anti-U94 immune response and opened the way to studies regarding the potential association between HHV-6 infection and disease. In fact, contrary to what was observed in an anti-HHV-6 response, which is generally unmodified in specific subpopulations because of high virus prevalence in the human population, a differential response toward U94 was observed in specific patients affected by some autoimmune diseases . Thus, the quantification of anti-U94 humoral and/or cellular responses may be used as a marker for an association between the disease and HHV-6 infection, as well as to monitor the effectiveness of eventual antiviral therapies.Finally, but perhaps first as to its importance in human biology, the U94 protein has been shown to possess key biological properties, some of which are directly correlated with HHV-6 pathogenesis (such as U94 action in the demyelination process). Among them, the ability of U94 to modulate the angiogenesis process, likely related to eventual vascular damage during natural virus infection, appears to have great potential as a therapeutic molecule due to the key role of neovascularization in tumor onset and progression. The results obtained by using this virus protein in vitro and in vivo are intriguing and promising and could open the way to new approaches in anticancer therapy. Based on the reports identifying the active domain of U94 in the N-terminal portion of the protein, it might be important to dissect the protein and identify the minimum active peptide toward the optimization of the anticancer effect of U94. A further interesting aspect is the inhibition of cholesterol synthesis, another pathway that deserves further investigation. Transcriptome and proteome approaches may be profitably used toward an understanding of the molecular pathways influenced by U94, possibly contributing to identifying new U94 action networks.In conclusion, the overall picture about U94 functions is still not complete, and the properties of U94 that have been uncovered in recent years have opened the way to finding the pieces of information still lacking, hopefully leading to the development of useful tools in the management of diseases of different origin. A patent for the use of U94 of HHV-6 and its derivatives to modulate HLA-G expression has been obtained by the authors based on the results summarized in this manuscript (Patent n° 102018000006137).


Despite a growing body of literature on HIV service costs in sub-Saharan Africa, only a few studies have estimated the facility-level cost of prevention of Mother-to-Child Transmission (PMTCT) services, and even fewer provide insights into the variation of PMTCT costs across facilities. In this study, we present the first empirical costs estimation of the accelerated program for the prevention of mother-to-child transmission of HIV in Zimbabwe and investigate the determinants of heterogeneity of the facility-level average cost per service. To understand such variation, we explored the association between average costs per service and supply-and demand-side characteristics, and quality of services. One aspect of the supply-side we explore carefully is the scale of production—which we define as the annual number of women tested or the yearly number of HIV-positive women on prophylaxis.We collected rich data on the costs and PMTCT services provided by 157 health facilities out of 699 catchment areas in five provinces in Zimbabwe for 2013. In each health facility, we measured total costs and the number of women covered with PMTCT services and estimated the average cost per woman tested and the average cost per woman on either ARV prophylaxis or ART. We refer to these facility-level average costs per service as unitary costs. We also collected information on potential determinants of the variation of unitary costs. On the supply-side, we gathered data on the scale of production, staff composition and on the types of antenatal and family planning services provided. On the demand side, we measured the total population at the catchment area and surveyed eligible pairs of mothers and infants about previous use of HIV testing and prenatal care, and on the HIV status of both mothers and infants. We explored the determinants of unitary cost variation using a two-stage linear regression strategy.The average annual total cost of the PMTCT program per facility was US$16,821 . The average cost per pregnant woman tested was US$80 (median US$47), and the average cost per HIV-positive pregnant woman initiated on ARV prophylaxis or treatment was US$786 annually (median US$420). We found substantial heterogeneity of unitary costs across facilities regardless of facility type. The scale of production was a strong predictor of unitary costs variation across facilities, with a negative and statistically significant correlation between the two variables (p<0.01).These findings are the first empirical estimations of PMTCT costs in Zimbabwe. Unitary costs were found to be heterogeneous across health facilities, with evidence consistent with economies of scale. Prevention of Mother-to-Child Transmission (PMTCT) is an integral component of HIV prevention programs in generalized epidemics. If implemented effectively, PMTCT has the potential to reduce HIV among infants , extend The ambitious target of EMTCT initially set for 2015 as a millennium development goal , requireThe literature evaluating efficiency and costs of HIV services has frequently focused on program- or national-level cost estimates, which often mask substantial heterogeneity at the clinic level. Although there is a growing body of literature on the per-client cost of HIV services in sub-Saharan Africa –21, onlyExamining variation across facilities as well as both the demand- and supply-side characteristics associated with it is critical to understand efficiency. For example, the ORPHEA study  examinedThe objective of this paper is twofold: 1) to estimate the facility-level total costs and the average cost per woman served (unitary costs) in the Accelerated National PMTCT Program. We measured services along two steps in the service cascade–HIV testing and counseling, and antiretroviral (ARV) prophylaxis or initiation of antiretroviral therapy (ART). 2) To explore determinants of unitary cost variability across facilities. We investigate three potential sources of variation. On the supply side, we explore the role of characteristics of the facilities, such as scale, the type of facility, the staff composition, location of the facilities, and availability of other related services nearby. On the demand side, we investigate the maternal HIV prevalence, MTCT rates, HIV free infant survival rate at 9–18 months, and uptake of HIV testing during pregnancy—all at the catchment area level. Finally, we also explore whether quality—as measured by the completeness of family planning, antenatal, and PMTCT services—is associated with unitary costs variation. We use data from a cross-sectional survey of 157 randomly selected facilities offering PMTCT services and their catchment areas, in five provinces in Zimbabwe.We analyzed 2013 data from the impact evaluation of Zimbabwe’s Accelerated National PMTCT Program. The project was funded by the Children’s Investment Fund Foundation together with Elizabeth Glaser Pediatric AIDS Foundation Zimbabwe. The impact evaluation combines cross-sectional surveys to determine the impact of Zimbabwe’s EMTCT program on MTCT as well as HIV-free survival among children aged 9 to 18 months. Previous results from this project have analyzed different aspects of the Program. For example, McCoy et al. (2015) examined the uptake of services and determined the factors associated with MTCT, maternal ART, and ARV prophylaxis . BuzdugaIn this analysis, we adopt a microeconomic lens to analyze efficiency. In the case of PMTCT services supplied by government facilities, scale  cannot be assumed to be exogenous–as is typically the case in the standard microeconomic model. The level of output produced by a health facility is determined as much by decisions made in the facilities, as by decisions made by patients. Health facilities choose not only how to allocate their resources, but also when to offer services, the quality of those services, and in general the type of experience they provide to patients. The results from those decisions in turn influence patients, who decide whether to demand those services or not, subject to their budget and other financial and non-financial constraints; and where to demand those services, constrained by the availability of nearby services. Thus, scale, measured as the number of services provided, is the result of decisions on the supply side by health providers, as well as decisions on the demand side by patients.Two critical elements on the demand side, which may influence efficiency, are size and complexity. Higher demand potentially leads to an expansion of scale, which in turn is associated with lower unitary costs because fixed costs are distributed across more patients and also because human resources operate closer to full capacity (less idle time). This gain in efficiency as a result of larger scale is known as economies of scale. On the other hand, populations with more complex needs may increase costs. For example, the most expensive drug combinations that include more than three different elements are recommended only to patients in the most advanced stage of the disease , or highIn the analysis presented in this paper, we used both types of characteristics of the demand–size and complexity, and we use the size of population on clinics’ catchment areas as an instrument for the endogenous measure of scale. For proxies of demand complexity, we used maternal HIV prevalence; rate of mother-to-child transmission of HIV; rate of HIV-free infant survival; and uptake of HIV testing during pregnancy.In 2010, the World Health Organization issued guidelines for EMTCT based on the CD4 count of HIV positive mothers. CD4 T-cells are a type of white cell part of the immune system that fights bacteria, viruses, etc. The CD4 count is a test that measures the number of T-cells to assess the immune system’s health. HIV-infected pregnant women with immune damage of less than 350 CD4 cells, were eligible for lifelong antiretroviral treatment (ART). On the other hand, pregnant women ineligible for ART (CD4>350) were recommended to receive one of two options of prophylactic regimens: Option A consisted of starting at 14 weeks of pregnancy through 7 days postpartum; or Option B, an ARV preventive regime beginning at 14 weeks of pregnancy and continuing until weaning . In 2013The Accelerated National PMTCT Program was implemented in 2011 by Zimbabwe's Ministry of Health and Child Care (MoHCC) based on PMTCT Option A from WHO's 2010 guidelines. Zimbabwe then switched to Option B+ in late 2013. , 30, 31.The sampling strategy followed a two-stage process. First, we randomly selected 157 out of 699 health facilities from five provinces that provided PMTCT services for the entirety of 2013 in Zimbabwe: Harare, Mashonaland West, Mashonaland Central, Manicaland, and Matabeleland South. These provinces were selected to include three of the four largest cities in Zimbabwe, rural communities with high and low HIV prevalence, representation of both major ethnic groups in Zimbabwe (Shona and Ndebele), and areas where detailed monitoring-and-evaluation data were being collected . Second,We estimated a sample size of 157 PMTCT clinic catchment areas. We expected to identify an average of 190 infants aged 9–18 months per catchment area of PMTCT clinic, assuming 10.5 living infants aged 9–18 months per 100 households and approximately 1,800 households per catchment area of a PMTCT clinic. If we identified all eligible infants and enrolled 1 in every four eligible infants, this would result in 47.5 living infants aged 9–18 months per catchment area. Our estimated sample size for the baseline community survey is approximately 7,800 infants  and their mothers or caregivers from 157 PMTCT catchment areas. However, those initial sample size calculations were revised upwards after incorporating new data on variability in the number of eligible mother-infant pairs and the underlying HIV prevalence across catchment areas. The overall size of the population recruited increasing from an estimated 7,800 to 9,087.The survey team administered a short questionnaire in each of the 157 health facilities. All data were collected on paper by a trained data collector and later entered into an Access™ database by a data entry operator. Researchers conducted periodic reviews of the data for completeness and consistency.We collected data on the type of facility visited, according to Zimbabwe’s classification of health centers. Our sample included ten different types of health facilities; however, for the analysis, we grouped them into hospitals and non-hospitals (See S 1 for a detailed description of the different types of facilities). PMTCT services costs in hospitals tend to be higher. One important reason is that hospitals are more complex facilities, given that they provide not only outpatient but also inpatient services. Hospitals also tend to include more specialized, therefore higher paid medical staff. Because of these reasons, one hypothesis regarding our study is that hospitals are less flexible in terms of allocation of resources compared to clinics. This lack of adaptability could decrease efficiency and increase costs.We adopted the perspective of service providers on the analysis. Therefore, we did not measure patients’ expenditures, such as out-of-pocket fees or transportation expenses. Moreover, only 4% of the women in the sample declared to have paid for PMTCT services.We used a retrospective microcosting approach to measure monthly quantities and prices of three essential input categories: personnel, ARV drugs, and HIV tests kits. These categories comprise the largest share of the total costs of PMTCT services, according to previous studies , 14, 33.We valued all inputs at market prices, including donations, adopting an economic rather than a financial costing approach. Given that all the units in our sample are government clinics, there is no variation in input prices or salaries across facilities. The Ministry of Health centrally establishes wages based on cadre categories and purchases all essential inputs (drugs and tests) through centralized procurement processes. Thus, the variation in unitary costs across facilities reflects differences in efficiency; the ability of clinics to produce services, given the resources at their disposal and given the demand characteristics they face.We collected data on the total annual outputs produced along two steps in the PMTCT service cascade–HIV testing and ARV/ART initiation. When these data were not available at the facilities, we collected them from the district health information system.We also assessed the time allocation of personnel providing PMTCT services through interviews with five randomly selected providers per facility, in which we asked them to report the time they spent working on PMTCT every day of the previous week. Self-reported time allocation to specific tasks has been found to overestimate effort allocated to particular services . We atteWe categorized staff in three types of nurses  and one broad category of health personnel which includes counselors, health promotion officers and other support staff (S 2). "Primary care nurse" was the category with the lowest average salary, and the second most common in the sample, after "general nurse."Additionally, we collected information on the scope and comprehensiveness of PMTCT and other related maternal services provided at the facilities, as a proxy for quality. Process quality, which measures the extent to which providers follow the processes outlined or explicitly listed in official guidelines, has been used previously as an indicator of the quality of health care . For exaThere is also evidence from less affluent countries; Das and Gertler (2007) document practice quality on six different studies in five low- and middle-income countries. They provide evidence on the large effect of process quality on health outcomes compared to those of availability or structural quality . Das andWe collected information on the availability of services offered at the facilities as a proxy for process quality. Specifically, we asked which services they provided out of a comprehensive list of family planning, ANC and ART/ARV prophylaxis services recommended by the government. For example, whether contraception services were offered during antenatal visits, after labor and delivery, during postnatal care visits, and during child immunization visits, and constructed a variable index for contraception services based on the number of recommended care practices undertaken in the facility. We computed similar variables for antenatal services and ARV prophylaxis or ART (see S 2 for the complete list of recommended processes used for each indicator).We also collected data on some characteristics of the demand as proxies for size and complexity. We surveyed mothers about their HIV status and estimated the maternal HIV prevalence; the rate of mother-to-child transmission of HIV; the rate of HIV-free infant survival; and the uptake of HIV testing during pregnancy. Finally, we asked each facility for the estimated total size of the population they served. All of these variables refer to the catchment area level.The survey team administered a household-level survey in 2014 to the selected sample of 9,087 mother-infant pairs. Mothers or caregivers 16 years old and older and their infants born between 9 and 18 months before the interview , were eligible to participate in the study. The data enumerator sought informed consent to complete the questionnaire and to collect dried blood samples (DBS) for HIV testing from the mother and her eligible infant. The mother could consent to participate in both, neither, or either the questionnaire and DBS.The questionnaire was administered using a Personal Digital Assistant (PDA) and captured the mother’s demographic characteristics, her experience with ANC and HIV testing, and more specifically, her experience with ANC during the pregnancy for the eligible child . Finallyj denotes total costs of facility j. Personnel costs were estimated by the sum of the number of hours hij each type of personnel i in a facility j worked on PMTCT services, multiplied by the hourly wage wi corresponding to provider type i. Recurrent costs (ART drugs and HIV test kits) were calculated as the sum of the k number of goods xjk multiplied by their prices pk.We calculated the total annual costs of PMTCT services for each facility as the sum of personnel and recurrent inputs costs used in 2013, as follows:jl along the cascade were defined as:jl is the 2013 annual number of outputs produced by facility j along the cascade indicator l, where l = 1 for number of pregnant women tested, l = 2 for HIV positive women on ARV prophylaxis or treatment.Then, the facility-level average unitary costs per output, ACThe objective of this costing approach is to explore the efficiency of the PMTCT program by looking at the heterogeneity of "unitary costs" across implementers. This approach provides insight on the efficiency of the program at the facility level and across the service cascade.We explored the facility-level variation of the unitary costs of PMTCT at two steps of the service cascade: HIV testing and ARV prophylaxis or ART. First, we present the dispersion of average unitary costs by type of facility to describe the variation across facilities. Then, we explore three potential determinants of the heterogeneity in unitary costs. On the supply side, we investigated the role of scale, staff categories, and facility type. On the demand side, we included in the analysis the prevalence of maternal HIV, the rates of mother-to-child transmission of HIV and HIV-free infant survival at 9–18 months, and the levels of uptake of HIV testing during pregnancy. Finally, we analyzed the role of the quality of services.We were interested in identifying how supply- and demand-side characteristics influence the variation of unitary costs across facilities, and in particular the role of the scale of PMTCT services. However, a simple linear regression could yield a biased parameter due to endogeneity bias since there may be unobservable or omitted characteristics simultaneously explaining costs and scale. To minimize this potential bias, we apply a two-stage least square model using demand size, measured by the size of the population at the catchment-area level, as an instrumental variable (IV). With this approach, we estimated first, the association between demand size and scale; and, second, the effect of scale and other supply-side characteristics on the unitary costs of PMTCT services.i represents the log-transformed unitary costs of PMTCT services (for each step of the cascade): the average cost per pregnant woman tested for HIV in facility i, or the average cost of an HIV-infected pregnant woman on ARV prophylaxis or ART in facility i. Scale is the endogenous variable represented by xi, ssi is a vector of s supply characteristics including a binary variable for type of facility , the proportion of primary care nurses with respect to the other types of staff; ddi is a vector of d demand-side characteristics, qqi is a vector of q process quality variables; and εi is the residual term.The second stage regression model is specified as follows:i) to obtain adjusted values of x (x*) and use them in the second stage of the analysis , and the average cost per HIV-infected pregnant woman receiving ART or ARV prophylaxis was $786  per year. The former ranged from $12 to $528, and the latter from $61 to $5,018.This variation in unitary costs across facilities is further illustrated in Healthcare costs data is usually skewed. To implement linear regression models, we transformed the data to natural logarithms and decrease skewness. This kind of transformation is a common approach in applied econometrics. Total population size in the catchment area satisfies the Stock-Yogo test for weak instruments as the F statistic for significance of the instruments in the first stage exceeds 10. The Durbin-Wu-Hausman test for over-identifying restrictions is satisfied only for cost per woman in ART but not for cost per woman tested implying that scale is not endogenous in regression 4 and OLS model 1 is valid. Second-stage estimates of the 2SLS model show a strong negative relationship between scale and unitary costs—Columns (4) and (6)—with slightly smaller coefficients than those from the naïve OLS estimations, which illustrates the relatively small magnitude of the bias introduced by the naïve models. According to the 2SLS results, an increase of 10% annual number of women served translated into an average 5.8% decrease in the cost per woman tested, and a 4.2% decrease in the cost per woman initiated on ART/ARV. Our identification strategy assumes that size of the population at the catchment area is a valid instrument, which means it only affects costs through scale. In order to test this assumption, we consider other paths through which population size may affect costs. For example, whether a catchment area is an urban area or not may affects costs. We tested this possibility and found that urbanicity and HIV test costs or ART costs are not correlated (0.01 and -0.01 respectively). Another reason for population size influencing costs other by scale could be that health professionals prefer to live in more populated areas, most likely urban areas, so there might be more competition to be relocated there. This may cause more concentration of higher skilled health providers in more populated areas which may increase the cost of health services. We explored this hypothesis and found that facilities in more populated areas have higher concentration of high skilled health providers ; and low concentration of low skilled providers (nurse aids -0.32). However, there is no association between proportion of skilled workers in the facility and costs (S4 File).We further explore the relationship of costs and scale with non-linear OLS models. In Results from Tables The data also confirmed the relationship between personnel categories and unitary costs; with lower costs linked to a higher proportion of primary care nurses. We found a statistical association between the type of facility and lower costs per woman tested. There was no significant difference in unitary costs per woman on ART between hospitals and non-hospitals, and we found no association between process quality of reproductive and maternal services and unitary costs.On the demand side, we found a significant association between maternal HIV prevalence and the proportion of women tested for HIV in prenatal care with higher HIV testing costs.Using data from a cross-sectional survey of 157 randomly selected health facilities offering PMTCT services in Zimbabwe, in this paper we presented the first empirical estimations of the PMTCT program costs along the service cascade in Zimbabwe, adopting the perspective of the government. We found that the average annual total cost of PMTCT per facility was $16,821 . The type of facility is a significant predictor, with an average of $20,786 in hospitals and $16,162 in non-hospitals. The average annual cost per service was $80 per pregnant woman tested for HIV , and $786 per HIV-infected pregnant woman on ARV prophylaxis or ART .Our results are comparable to previously published estimates. In a study conducted in four African countries in 2013 , found tA unique feature of our study is the large sample size of facilities included in the analysis, which allowed us to analyze the technical efficiency of PMTCT service provision in Zimbabwe. Our results demonstrate the high variability of facility-level unitary costs, a finding consistent with previous research. For example, a study that identified significant heterogeneity in PMTCT unitary costs in Kenya, Nigeria, Rwanda, South Africa, and Zambia with sample size similar to ours, and suggested potential gains from more efficient delivery of services , 14.In this study, we explored the relationship between supply-side characteristics, demand size and complexity, and unitary costs. Scale of services was the primary predictor of unitary cost variability, a link that has been documented previously , 21, 40.Another supply-side determinant of efficiency identified in this study was the type of facility. Hospitals were found to be 43% more expensive for HIV testing than clinics, on average. While our data do not allow us to identify the reasons behind this difference, one hypothesis is that relatively sophisticated facilities necessarily provide this simple service at a higher cost than clinics. Another possibility is that the quality of testing services in hospitals is higher with better content, longer duration, and more skilled labor. Our data show that facilities relying more heavily on primary care nurses were 40% less expensive for HIV testing than other using personnel with higher salaries. We did not see a correlation between the quality of services and unitary costs. We found no difference between hospitals and non-hospitals in the cost per woman on ARV prophylaxis or ART.On the demand side, our results did not suggest a relationship between the complexity of the demand and unitary costs. However, we did observe a strong correlation between demand size and scale. Our results predicted a 6% increase in the number of women on ART for every 10% increase in population size.In this study, we use instrumental variables as a strategy to estimate the effect of scale on unitary costs of PMTCT services. As mentioned before, previous studies have documented this relationship and found evidence consistent with the existence of economies of scale. However, to our knowledge, this is the first study that uses an econometric strategy to disentangle the endogenous relationship between scale and unitary costs. We instrumented scale with population size at the facility catchment area level. We found that while population and scale are strongly correlated, population and costs are not, which is consistent with the assumptions of our identification strategy. Our results suggest then the presence of economies of scale in the production of PMTCT services in Zimbabwe.Readers should interpret our results in light of the limitations of the study. First, the data collected on time allocation by staff was self-reported, which has been documented to be biased upwards when compared to more reliable, observation-based methods, such as time-motion studies , 22, 41.Second, our measurement of costs included the major costs categories, namely, staff, ARV drugs, and HIV tests kits. We left out costs associated with recurrent services, such as utilities and fuel, capital costs like buildings and equipment, and costs related to supervision and training. While the relative contribution of these categories to total PMTCT costs has been documented to be less than 10% , 14, 33,Our indices of process quality focus on completeness or comprehensiveness of services. Although previous studies have found associations between this type of quality proxy and health outcomes and patient's perceptions , 37, 38,Although the validity of an instrumental variable cannot be fully statistically tested, we tried to verify the two conditions for the instrumental variable model to be valid. We found a strong correlation between population and scale and explored potential channels through which population can affect costs other than scale and did not find any. These results support our choice of instrumental variable.We found a strong relationship between the scale of services and unitary costs, consistent with the existence of economies of scale and robust after correcting for potential endogeneity bias. However, the policy implication of this finding should not be to provide PMTCT services only in large facilities or in catchment areas with large populations. This interpretation could have undesirable equity consequences on communities in smaller locations. The policy implication should be instead to focus efforts on understanding the differences between inefficient and efficient small facilities—as well as between inefficient and efficient large facilities. In other words, efforts on identifying other determinants of efficiency while accounting for scale effects would be valuable. For example, our results suggest that less sophisticated facilities and staff composition also have efficiency implications. Another policy consequence of the scale effect is for programs to make the appropriate budget planning to adjust for the higher cost per patient expected in smaller facilities. Finally, HIV programs could also consider merging small facilities when doing so could represent gains in efficiency without compromising access to services by the population.Despite the limitations of the study, our results provide robust cost estimates of the PMTCT program in Zimbabwe using data from a cross-sectional survey of 157 randomly selected health facilities. Furthermore, we found that a large proportion of heterogeneity in unitary costs is explained by scale, which, in turn, is determined by population size in the catchment area. Although our results identify strong associations and control for potential biases, the nature of our data does not allow us to establish robust causal relationships. Program designers nonetheless can draw lessons to improve the efficiency of PMTCT programs without compromising equity.S1 File(DOCX)Click here for additional data file.S2 File(DOCX)Click here for additional data file.S3 File(DOCX)Click here for additional data file.S4 File(DOCX)Click here for additional data file.


In India, under the National Tuberculosis Elimination Programme, the government provides free treatment for multidrug-resistant tuberculosis; however, many patients seek care elsewhere, which is costly. To determine those out-of-pocket expenses, we interviewed 40 presumptive patients and found that they spent more than their median annual income before registering for the government program. In India, the annual economic loss resulting from tuberculosis (TB) is US $3 billion , line probe assay, or culture, per PMDT guidelines (>15 years of age) with MDR TB who were registered under PMDT during August 2016–April 2017. By using a valid, pretested, semistructured tool, we interviewed patients about various costs incurred by themselves, their families, or both, from the time they became a presumptive MDR TB patient until they underwent pretreatment evaluation at PMDT. Information about various costs reported by patients was validated with bills, if available. We used the following cost categories: direct medical, direct nonmedical, indirect, and coping. Direct medical costs are expenses incurred during diagnosis and treatment of illness; direct nonmedical costs are costs of food, accommodations, and additional nutrition/supplements; indirect costs are the loss of wages because of illness; and coping costs are the costs of coping mechanisms   . Of the orrowed) . Ethics https://www.epidata.dk) and analyzed by using SPSS Statistics 25.0 (https://www.ibm.com) and EpiData analysis 2.2.2.183 software. Direct and indirect costs were summarized as median and interquartile ranges (IQRs). Categorical variables were expressed in proportions. Costs were collected by using Indian rupees (INR) converted to United States dollars (USD) based on the 2016 conversion rate (1 USD = 66.3731 INR). To compare the costs across different countries, we first converted the reported costs (USD) from other studies to local currency for the reported year, then adjusted them for inflation year by year until 2016 (Data were double entered in EpiData version 3.1 software (til 2016 .We included 40 of the 63 registered patients in the study. Median (IQR) age of participants was 39 (29–50) years. Most patients were male , and most lived in rural areas . Median (IQR) reported patient family income was $608 ($228–$912)/year. Of the 40 patients, 39 (97%) had pulmonary MDR TB and 24 (60%) had approached the private healthcare sector for their first clinical encounter .The median (IQR) pretreatment out-of-pocket expenses incurred by patients were $171 ($72–$432) total, $105 ($49–$306) direct, and $51 ($2–$306) indirect. Within direct costs, direct nonmedical costs ($51) were more than direct medical costs ($37). Of the direct nonmedical costs, most was spent on food ($35). Most of the direct medical costs were for diagnostic investigation ($18) and treatment ($15) .The median total pretreatment out-of-pocket expense incurred by patients in our study is similar to that found in a study in Peru ($210) after adjusting for inflation rate and cost conversion (The median indirect out-of-pocket expense incurred by patients in India was $51 ($2–$306). This finding contrasts with those of studies in Ethiopia and Indonesia, where indirect pretreatment costs after adjustment for annual inflation were substantially lower  . The adjusted indirect expenses were 10 times greater ($51 vs. $578)  Table.In addition, 18 (45%) patients in the study lost their job because of the disease and had to borrow money for disease management and daily household needs before receiving accurate diagnosis and appropriate treatment. The percentages of persons with job losses were substantially lower than those reported for Peru (90%) and Ethiopia (72%) but similar to those for Indonesia (53%) . Wingfield et al. reported a median debt of $435 and a loss of income of $2,450 before diagnosis for patients in Peru  of the patients, approximately two thirds, had already incurred catastrophic expenses before they were registered for MDR TB treatment.Our study appraised the costs expended by MDR TB patients from a single PMDT center. Determination of a complete estimate of costs borne by all MDR TB patients in India would require a comprehensive study conducted at the community level and inclusion of patients receiving treatment from the public and private healthcare sectors.New strategies that systematically engage private providers are needed to reduce the cost burden surrounding diagnosis for vulnerable patients. The government of India may consider widening the spectrum of free services before patient enrollment in a government-monitored treatment program channeled through the private sector.Additional methods and results of study for pretreatment out-of-pocket expenses for presumptive multidrug-resistant tuberculosis patients, India, 2016–2017.


Influenza viruses are presumed, but not conclusively known, to spread among humans by several possible routes. We provide evidence of a mode of transmission seldom considered for influenza: airborne virus transport on microscopic particles called “aerosolized fomites.” In the guinea pig model of influenza virus transmission, we show that the airborne particulates produced by infected animals are mainly non-respiratory in origin. Surprisingly, we find that an uninfected, virus-immune guinea pig whose body is contaminated with influenza virus can transmit the virus through the air to a susceptible partner in a separate cage. We further demonstrate that aerosolized fomites can be generated from inanimate objects, such as by manually rubbing a paper tissue contaminated with influenza virus. Our data suggest that aerosolized fomites may contribute to influenza virus transmission in animal models of human influenza, if not among humans themselves, with important but understudied implications for public health. Influenza viruses are believed to transmit through the air as respiratory droplets or aerosols. In the guinea pig model, Asadi et al. show that influenza virus can also be transmitted as aerosolized fomites, which are microscopic dust particles stirred up from a virus-contaminated environment. Precisely how influenza spreads among humans has long been a matter of debate11; however, there is broad agreement about the possible modes of transmission between humans11. Direct or indirect contact modes require a susceptible person to self-inoculate by, for instance, touching one’s nose with a virus-contaminated hand; “direct” indicates that person-to-person contact transfers the virus between infected and susceptible hosts, whereas “indirect” implies transmission via a fomite, which is an object like a doorknob or toy that has been contaminated with infectious virus9. Airborne transmission may occur by two modes, either by sprays of virus-laden respiratory droplets, such as from a cough or sneeze, impacting immediately onto the respiratory mucosa of a susceptible individual, or by the eventual inhalation of droplet nuclei, microscopic aerosol particles consisting of the residual solid cores of evaporated respiratory droplets9. The relative contribution of each of these transmission modes remains unknown, and viral, host, or environmental factors may affect which modes are favored in different settings13.Seasonal epidemic influenza causes hundreds of thousands of deaths worldwide every year15. Animal models of influenza virus transmission are often used to try to elucidate these uncertainties, and to test the efficacy of vaccines13, under controlled laboratory conditions. Typically, in modeling airborne influenza virus transmission, a virus-donor animal, inoculated with an influenza virus, and a virus-recipient animal, naive to influenza virus, are physically separated in cages that share a common air space, such that infectious particles generated by the donor animal must travel through the air to infect the susceptible recipient16. However, the physical nature of these infectious particles is just beginning to be elucidated22; thus, in this work we seek to characterize the airborne particles that pass between inoculated donor and susceptible recipient in the guinea pig model of influenza virus transmission. We first show that the vast majority of airborne particulates emitted from a guinea pig cage are non-respiratory in origin and thus presumably environmental dust. We then demonstrate that infected guinea pigs heavily contaminate their fur and surrounding environment with virus, and we further establish that if these dust particulates become contaminated with influenza virus they can serve as aerosolized fomites that carry the virus to a susceptible guinea pig through the air. Finally, we show that aerosolized fomites can be generated from inanimate objects, such as by rubbing a virus-contaminated paper tissue. We conclude by discussing the implications of aerosolized fomites for respiratory virus transmission in other animal models and between humans.Uncertainty surrounding the modes by which influenza virus transmits among humans under different conditions hinders the assessment of non-pharmaceutical interventions designed to prevent influenza’s spread−1, and observed that the particle count spikes were almost entirely coincident with guinea pig motion  that enumerates particles in the size range of 0.3–20 μm. A camera placed above the cage simultaneously captured guinea pig movement over time Fig. . We thenion Fig. . Plottinion Fig. , suggestion Fig. . Mobile ion Fig. .Fig. 1Gu−1 prior to inoculation with influenza virus  (Pan99) virus only slightly increased particle emission by anesthetized, stationary guinea pigs, with up to 0.5 particles s−1 measured on days 2–3 post-inoculation from two of the three animals . Likewise, the size distributions of the particles emitted by euthanized animals were similar to the anesthetized animals, with 69% of the particles between 0.3 and 0.5 μm , and virus remained cultivable from swabs until at least 3 dpi Fig. . No meas24. We then paired the contaminated virus-donor animals with susceptible virus-recipient animals in cages that only permit transmission by airborne routes  per 30-min sample (1.2 FFU min−1), similar to our tissue experiments; the highest exhalation rate was 1100 FFU per 30 min (37 FFU min−1)20. Ultimately, the clinical import of any quantity of aerosolized fomites depends entirely on their infectiousness in the susceptible human host who inhales them. The human infectious dose for Pan99 aerosolized from paper tissues is unknown, but for an influenza A (H2N2) virus in liquid solution, aerosolized into similarly sized particles (1–3 µm), inhalation of ~2 pfu or fewer (0.6 to 3 TCID50) was found to be sufficient to initiate human infection26. Thus, we conclude that human infection by aerosolized fomites generated from an inanimate source like a virus-contaminated tissue is possible, though it remains to be demonstrated experimentally or empirically.In vitro, we demonstrate that an influenza A virus, dried on paper tissues for 30–45 min at ambient room temperature and humidity, retained infectiousness on aerosolized fomites that were collected from the air with a bioaerosol sampler and subsequently grown in cell culture. The quantity of aerosolized fomites generated by this method, 1–5 pfu min27  and 2 transmission events in 3 pairs of ferrets28 . These results suggest that influenza virus transmission via aerosolized fomites may be less efficient than transmission by respiratory droplets or droplet nuclei, under the conditions tested. An alternate interpretation, however, is that the immune, virus-painted donor guinea pigs contaminated their environment less effectively than the intranasally inoculated guinea pigs, thus decreasing the number of aerosolized fomites that could be produced under these conditions. The intranasally inoculated animals had the opportunity to continuously re-contaminate their environment as the virus replicated in their respiratory tracts, before their immune systems suppressed it. In contrast, the immune, virus-contaminated animals were contaminated only once, and viable influenza virus was not replenished. While the swabs from the animals’ bodies yielded comparable viable virus titers on days 2 and 4 post-inoculation or post-contamination, in the range of hundreds of pfu ml−1 of swab eluate, we recovered substantially more viable influenza virus from the cage walls of the intranasally inoculated animals than the immune, contaminated ones, a discrepancy that is as yet unexplained. Virus deposition by inoculated guinea pigs, possibly via direct nose and mouth contact, may be more efficient than the transfer of dried virus from the bodies of contaminated guinea pigs or its retention on the cage walls. Respiratory mucus may also have a preservative effect on non-porous surfaces, such that deposited virus retains its infectivity longer. These hypotheses remain open for future research. However, our results do serve as a clear proof of principle that influenza virus can transmit through the air when carried on micron-sized, non-respiratory particulate matter from the environment.In vivo, we show that guinea pigs painted with influenza virus harbored viable virus on their bodies for up to 4 days post-contamination, which was subsequently transmitted through the air to infect 3 of 12 virus-susceptible partner animals housed in separate cages . This transmission rate is lower than previously observed, under similar environmental conditions, with the same influenza virus isolate after intranasal inoculation into donor animals: 7 transmission events in 8 pairs of guinea pigs29 and experimentally explored30 in the past, and other respiratory pathogens are known or suspected to initiate human infection in this manner32. However, current opinion about influenza virus transmission, both in humans11 and in animal models33, appears to presume that airborne infectious virus derives solely from exhaled, coughed, or sneezed respiratory particles, or occasionally from aerosol-generating medical procedures11. We now provide direct experimental evidence that the airborne particles mediating mammalian influenza virus transmission need not be directly emitted into the air from the respiratory tract of an infectious host; rather, aerosolized fomites from a virus-contaminated environment can also spread influenza viruses through the air.Influenza virus transport on dust has been infrequently hypothesized30, raising the possibility that aerosolized fomites, and not solely expiratory particles, may contribute to airborne virus transmission in other animal species. Though only experimentally demonstrated in the guinea pig model of influenza virus transmission to date, aerosolized fomites could plausibly contribute to airborne transmission in other animal models, including not only ferrets but also hamsters35, swine37, and mice39. This possibility should be experimentally explored in a rigorous fashion, because the confidence with which we can extrapolate animal data to human influenza rests on the degree to which animal models are representative of human influenza. If aerosolized fomites contribute differently to airborne influenza virus transmission in animal models and humans, it would be an important caveat in generalizing animal data to public health.As we have demonstrated in guinea pigs, infected ferrets have been shown to contaminate dust in their environment with infectious influenza virus41. In particular, mutations that increase the thermal and acid stability of the receptor-binding hemagglutinin (HA) protein enhance the transmissibility of H5N1 viruses in ferrets42. Linster et al.40 hypothesized that, although the mutations that enhanced “airborne transmission between ferrets may be related to the pH of fusion or thermostability, these properties may merely be a surrogate for another—as yet unknown—phenotype, such as stability of HA in aerosols, resistance to drought, stability in mucus, or altered pH in the host environment”40. Subsequently, when aerosolized under laboratory conditions, swine influenza viruses that transmit efficiently by air among ferrets were shown to remain infectious in aerosols longer than viruses that transmit less efficiently43. When the airborne virus source was an infected ferret, a human influenza virus isolate from the 2009 H1N1 pandemic demonstrated enhanced airborne survival, relative to a variant virus encoding a destabilizing HA mutation that increases its pH of fusion44. These data suggest that the stability of the HA protein in the environment is one critical factor in influenza virus transmissibility by airborne routes.Our data do not explain why certain strains of influenza virus transmit with poor efficiency via airborne routes, but the literature offers some clues. Avian influenza A viruses of the H5N1 subtype have been found to replicate efficiently in but transmit poorly among both ferrets and guinea pigs, and key amino acids in a few viral proteins have been implicated in determining airborne transmissibility in these animal models46, while ferrets do47. Ferrets have been shown to transmit fewer strains of influenza B virus by airborne routes48 than guinea pigs50. But both influenza A (H1N1) and B viruses have transmitted well enough among people to become endemic in humans. Given the overall complexity of influenza virus transmission, it is unlikely that the presence of aerosolized fomites or their environmental stability entirely explains why some influenza viruses transmit through the air efficiently while others do not. More likely, airborne transmission efficiency depends variably on multiple factors: the infectiousness of the virus donor, the susceptibility of the virus recipient, and the stability of the virus in the environment between them. The transmission chain has many links, any of which could be a weak link that precludes efficient transmission, whether by aerosolized fomites or any other route. Despite the many remaining uncertainties surrounding influenza virus transmission in both humans and animal models, however, our data do indicate that aerosolized fomites may contribute, under specific circumstances that remain to be fully elucidated, to airborne influenza virus transmission in an animal model.However, species also has a role in the transmissibility of different influenza virus strains. Guinea pigs do not transmit pre-2009 H1N1 viruses efficiently12 by the airborne route. During the COVID-19 pandemic in China, air sampling in various hospital locations found the highest airborne genome counts of SARS-CoV-2 in rooms where health care workers doffed their personal protective equipment (PPE), hinting that virus was possibly being aerosolized from contaminated clothing as it was removed51. In light of our experiments, we conclude that the contribution of aerosolized fomites to respiratory virus transmission in both humans and animal models requires further scientific consideration and rigorous investigation.To our knowledge, no experimental evidence exists to establish that the airborne transmission of influenza viruses between experimental animals, or even between humans, occurs entirely via exhaled respiratory particles, as is commonly presumed. Our experimental data confirm that influenza virus transmission by aerosolized fomites is, at minimum, biologically plausible, and possibly generalizable to other respiratory viruses that transmit preferentially or opportunistically52 (Millipore Sigma) in Dulbecco’s Modified Eagle Medium  supplemented with 10% fetal calf serum and 1 mg ml–1 of G418 selective antibiotic  at 37 °C and 5% CO252. Stock virus was grown in the allantoic fluid of 10-day-old embryonated chicken eggs for 48 h at 37 °C53 and then frozen in aliquots at −80 °C.Influenza A/Panama/2007/1999 (H3N2) virus (Pan99) was cultured in Madin Darby canine kidney (MDCK)-SIAT1 cells−1) administered intramuscularly. For intranasal inoculation, Pan99 stock virus was diluted in PBS supplemented with antibiotics  (PBS + P/S). An inoculum of 104 plaque-forming units (pfu) in 300 μl was instilled intranasally by applying 150 μl to each nostril54. Inoculated guinea pigs were placed supine, in a nose-up position, during recovery from anesthesia (~30–45 min). All procedures were performed in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals55, and the research protocol was approved by the Icahn School of Medicine at Mount Sinai Institutional Animal Care and Use Committee (IACUC protocol #2014-0178).Five- to six-week-old female Hartley strain guinea pigs weighing 350–400 g were obtained from Charles River Laboratories. Animals were allowed access to food and water ad libitum and kept on a 12-h light-dark cycle. Prior to intranasal virus inoculation, guinea pigs were anesthetized with a mixture of ketamine (30 mg/kg of body weight) and xylazine  mounted above the cage recorded guinea pig movement at 1 image s−1 using iSpy (64-bit) software, version 7.2.1.0. Three healthy guinea pigs were placed individually inside the measurement cage with granulated dry corncob (CC) bedding   with an airtight, transparent Plexiglas lid, was modified to be connected to an aerodynamic particle sizer  with conductive silicone tubing (inner diameter of 0.95 cm and length of 40 cm) Fig. . The APS1)” Fig. . To asse1)” Fig. , the par2 inhalation under ketamine and xylazine anesthesia. Death was confirmed by physical examination57 before placing them individually inside the aluminum sleeve. APS measurements were taken for 30 min per guinea pig. The time-averaged particle emission rates, The APS measurement cage was also used to quantify the expiratory particles from three anesthetized guinea pigs prior to and after intranasal inoculation with Pan99. To minimize non-respiratory background particulates, each anesthetized guinea pig was placed in a closed aluminum sleeve , Fig.  were cal5), Fig.  indicatiEach APS experiment was performed once per condition, on each of three individual guinea pigs. For the awake, mobile guinea pigs, the variable experimental condition was bedding type . For stationary guinea pigs, the variable in the experimental conditions were pre-infection vs. post-infection with Pan99 virus and anesthetized vs. euthanized guinea pigs. Measurements on anesthetized guinea pigs were performed on 4 different days , and once with the euthanized guinea pigs (one 30-min measurement per guinea pig).2 openings) closed off both sides of the air conduit to prevent contact between guinea pigs while allowing airflow. The air was drawn through the transmission cage unit in a unidirectional manner, from donor guinea pig upstream toward the recipient guinea pig downstream  were used for cage bedding, and guinea pig chow and water were also supplied in the cage.Transmission experiments were performed at constant temperature and relative humidity (RH) in environmentally controlled chambers  set at 20 °C and 20% RH. Each transmission experiment replicate comprised four pairs of guinea pigs, each with a virus-immune donor, contaminated with influenza virus, paired with a virus-naive recipient. Each transmission pair was housed in a custom-fabricated cage unit consisting of two standard polycarbonate animal cages (each 26.7 cm × 48.3 cm × 20.3 cm) joined together by a stainless-steel air conduit (7.6 cm × 34.3 cm × 14 cm). Two panels of wire mesh  compartment of a transmission cage unit and allowed to dry at regulated temperature (20 °C) and humidity (20% RH), with the cage fans off, prior to placing an influenza virus-naive guinea pig into the recipient (downstream) compartment. Recipient guinea pigs were kept in a separate room in the animal vivarium during the application of influenza virus to the immune donor guinea pigs, and gloves were changed before handling the recipient guinea pigs to place them into the transmission cage unit. Transmission pairs were kept together for a total of 7 days, and nasal washing was performed on days 2, 4, and 6 post-contamination by instilling a total of 1 ml of PBS + P/S into both nares and allowing it to drain onto a sterile Petri dish.Donor guinea pigs had been previously infected with Pan99 at least 6 weeks prior to these experiments. On day 0, 10 ml of Pan99 virus at a concentration of 10Samples were collected in 1.5-ml tubes on ice, centrifuged to pellet debris, and stored at −80 °C until titration by plaque assay. Between transmission experiments, polar fleece bedding covers were laundered through two complete hot-water washer cycles, first with a dye- and perfume-free detergent  and then again without detergent. Covers were tumble-dried at the high-heat setting and then placed over new absorbent pads . Pads and polar fleece covers were autoclaved (15-min dry cycle) before use.Assessment of the duration of virus viability on guinea pigs’ bodies was performed by swabbing fur, ears and paws with sterile cotton tipped applicators  wetted in 1 ml of PBS + P/S and then eluted in the same solution after swabbing. Cage walls were swabbed in the same manner. Swab elution samples were kept on ice until centrifugation to pellet debris and were then titrated by plaque assay immediately afterwards.Intranasally inoculated guinea pigs were housed in cages with corncob (CC) bedding during the environmental swabbing experiment. Immune, virus-contaminated guinea pigs were swabbed during the transmission experiments in which they were the virus-donor animals. They were housed in the transmission cage units with polar fleece (PF) bedding, as described above.The APS was used to measure the emission rate and size distribution of aerosolized fomites between 0.3 and 20 μm in diameter generated from three different kinds of paper tissues, including lab wipes , paper towels , and toilet paper . A stainless-steel funnel was connected to the APS inside a biosafety cabinet (BSC) with conductive silicone tubing (inner diameter of 0.95 cm and length of 15 cm) and 2-min samples were collected; each tissue was rubbed for 10 s in front of the funnel followed by 30 s rest, repeated three times, for a total of 30 s of particle generation  was used to collect and enumerate infectious aerosolized fomites. The BioSpot collects particles in the size range of 10 nm to 10 μm, without differentiation by size, by enlarging particles through condensation of water onto them. We connected a stainless-steel funnel to the BioSpot bioaerosol collector with conductive silicone tubing (inner diameter of 0.95 cm and length of 80 cm). The funnel was placed in the BSC for each 10-min sampling period. Tissues were rubbed in front of the funnel for 8 min, followed by a 2-min wash-out with air drawn from the BSC in the absence of particle production. The positive control was Pan99 virus in PBS + 0.3% BSA + P/S/A (200 pfu ml–1) aerosolized at 0.42 ml min–1 by a nebulizer  positioned in front of the funnel, and the negative control was air drawn from the empty BSC. The BioSpot air sample flow rate was 8 L min–1, and airborne particles, enlarged via condensation, were collected into 2 ml of phosphate-buffered saline (PBS) supplemented with 0.5% bovine serum albumin  and penicillin, streptomycin, and amphotericin  in 35 mm polystyrene Petri dishes (VWR). After collection, samples were transferred into sterile 5-ml tubes and kept on ice until titration.To generate infectious aerosolized fomites, Pan99 stock virus was diluted in PBS + 0.5% BSA + P/S/A and was added drop-wise to lab wipes, paper towels, and toilet paper, at 3.6 × 1060. Viruses were serially diluted in PBS + 0.3% BSA + P/S/A, inoculated onto cell monolayers, and then incubated at 37 °C and 5% CO2 for 1 h. After incubation, monolayers were overlaid with 1.3% microcrystalline cellulose  in plaque assay medium, consisting of Minimum Essential Medium (MEM) supplemented with 1× Ham’s F-12K nutrient mix, 1.2% sodium bicarbonate, 100 mM HEPES buffer, 20 mM L-glutamine, and P/S/A , with 0.3% BSA and 1 μg ml−1 of tosylsulfonyl phenylalanyl chloromethyl ketone (TPCK)-treated trypsin (Thermo Scientific). Plates were incubated at 37 °C and 5% CO2 for 3 days. Cells were then fixed with 4% formaldehyde, and plaques were visualized with a crystal violet counterstain. BioSpot bioaerosol collection samples were titrated, undiluted, by inoculation onto MDCK-SIAT1 cells that had been grown to confluence in 35-mm-tissue culture dishes . Dishes were spinoculated (300 × g) for 60 min at room temperature, in stacks of four dishes, in the A-4-44 rotor of an Eppendorf 5804R centrifuge. The inocula were then aspirated, and dishes were overlaid with plaque assay medium, incubated, fixed, and stained as described above. Viral titers calculated from plaque assay counts were compiled in Excel for Mac 2011 version 14.7.3 (Microsoft Corporation) for import into MATLAB .The virus titers of Pan99 stock aliquots and guinea pig nasal washes and body swab eluates were determined by plaque assay of 10-fold serial dilutions on MDCK-SIAT1 cells, grown in confluent monolayers in 6-well platesp-value , and a Bernoulli likelihood function were used to obtain a 95% credible interval for the posterior distribution of the transmission probability θ64 given each of these data sets.The power law fit of the APS-quantified, time-averaged particle emission rate over 1 min lue Fig. , were peigs Fig. . Data weFurther information on research design is available in the Supplementary InformationPeer Review FileDescription of Additional Supplementary InformationSupplementary Movie 1Supplementary Movie 2Reporting Summary


Transmission of severe acute respiratory coronavirus 2 (SARS-CoV-2) mainly occurs through direct contact with an infected person via droplets. A potential role of contaminated surfaces in SARS-CoV-2 transmission has been suggested since the virus has been extensively detected on environmental surfaces. These findings have driven the investigation of virus stability on surfaces under several conditions. However, it remains unclear how long the infectious virus survives on surfaces under different climate conditions, which could play a role in predicting the seasonality of SARS-CoV-2. Therefore, the aim of this study was to estimate the virus stability and its biological half-life on various types of surfaces under indoor and seasonal climate conditions. This study revealed that SARS-CoV-2 survived the longest on surfaces under winter conditions, with a survival post-contamination on most surfaces up to 21 days, followed by spring/fall conditions, with a survival up to 7 days. Infectious virus was isolated up to 4 days post-contamination under indoor conditions, whereas no infectious virus was found at 3 days post-contamination under summer conditions. Our study demonstrates the remarkable persistence of SARS-CoV-2 on many different common surfaces, especially under winter conditions, and raises awareness to the potential risk of contaminated surfaces to spread the virus. Severe acute respiratory coronavirus 2 (SARS-CoV-2), which first emerged in December 2019 in a wet market in Wuhan, China, is responsible for the current pandemic. Although transmission of SARS-CoV-2 mainly occurs through infectious droplets or close contact with an infected person, virus droplets can survive and remain infectious on inanimate surfaces, which can contribute to the spread of the virus ,4,5. How4 50% tissue culture infectious dose (TCID50) of SARS-CoV-2. After 4.5 h of incubation, virus titers on surfaces ranged from 102.4 to 102.7 TCID50 on cloth and 103.3 to 104.2 TCID50 on other materials; these virus titers served as the starting titers for the linear regression model. Under indoor conditions, infectious virus was recovered from cloth up to 1 dpc; from concrete, polypropylene, stainless steel, and galvanized steel up to 3 dpc; and from nitrile gloves, Tyvek, N95 mask, Styrofoam, cardboard, rubber, and glass up to 4 dpc. In contrast, viable virus disappeared quickly under summer conditions and was undetectable on cloth, cardboard, concrete, and stainless steel at 2 dpc, and on nitrile gloves, Tyvek, N95 mask, Styrofoam, rubber, glass, polypropylene, and galvanized steel at 3 dpc. However, the virus titer only slowly decayed over time under spring/fall conditions. Virus titers on surfaces ranged from 101.1 to 102.3 TCID50 at 7 dpc, except for cloth, with virus only detectable up to 3 dpc. Under winter conditions, we observed the longest survival time. The virus was able to survive up to 15 dpc on cloth and 21 dpc on most of the other surfaces. The half-life of SARS-CoV-2 on surfaces ranged from 3.5 to 12.86 h, 2.54 to 5.58 h, 17.11 to 31.82 h, and 47.94 to 121.78 h under indoor, summer, spring/fall, and winter conditions, respectively  at 25 °C/70% relative humidity (RH) . Surfaceectively . The virPotential modes of transmission of SARS-CoV-2 include direct contact with an infected person via droplets, inhalation of aerosol, or exposure to infectious body fluids or contaminated surfaces (fomite). To date, there has been no scientific report that has demonstrated SARS-CoV-2 infection via contaminated surfaces. However, the role of fomites in transmission of SARS-CoV-2 is debated because the virus has been detected on environmental surfaces as well as personal protective equipment in hospitals and households ,7. In ad5 TCID50/mL virus inoculum (isolate USA-WA1/2020) and described the SARS-CoV-2 half-life, which ranges from 3.46 to 6.81 h at 22 °C/40% RH [7.8 TCID50/mL) to glass, banknotes, inner and outer mask layers, polypropylene, and stainless steel [Van Doremalen et al. contaminated cardboard, plastics, and stainless steel with 50 μL of 10ss steel .Our study showed a remarkable persistence of infectious SARS-CoV-2 on various types of surfaces, especially under winter climate conditions. However, virus stability was highly dependent on the substrate as well as temperature and humidity. Previous studies showed reduced virus stability in human nasal mucus and sputum when compared to culture medium  even at There are certain limitations to this study. First, we used the USA-WA1/2020 isolate, which was isolated in January 2020 from the first COVID-19 patient in the US. Since its first emergence at the end of 2019 and the subsequent global spread, SARS-CoV-2 has continuously evolved and accumulated mutations, which might have an impact on the environmental stability. Furthermore, the experimental design with a pre-determined time of termination (max. 21 days) resulted in samples  which were harvested before the disappearance of infectious virus. Nonetheless, we were still able to successfully calculate the biological half-life of SARS-CoV-2 on surfaces and determine the influence of seasonality on virus decay. Lastly, the SARS-CoV-2 inoculum in this study was prepared in cell culture medium used to grow the susceptible cell line (VeroE6 cells); however, under natural conditions, the infectious virus is excreted in biological fluids  from infected humans and animals. Therefore, the virus stability might be different in various types of biological fluids when compared to cell culture medium, which was used in this study.In conclusion, our study determines the biological half-life of SARS-CoV-2 on diverse surfaces under different climatic conditions, which correlates to the potential risk of contaminated surfaces to spread the virus. It clearly demonstrates that the virus is more resistant under winter and spring/fall but not summer conditions. Therefore, the practice of good personal hygiene and regular disinfection of potentially contaminated surfaces remains a critical tool to minimize the risk of infection through contaminated surfaces.SARS-CoV-2 stability was tested on 12 surfaces including nitrile glove , Tyvek , N95 mask , cloth , Styrofoam , cardboard , concrete , rubber , glass , polypropylene , stainless steel , and galvanized steel . Materials were cut into small pieces, washed, dried, and autoclaved . To make concrete, the coarse aggregate was removed by a strainer, and the fine aggregate was mixed with water according to the manufacturer’s instruction. The mixture was then poured into a silicone mold and air-dried in a biosafety cabinet overnight. 4 TCID50 SARS-CoV-2 (strain USA-WA1/2020) in Dulbecco’s Modified Eagle’s Medium (DMEM) with 5% FBS was added onto each material. The positive control had the same amount of virus in 2 mL medium in a sealed 2 mL tube. The virus was air-dried inside a biosafety cabinet for 4.5 h at room temperature and room relative humidity (RH). The plate with the virus-contaminated material was then incubated under four different conditions in an environmental chamber : 21 °C/60% RH, 25 °C/70% RH, 13 °C/66% RH, and 5 °C/75% RH, environmental conditions simulating indoor setting, summer, spring/fall, and winter conditions for the Midwestern US, respectively. To calculate the average temperature and RH for each season, maximum and minimum temperature and relative humidity (RH) data at Manhattan, Kansas, was acquired from the National Service Forecast Office (https://w2.weather.gov/climate/index.php?wfo=top accessed on 11 May 2020) (10(2)/slope in GraphPad Prism 9, San Diego, CA, USA. To determine the seasonal pattern of stability, analysis of variation (ANOVA) and subsequent Tukey’s multiple pairwise comparisons were performed to compare the slopes of linear regression under summer, spring/fall, and winter conditions.Each material surface was placed in a 6-well or 12-well plate, and 50 μL of virus inoculum consisting of 5 × 10ay 2020) . Climate


Nairoviridae, was first isolated in the 1970s from vespertilionid bats in Central Asia. Issyk-Kul virus has been described as human-pathogenic virus, causing febrile outbreaks in humans with headaches, myalgia and nausea. Here we describe the detection of a novel strain of Issyk-Kul virus from Eptesicus nilssonii in Germany. This finding indicates for the first time the prevalence of these zoonotic viruses in Europe.Bats have been gaining attention as potential reservoir hosts of numerous viruses pathogenic to animals and man. Issyk-Kul virus, a member of the family  Numerous studies have been focusing on the identification of other highly pathogenic viruses possibly transmitted by bats, and a plethora of novel viruses was described6. Subsequently, bats are increasingly being recognized as potential reservoir host of other viruses (i.e. bunyaviruses) that may also have the ability to cause severe disease in humans. Within the order Bunyavirales, the family Nairoviridae contains the genus Orthonairovirus, named after the Nairobi sheep disease orthonairovirus (NSDV) species7. NSDV and other members of the genus, like Crimean Congo Hemorrhagic Fever virus, Dugbe virus and Ganjam virus, are highly pathogenic to animals and humans8. Orthonairoviruses are often transmitted by ticks, although the vertebrate reservoir host of the viruses remains unknown; the virus has not been found in wild ruminants or other animals in enzootic areas. A bat nairovirus, Ahun nairovirus, has been detected in lung tissues of one Pipistrellus pipistrellus and one Myotis mystacinus10. Phylogenetically, Ahun nairovirus appears as a new clade, distinct from other orthonairoviruses10. Another bat orthonairovirus, identified by metagenomics, originates from fecal samples of Molossus molossus bats from French Guiana11. In a comprehensive study the virome of European bats in nine pools was sequenced and, besides three novel nairoviruses, numerous viruses were identified12. However, in this manuscript we describe the further characterization of a novel Issyk-Kul virus strain PbGER (ISKV PbGER), from German bats by virome sequencing by annotation and phylogeny.Bats have been identified as reservoir hosts of several highly pathogenic viruses such as Hendra virus, Nipah virus and SARS virusEptesicus nilssonii) showing histopathological alterations were pooled and purified using the TUViD-VM protocol and subjected to virome sequencing13. The total read number after sequencing was 9,806,241 reads of which 7970 were specifically mapping to Issyk-Kul virus (ISKV), a member of the genus Orthonairoviridae.Organ tissue homogenates from 12 bats (The identified virus sequences shared the highest identity with the strain Issyk-Kul virus KR709221 and were confirmed by sequence assembly and comparison to Issyk-Kul virus reference strain KR709221. The virus was named Issyk-Kul virus strain PbGER (ISKV PbGER) after the origin of the bats in Prackenbach, Germany. Individual bats were tested back for ISKV PbGER with newly designed specific primers and confirmed by conventional PCR and Sanger sequencing in tissues of nine individual bats. To extend the ISKV PbGER L segment, primer walking was utilized. Initially the assembled sequence of the L segment was interrupted by 35 gaps. The longest continuous part of the sequence had a length of 1281 nt . In this study, we were able to reduce the number of gaps to 10 and recover 93.5% of the L segment with an identity of 95.1% nt to strain LEIV-315K (KR709221) by using primer walking and Sanger Sequencing. The longest continuous part of the L segment sequence is now 6154 nt long (MN851301).Assembly of the M segment resulted in 89% of the segment length with 87% nt % identity to strain LEIV-315K KR709221). For the S segment, 98% of the whole length and an identity of 89.2 nt % with LEIV-315K (KR709221) was reached  is displayed in Fig. 14. In this study a novel nairoviral (ISKV PbGER) sequence was detected and further characterized. ISKV PbGER shows the highest identity with Issyk-Kul virus strain LEIV-315K . Nine out of twelve Eptesicus nilssonii bats sequenced, proved to be infected with ISKV PbGER  in Kazakhstan, is very close related to ISKV PbGER18. This was also seen for the L segment in Fig. The majority of nairoviruses is transmitted by ticks several of which are capable of causing severe disease in humans and animalsER Table . The phyEptesicus nilsonii. Eptesicus nilsonii is a common bat distributed throughout Asia and Europe (including the polar regions). In Scandinavia they are even the most frequent bat species19. They are dependent on humid habitats in close proximity to fresh water. In winter, they hibernate on heated attics and wall claddings of human dwellings. Issyk-Kul virus is described to be transmissible by bat urin and feces. Taking all this information’s into account, there is a potential for bat to human contact.Issyk-Kul virus is a known zoonotic virus described already more than 40 years ago in Central Asia. Our findings show for the first time the abundance of this virus in Europe and within the species, Eptesicus nilssonii might also have the potential of zoonotic transmission to humans and should be subject of further investigation. Our findings support the theory that bat species may share a long evolutionary history with orthonairoviruses and may have continuing cycles of transmission because of preying on varying tick species.Furthermore, these findings indicate that the described ISKV PbGE(R from http://ec.europa.eu/environment/nature/legislation/habitatsdirective) and through the Agreement on the Conservation of Populations of European Bats (www.eurobats.org), investigative research requires special permission by local government bodies20. We herewith confirm, that all permits to investigate carcasses of deceased bats and all experimental protocols on the dead bats were approved by the respective local governmental authorities . We herewith further confirm that all experiments were performed in accordance with relevant guidelines and regulations. As part of a study on diseases in native bats20, organs of 12 Eptesicus nilssonii bats showing histo-pathological alterations were examined. The pool consists of the following individuals and organs . If bats died in care or had to be euthanized for medical reasons, the carcasses were immediately stored at − 20 °C and transferred to the Leibniz Institute for Zoo and Wildlife Research, Berlin, Germany, for pathological and bacteriological examination23. Aliquots of the individual organs were divided between one tube with RNAlater®  for RNA extraction and one tube native frozen at − 80 °C and sent to the Robert Koch Institute for virological examination12. Samples were pooled and viruses purified using the TUViD-VM protocol and subsequently prepared for sequencing. Libraries were built as sequencing was performed as described before12.Several bats were found to have alterations that may be related to viral infections . Animals were found dead, injured or moribund near roosting sites or human habitations12. All work was performed at Biosafety level-2 conditions with appropriate precautions. After obtaining the result that the novel strain is Issyk-Kul virus (rated BSL-3 in Germany), further work on the original material was restricted to BSL-3. Homogenized organs [n = 31] with potentially virus-related histopathological changes (data available on request) were pooled , Sanger sequenced and compared to the original sequence obtained from NGS. For ISKV PbGER a real-time PCR was designed to test the individual organs of all Eptesicus nilssonii bats involved in the initial study. Primers, probes, mastermix and cycling conditions were as follows: ISKV PbGER-F (5′-GTGGAAGAATAACGCACCCCATAT-3′), ISKV PbGER-R (5′-ATGCACCAGGTCGTTTGTGGT-3′), Probe: ISKV PbGER-P FAM-TCCACTGAAGGCTGGTAGGA. The mastermix consisted of 10 × buffer (Platinum Taq Kit), 1 µl of dNTPs , 0.75 µl of primer F (10 µM), 0.75 µl of primer R (10 µM), 0.25 µl of probe (10 µM), 3 µl of MgCl2 , 14.55 µl of H2O (Platinum Taq Kit) and 5 µl of cDNA. Cycling conditions were 600 s at 95 °C followed by 45 cycles of 15 s at 95 °C and 30 s at 60 °C.Raw data was processed as described before24. Model of evolution was predicted via JModelTest and the model with the best AIC score was picked25. The actual reconstruction was performed via the Bayesian MCMC approach using MrBayes 26. Reconstructed tree was visualized using FigTree and posterior probabilities were depicted for each node (http://tree.bio.ed.ac.uk/software/figtree/).Final sequences obtained from data analysis and Sanger sequencing were used to reconstruct phylogenetic trees. Sequences were aligned to type species of the respective viral family (ICTV database) and other related viruses of interest via ClustalW. Alignment quality was checked with the online tool T-CoffeeSupplementary Infomations.