Bunyavirales infect a wide variety of host species, including plants, animals and humans, and pose a threat to public health. Major families in this order have tri-segmented negative-sense RNA genomes, the 5′ and 3′ ends of which form complementary strands that serve as a replication promoter. Elucidation of the mechanisms by which viral polymerases recognize the promoter to initiate RNA synthesis is important for understanding viral replication and pathogenesis, and developing antivirals. A list of replication promoter configuration patterns may provide details on the differences in the replication mechanisms among bunyaviruses. By using public sequence data of all known bunyavirus species, we constructed a comprehensive list of the replication promoters comprising 40 nucleotides in both the 5′ and 3′ ends of the genome that form a specific complementary strand. Among tri-segmented bunyaviruses, members of the family Nairoviridae, including the highly pathogenic Crimean-Congo hemorrhagic fever virus, have evolved a GC-rich promoter structure differing from that of other families. The unique promoter structure might be related to the large genome size of the family Nairoviridae among bunyaviruses, and the large genome architecture might confer pathogenic advantages. The promoter list provided in this report is useful for predicting the virus family-specific replication mechanisms of bunyaviruses.Members of the order  Bunyavirales is a new order proposed by the International Committee on Taxonomy of Viruses (ICTV) in 2016. It consists of 12 families of divergent viruses: Arenaviridae, Cruliviridae, Fimoviridae, Hantaviridae, Leishbuviridae, Mypoviridae, Nairoviridae, Peribunyaviridae, Phasmaviridae, Phenuiviridae, Tospoviridae, and Wupedeviridae. Members of Bunyavirales have a segmented single-stranded negative-sense or ambisense RNA genome. The families Arenaviridae, Hantaviridae, Nairoviridae, Peribunyaviridae, and Phenuiviridae include several important pathogens that can cause severe diseases in animals, including humans, while the families Fimoviridae, Phasmaviridae, Phenuiviridae, and Tospoviridae include pathogens associated with plant diseases.i.e., the small (S), medium (M), and large (L) segments, based on their relative sizes. Each segment acts as a template for the replication of a positive-sense antigenome, and for the transcription of mRNA. The S segment encodes the nucleocapsid protein (NP), the M segment encodes a glycosylated polyprotein precursor (GPC) that is cleaved into envelope spike proteins Gn and Gc, and the L segment encodes the L protein, an RNA-dependent RNA polymerase (RdRp) responsible for the transcription and replication of the three RNA segments. The RNA synthesis activities of the three RNA segments are regulated by nucleotide (nt) sequences within the 3′ and 5′ untranslated regions (UTRs), which flank the S, M, and L open reading frames. The terminal nts of 3′ and 5′ UTRs exhibit complementarily, and such sequences have been shown to bind to, and influence the activity of, viral RdRp, promoting transcription to yield a 5′-capped mRNA by using cleaved host mRNA as a primer, and replication that results in the synthesis of a full-length copy of the genome template.Major groups of bunyaviruses possess tri-segmented negative-sense RNA genomes, and share the same genetic organization consisting of three segments, i.e., promoter element 1 (PE1), the genomic extreme complement region, and PE2, the complement region located behind PE1, which was first described in Bunyamwera virus (BUNV) of the family Peribunyaviridae2. PE1 comprises approximately 10–15 nts located at the extreme termini of the genome that are strictly conserved among all three segments. These nts have been shown to interact with L protein at separate sites in La Crosse virus (LACV) of the family Peribunyaviridae4. PE2 comprises segment-specific nts at subsequent positions that are required to form canonical Watson–Crick base-pairing with corresponding nts at the opposite end of the template5. These RdRp-RNA and RNA-RNA interactions are thought to account for the pseudocircular form of viral ribonucleoprotein complexes7. In BUNV, sequence changes within PE1 have a significant effect on promoter function, but adjacent nts within PE2 are highly resistant to sequence changes, provided that their interterminal Watson–Crick base-pairing potential is maintained1. For the family Nairoviridae, little is known on the roles of the 3′- and 5′-terminal UTRs in regulating RNA synthesis. As with other Bunyavirales members, the UTRs of all nairoviruses comprise highly conserved terminal proximal nts (PE1) shared by all three segments, followed by less conserved regions that are segment-specific nts (PE2). The importance of these segment-specific nts in RNA synthesis has been partially examined using a minigenome reporter assay in non-pathogenic Hazara virus (HAZV), which is closely related to Crimean-Congo hemorrhagic fever virus (CCHFV)8. PE1 and PE2 were found to be separated by a spacer region, which exhibited a critical requirement to be short in length and lack base-pairing ability. Taken together, the accumulated data indicate that the promoter structure of bunyaviruses differs among virus families. Understanding these properties is essential for developing antivirals targeting viral RNA synthesis and processing.Bunyavirus promoters are composed of two promoter elements, Nairoviridae differ from those of other virus families, and have characteristics unique to the family, which has a large genome size.To characterize the promoter structure of diverse bunyaviruses, we constructed a list of all viral promoters that exhibit complementarity and each of the nt counts within the first 40 nts of both the 5′ and 3′ (anti)genomic ends. We found that the promoters of the family Bunyavirales, including the families Arenaviridae, Cruliviridae, Fimoviridae, Hantaviridae, Leishbuviridae, Mypoviridae, Nairoviridae, Peribunyaviridae, Phasmaviridae, Phenuiviridae, Tospoviridae, and Wupedeviridae, which are tri-segmented or multi-segmented viruses, as summarized in Fig. https://ictv.global/taxonomy) was analyzed. Because there are incomplete genome sequences in the National Center for Biotechnology Information (NCBI) database that do not precisely cover the genome extremes, we selected viral sequences with complete complementarity in the terminal + 1 to + 3 nts (some exceptions with non-complementary + 1 nt are included). The complement structure of 5′ and 3′ genomic ends (positive-sense form) as well as the genome length, counts of G:C/A:U complementarity, and counts of each nts  in the promoter region were calculated by using an automatic calculating system based on an Excel file (Supplementary Table Arenaviridae (2 species), Cruliviridae (3 species), Hantaviridae (24 species), Mypoviridae (1 species), Nairoviridae (20 species), Peribunyaviridae (56 species), Phasmaviridae (2 species), Phenuiviridae (47 species), Tospoviridae (19 species) and Wupedeviridae (1 species); multi-segmented bunyaviruses Fimoviridae (17 species) and Phenuiviridae (14 species); and di-segmented bunyavirus Arenaviridae  in Hantaviridae and Nairoviridae. These promoters were further categorized into those starting with a tri-nt repeat (5′-AGUAGU and 5′-UAGUAG) and those starting with a di-nt repeat  . It has been reported that HAZV has a promoter composed of two complementary regions of PE1 and PE2 separated by a spacer region formed by non-complementary sequences at the 13–16 nt position8. We found the same feature in most virus species of the Nairoviridae family  Fig. A,B. We nUC) Fig. A. The coNairoviridae than in those of other virus families -4 pathogens that cause hemorrhagic fever in humans, such as CCHFV11. We hypothesized that the high pathogenicity of this virus family in mammals may be related to its large genome size. We examined the length of the available sequences annotated to “Nairoviridae” in the NCBI database. We first selected virus genome sequences possessing 5′-UCUC—GAGA-3′ ends, which are the most conserved genomic end sequences in nairoviruses, in the L, M and S segments. The lengths of these sequences are shown in Fig. Nairoviridae contains two highly pathogenic viruses in mammals, i.e., CCHFV and Nairobi sheep disease virus (NSDV), which have a mortality rate of 30% and 90% in humans and small ruminants, respectively13. The length of the sequences of CCHFV and NSDV are shown as red and yellow bars, respectively, in Fig. Among tri-segmented viruses belonging to the order Bunyavirales. Our analysis focused on five major tri-segmented virus families, and the results indicated that the genomes can be divided into two categories: those with a promoter starting with A  and those with a promoter starting with U  of the families Peribunyaviridae and Phenuiviridae, respectively, contain a 5′-triphosphate end that starts with A (5′-pppA)16. The 5′-pppA is generated by viral RdRp that recognizes the opposite U as the template. As seen in several segmented and non-segmented RNA viral polymerases19, bunyaviral RdRp synthesizes RNA from an internal nt, and not from the terminus of the template. In LACV, RNA synthesis is initiated with A using the U at the + 4 position of the antigenome (3′-UCAUCA) as the template during genome replication4. The elongated product, 5′-pppAGU, is realigned to the + 1 to + 3 position of the antigenome template (3′-UCAUCA), and is further elongated to generate 5′-pppAGUAGU. Accordingly, the position of U responsible for RNA synthesis initiation is presumably the + 3 position in the Phenuiviridae (3′-UGUG) and Tospoviridae (3′-UCUC) antigenomes. This indicates that the 5′-pppAC and 5′-pppAG products realign to the 3′-UGUG and 3′-UCUC of the antigenomes, respectively, and are further elongated to generate 5′-pppACAC and 5′-pppAGAG, respectively, which are precise complementary chains of the antigenome templates. In contrast, the genomes of Hantaan virus (HTNV) in the family Hantaviridae, and CCHFV in the family Nairoviridae contain a 5′-monophosphate end20, suggesting an unconventional RNA processing event during replication. In HTNV, RNA synthesis is initiated with an internal G at the + 3 position by using a C of the 3′-AUCAUC of the antigenome as the template20. Subsequently, the elongated 5′-pppGUA product realigns to the 3′-AUCAUC to further produce 5′-pppGUAGUA. Then, the extreme 5′-pppG is removed by an endoribonuclease activity of viral RdRp to produce 5′-pUAGUA (5′-monophosphate end)20. The endoribonuclease activity of RdRp is responsible for the cap snatching that cleaves the 5′ end of the host mRNA for use as a transcription primer21. The -1 position of viral mRNA of HTNV is G, indicating that viral RdRp can cleave host mRNA after the G nt (cleave GpN to produce G/pN) during transcription. In Nairoviridae, the − 1 position of viral mRNA is C23, and it is also generated via the cap-snatching mechanism. Similar to the RNA synthesis in Hantaviridae, it is supposed that nairoviral RNA synthesis is internally initiated with 5′-pppC at the + 2 position by using the G of 3′-AGA of the antigenome as the template. Subsequently, the 5′-pppCU product would realign to the 3′-AGA, and be further elongated to generate 5′-pppCUCU. The 5′-pppC would then be removed, resulting in the production of a 5′-monophosphate end. Therefore, although the hantaviral RdRp is a conventional enzyme that initiates RNA synthesis with a purine (G), the nairoviral RdRp is considered to be an unconventional enzyme that can initiate with a pyrimidine (C). Such a difference may be important for the targeting of novel antivirals specific for nairoviral diseases.In this study, we tabulated the replication promoter structures of all known virus species in the order 24. The repeats can determine the initiation site for RdRp , which is important for the prime-realign RNA synthesis mechanism. The biological significance of the internal position of RNA synthesis initiation is unclear. It is likely that the di-nt repeat is restricted to virus families possessing an ambisense genome, such as Phenuiviridae and Tospoviridae, as well as Nairoviridae (for which only CCHFV has been reported)25. This suggests that the ambisense coding property may be related to the di-nt repetition in the genomic ends. If this is true, analysis of genomic end repetition patterns may enable the elucidation of new transcripts in various bunyaviral genomes.Our analysis additionally confirmed that most bunyaviral genomes begin with a di-or tri-nt repeat Fig. , which hNairoviridae promoter appears to have high G:C complementarity in the 17 to 21-nts region  in each promoter region do not differ significantly among the L, M, and S segments in all virus families, except for dae Fig. B. It is ent Fig. B. This sely Fig. B. The naely Fig. B. This lNairoviridae is so large. Nairoviridae is the only tri-segmented virus family that includes hemorrhagic fever viruses classified as BSL-4 pathogens, such as CCHFV. We hypothesized that the large genome size of the family Nairoviridae may be related to its high pathogenesis in mammals. Although the length of the L segment in Nairoviridae is the longest among all bunyaviruses, it is not particularly long among the highly pathogenic viruses in this family , virion envelope glycoprotein Gn, and virion envelope glycoprotein Gc27. We showed that although the sizes of Gn and Gc are similar among virus species, those of the O-glycosylation sites and GP38-like protein are different; in particular, they are larger in CCHFV and NSDV  on December 7th, 2021. The complete sequences of the L, M and S segments of bunyaviruses available on the NCBI associated with the GenBank accession numbers listed in Supplementary Table https://weblogo.berkeley.edu/logo.cgi).In total, 590 bunyavirus species were registered in the ICTV list .Sequences annotated as “29, SignalP-6.0 for the signal cleavage site30, and NetOGlyc-4.0 for the O-linked glycosylation sites31. Data on the glycoprotein sequences were collected from UniProt (https://www.ebi.ac.uk/uniprot/index). The UniProt accession numbers were: CCHFV, Q8JSZ3; NSDV, A0A0A7H8l1; Dugbe virus, Q02004; Tofla virus, A0A0U5AG15; HAZV, A6XIP3; and Erve virus, J3S7E1. GP38-like regions were found using the Protein Basic Local Alignment Search Tool (BLASTp) based on the amino acid sequences of the CCHFV and Dugbe virus GPC.The structural characteristics of the nairovirus glycoprotein were predicted using TMHMM-2.0 for the transmembrane proteint-test or one-way analysis of variance followed by Tukey’s test, as appropriate.Statistical analyses were performed with Prism software . Statistical significance was assigned when p values were < 0.05. Inferential statistical analysis was performed by a two-tailed unpaired Student’s Supplementary Table 1.Supplementary Table 2.Supplementary Table 3.Supplementary Figures.


Rabies causes approximately 60,000 casualties annually and has a case fatality rate approaching 100% once clinical signs occur. The glycoprotein on the surface of the virion is important for the host immune response and facilitates interaction of the virion with host cell receptors. Nicotinic acetylcholine receptors were the first receptors identified as a molecular target for the rabies virus. Additional targets, including neural cell adhesion molecule, p75 neurotrophin receptor, metabotropic glutamate receptor subtype 2, and integrin β1, have been added to the list, all of which can mediate viral entry into the cell. Multiple receptors and different subtypes of nicotinic acetylcholine receptors result in a complex picture of virus-receptor interactions. In addition, some data suggest that the rabies virus glycoprotein inhibits cell signaling events mediated by various nicotinic receptor subtypes that have been implicated in altering behavior in unaffected animals. This review focuses on interactions between the rabies virus glycoprotein and nicotinic receptors and proposes possible functional consequences, including behavioral modifications and therapeutic approaches for future research.  •Rabies virus glycoprotein ectodomain binds to nAChRs.•Glycoprotein and nAChR-selective α-neurotoxins share regions of sequence homology.•Peptide of the rabies virus glycoprotein modifies behavior in mice.•Nicotine and genetic deletion of α7 nAChRs modulates pathological aggression.•We hypothesize rabies virus glycoprotein-nAChR interactions modulate behavior. Rabies virus, Rabies virus glycoprotein, Nicotinic acetylcholine receptor, Behavior, nAChR. As mostRhabdoviridae family. The virus is enveloped and has a single-stranded, negative-sense RNA genome, containing five genes which code for nucleoprotein (N), phosphoprotein (P), matrix protein (M), glycoprotein (G), and a viral RNA polymerase (L) . The RABV glycoprotein (RGP), which interacts with cellular targets, is arranged on the surface of virus particles forming trimers, containing cytoplasmic, transmembrane, and ectodomains .-68.66-68Loop II of α-bungarotoxin and α-cobratoxin have approximately 50% sequence homology to the RGP neurotoxin-like domain . CirculaTorpedo receptor, α-bungarotoxin loop II reaches under loop C, penetrating deeply into the ACh binding pocket .No data was used for the research described in the article.The authors declare no conflict of interest.No additional information is available for this paper.


Three major diseases in Japan, cancer, heart disease, and cerebrovascular disease (CVD) are the leading causes of death in Japan. This study aimed to clarify the social burden of these diseases, including long-term care (LTC), and to predict future trends.The comprehensive cost of illness (C-COI), a modification of the cost of illness (COI), was used to estimate the social burden of the three major diseases in Japan. The C-COI can macroscopically estimate both direct and indirect costs, including the LTC. A new method for future projections of the C-COI was developed according to the method for future projections of the COI. All data sources were government statistics.The C-COI of cancer, heart diseases, and CVD in 2017 amounted to 11.0 trillion JPY, 5.3 trillion JPY, and 6.5 trillion JPY, respectively. The projected future C-COI in 2029 was 10.3 trillion JPY, 5.3 trillion JPY, and 4.4 trillion JPY, respectively. In 2029, the LTC costs accounted for 4.4%, 12.8%, and 44.1% of the total C-COI, respectively. Informal care costs are projected to be approximately 1.7 times higher, assuming that all family caregivers will be replaced by professional caregivers in 2029.Indirect costs for all three diseases were projected to decrease owing to aging of the patient. In contrast to the other two diseases, the LTC cost of CVD accounted for a large proportion of the burden. The burden of CVD is expected to decrease in the future, but informal care by older family caregivers is suggested to reach its limits. In the future, the focus of resource allocation should shift from medical care to LTC, especially support for family caregivers. A method of future projections for the social burden based on the C-COI was considered effective for identifying issues for healthcare policy in the context of the times. The three major diseases in Japan that have been the leading causes of death since the 1950s are cancer , heart disease , and cerebrovascular disease (CVD) (I60–I69). Each of these diseases is strongly associated with aging. Since 1981, the consistently increasing incidence of cancer has been a preponderant cause of death in Japan. The second leading cause of death is heart disease, which is also increasing. In 2019, CVD was ranked fourth among the causes of death. It was the most common cause of death until the 1970s but has been declining since then. The deaths caused by these diseases accounted for 27.3%, 15.0%, and 8.8% of all deaths, respectively; the national medical expenses accounted for 4.0 trillion yen , 2.0 trillion yen (18.6 billion dollars), and 1.8 trillion yen (13.4 billion dollars) in 2019, respectively MDC, MbC, and MtC were the “medical costs” included in the original COI; FCC and ICC were the additional "LTC costs." MDC and FCC were direct costs, and MbC, MtC, and ICC were indirect costs.In Japan, almost all medical costs, including those incurred in private hospitals, are covered by universal health coverage (UHC) and the associated patients’ out-of-pocket expenses. Therefore, this study defines MDC as the sum of outpatient and inpatient costs, which are both covered by UHC and out-of-pocket expenses. They can be calculated directly using government statistics related to UHC.Since Japan introduced a public LTC insurance system in 2000, statistical data related to the system have been available for estimating LTC costs . All oldFCC = NLR × LCyMbC is the labor value lost during hospitalization or outpatient visits. It was calculated by multiplying the number of days of outpatient visits or hospitalization by the labor value by the sex–age group. Labor value includes both the value of domestic work and salary income. The calculation assumed that the patient had missed one day of work due to hospitalization and half a day for outpatient visits.MbC = THD × LVd + TOVy/2 × LVdTHD = HP × ALOSMtC was evaluated as the loss of human capital  by multiplying the number of deaths from a disease by the lifetime labor value by the gender-age group. Lifetime labor value refers to the income that a patient who died from a disease would have earned in case they had survived to the average life span; it is calculated as the present value by accumulating the average income of the gender-age group. The discount rate for calculating the present value was 2% as recommended by Japan’s economic valuation guidelines .MtC = NDy × LVlICC is a production value lost when people spend their time as a "family caregiver." The opportunity cost approach (OCA) was used for past ICC estimations. ICC by OCA was defined as the loss of labor value of family caregivers considering sex and age, which is similar to MbC.ICC (OCA) = NFC × ATCd × 365 × LVhFor future projections of medical costs, time-series forecasting with regression models, as established in previous studies, was used –20. The The future MDC was calculated as follows:MDC (future) = TOVy × OUC + THD × IUCAs mentioned earlier, the principle is to use a logarithmic approximation for variables with an upward trend and an exponential approximation for variables with a downward trend. However, unlike other variables, OUC and IUC were considered to have few factors that would reach their limits. In particular, the IUC for all three diseases has shown an exponential increase in recent years. Consequently, an exponential approximation was chosen. For the OUC, a linear approximation was selected considering past trends.In this study, a new method was developed to project future LTC costs using time-series forecasting with a regression model. NLR, LCy, NFC, and ATCd were predicted using linear or logarithmic/exponential approximations, according to rules similar to those of medical costs. These variables were predicted by not only gender and age group but also nursing care level, as a ratio to the population. The labor value was highly dependent on economic conditions and was not predicted using this estimate. Therefore, the LVd, LVl, and LVh were not predicted. Values obtained in 2017 were used in this study.Future projections of the ICC were made using not only the OCA, the standard method, but also the replacement approach (RA). In the RA, the average wage of professional caregivers was used instead of the lost family labor value through care.ICC (RA) = NFC × ATCd × 365 × AWPC[AWPC: Average wage of professional caregivers]The study protocol was reviewed by the Ethical Committee of the Toho University School of Medicine. The committee confirmed that no approval was necessary for this kind of study in Japan owing to the anonymous nature of the data (reference number A19034).Regarding future projections and time trend, the total C-COI of cancer increased slightly until 2017, after which it was expected to show a slight decrease until at least 2029. It has been projected that the MDC of cancer would continue to increase, but the MbC and MtC declines would outweigh them in the future. It was estimated that the total C-COI of heart disease would continue to increase slightly. The breakdown was predicted to be an increase in MDC and slight decrease in MbC and MtC. Previously, the LTC costs for heart diseases have been on the rise but were estimated to level off in future projections. The total C-COI of CVD increased in 2014 compared with that in 2008. However, it began to decrease in 2017 and was predicted to continue to decrease thereafter. In future projections, all five CVD components are estimated to decrease. Regarding the details of the change in the LTC costs of CVD, both FCC and ICC showed an upward trend until 2014. However, it began to decrease in 2017.The future projections for the ICC of CVD using RA were 1.58 to 1.66 times larger than those using OCA. Consequently, the ICC projected using RA accounted for 61.1%–61.8% of the LTC costs.In this study, past estimations and future projections of the C-COI for the three major diseases in Japan were made using government statistics. Trends in the total amount and breakdown of C-COI showed different trends for each of the three diseases. The COI of cancer and heart diseases has been increasing to date, but the C-COI of cancer is predicted to start declining in the future. The C-COI of CVD has remained unchanged to date, but future projections suggest that it will decrease. The LTC costs accounted for a small proportion of the total C-COI of cancer and heart diseases but approximately half of the C-COI of CVD.The C-COI for cancer was on the rise until 2017. The main contributor to this increase was the MDC. The C-COI of heart disease was estimated to continue to increase consistently from 2008 to 2019. However, MtC, the largest component of the C-COI of heart diseases, is estimated to start declining in 2023, even though the number of deaths will continue to increase. The decline in MtC is thought to be due to an increase in the age of death, showing a trend similar to that of cancer. As the average age of death for heart disease is higher than that for cancer , the humCVD had a notably different proportion of components compared to the other two diseases. Estimates using OCA, the basic method, show that LTC costs account for approximately half of the C-COI, and MtC accounts for a smaller proportion of the C-COI than the other two diseases. This difference may reflect the long-term sequelae of CVD . AccordiA breakdown of the LTC costs for CVD shows that informal care currently contributes to the same burden as formal care in monetary terms. In 2000, Japan introduced a public LTC insurance system in addition to conventional UHC to socialize the LTC that the family had been conducting until then. Nevertheless, it became clear that approximately half of the LTC costs incurred owing to CVD were sustained by family care. Family members providing informal care in Japan are also aging. According to the Comprehensive Survey of Living Conditions, the average age of family caregivers in 2016 was 65.5 years, and the proportion of those aged 60 years and over reached 70% . Table 2These methods for future projections have some limitations. In this study, future projection of each variable was performed using linear or logarithmic/exponential approximations. No social factors were expected to suppress the increase in unit costs for medical care. Therefore, future projections were made under the assumption that the IUC and OUC would consistently increase. In particular, the IUC, which has a trade-off relationship with ALOS, was assumed to show an exponential increase. In recent years, although ALOS has been rapidly decreasing owing to the policy of the Japanese government , the proIn addition, it was difficult to correct errors and misclassifications in the government statistical data. For example, it cannot be ruled out that deaths due to comorbidities were misclassified as deaths from the three major diseases. In this study, data from vital statistics were used to identify the causes of death. The vital statistics are compiled on the basis of death certificates issued by physicians who know the course of the patient leading to death, except in some exceptional cases. Japanese death certificates are formatted to provide a sequential and detailed description of the causal factors leading to death, which is expected to produce relatively accurate cause-of-death statistics . All staIn this study, as data from public LTC insurance are used to estimate LTC costs, it is not possible to consider patients who have sequelae from disease but do not use the LTC insurance system. Public LTC insurance is supposed to be secured by all older people aged 65 and over, but to actually receive the subsidy, an application must be made. By applying to the local government and receiving certification, 90% of the fees for LTC services can be subsidized by the insurance system. Those who need LTC but do not use public LTC insurance are likely to be limited to people with exceptional circumstances, such as ignorance of the system or a lack of capacity to apply. Therefore, this limitation was not expected to majorly impact the overall projection results. Although it is difficult to ascertain the number of such people since the establishment of public LTC insurance, the proportion of the older adult population using LTC insurance has been on the rise, particularly among people with mild disabilities . This maIn addition to the C-COI method, numerous attempts have been made to explore the future value of the social burden of the disease. For example, previous studies have shown that public health expenditure is procyclically influenced by the macroeconomic environment and exhibits cyclical behavior –40. UsinAttempts to estimate the burden of LTC, including informal care, have also been made worldwide –50. ThesThe C-COI method was used to visualize the burden of medical care and LTC, including indirect costs, from the past to the future. The C-COI of the three major diseases differed greatly in their total amount, components, and trends over time. For cancer and heart diseases, MDC is projected to continue to increase, whereas MbC and MtC are projected to decrease due to increases in the age of onset and death. This suggests that the total social burden of cancer, which is the leading cause of death in Japan, will decline in the future. The proportion of LTC costs in the C-COI of CVD, which has long been a major health issue in Japan as a chronic disease causing long-term sequelae, remains very high compared to that of the other two diseases; however, the overall social burden is expected to decrease in the future. At the same time, further aging of family caregivers could lead to the collapse of the current family care system and increase costs through a substantial increase in the demand for professional caregivers. Policies may be needed to shift resource allocation from medical care to LTC.


Molecular biology amplification enables sensitive detection of most respiratory viruses through nasopharyngeal swabbing. We developed an innovative approach to detect viral genomes on used facial tissues. In 2 communities of children, used tissues were collected once weekly for 1 year. Pooled analysis of tissues enabled detection of successive virus circulation in 4 age groups over time and forecasted by several weeks the circulation of influenza in the general population. At the individual level, in a proof-of-concept study of 30 volunteers with influenza-like signs/symptoms, we identified common respiratory viruses. The signals for SARS-CoV-2 obtained in parallel from 15 facial tissues and swab samples were similar and often higher for the tissues (11/15). Individual analysis of tissues offers a noninvasive, sensitive, and affordable alternative to self-sampling without a medical care requirement. Pooled analyses may be used to detect virus spread in specific communities, predict seasonal epidemics, and alert the population to viral infections. Respiratory viral infections (RVIs) are the most common virus-induced pathologies. Associated signs/symptoms range from common colds with simple rhinorrhea without fever to acute respiratory distress syndrome sometimes leading to death  based on a standard nasal swab sample tested by using the same diagnostic assay or had respiratory signs/symptoms consistent with an upper respiratory tract viral infection.All participants were informed about the purpose of the study. Because nose blowing is an individual act, participation was intentional and voluntary. Most volunteers were either family members, professionals working in the laboratory, or their relatives. This type of research is in agreement with the Jardé law on biomedical research and does not require additional authorization. Nevertheless, we obtained approval from the ethics committee of the University of Rennes.maternelle), from week 37 of 2019 through week 10 of 2020. For both locations, parents were informed of the study, and results were regularly communicated by the directors on an informal basis.We conducted the pooled collection study in 2 parts. The first part was conducted in a daycare center located within the Centre Hospitalier Universitaire , which hosted mostly employees’ children. Collection of used facial tissues started on week 40 of 2018 and continued through week 15 of 2019. The second part was conducted in a preschool  and oldest (19 months–4 years of age). The daycare staff collected all tissues during the week, and the container was collected each Friday; tissues were used for either nose blowing or, for the younger children, nose washing with a small amount of physiologic solution. For the second part of the study, the preschool comprised 2 classes of 20 pupils each: the first (3 years of age) and the second (4 years of age) grades. Tissues were used only for nose blowing, and the children were asked to throw their tissues in the specific container instead of the garbage. Each Friday, the filled containers were collected and clean empty containers were delivered for the following week.For 15 volunteers with COVID-19 diagnosed by reverse transcription PCR of a standard nasopharyngeal swab sample, results obtained the same day on individual facial tissues enabled comparison of cycle threshold (Ct) values between both matrices. To enable documentation of secretion kinetics over 5 days, 2 volunteers, after COVID-19 diagnosis, agreed to provide daily nose discharge from nasopharyngeal swab samples.https://www.thermofisher.com) per tissue. We collected the saturated liquid after the pooled tissues had been gently soaked for few minutes and then pressed with a home-brew device. For individual collections, we introduced the used tissues into a 60-mL syringe soaked with 7 mL of DPBS. We then firmly pressed the syringe plunger onto the soaked tissue and, depending on the tissue absorbing capacity, regularly obtained 5 mL of residual liquid.To determine the volume of soaking buffer needed, each week, we counted all tissues from each container. Eventually, we chose a volume of 7 mL of Dulbecco’s phosphate-buffered saline  on the sample of collected liquid, according to the manufacturer’s recommendations. The kit detects influenza A virus; influenza A(H1) virus; influenza A(H1N1)pdm09 virus; influenza A(H3) virus; influenza B virus; respiratory syncytial viruses A and B; adenovirus; enterovirus; human metapneumovirus (hMPV); parainfluenza viruses 1–4; bocaviruses 1–4; coronaviruses 229E, NL63, and OC43; and human rhinovirus. For individual testing, and particularly to detect SARS-CoV-2 RNA, we used TaqPath , Alinity , GeneXpert , and FilmArray . To detect cytomegalovirus (CMV), Epstein-Barr virus, and human parvovirus B19 genome, as part of our routine daily workflow we used RealStar assays . All products were used as recommended by the manufacturers. For the kinetic study on SARS-CoV-2–positive samples and the comparison of signals between nasopharyngeal swab samples and tissues, we obtained all Ct values from a SARS-CoV-2 AMP Kit automatized on Alinity. This test amplifies 2 genome target genes (RNA polymerase and nucleocapsid), and the obtained signal is provided as a single Ct. Concordance between the signals obtained by the different commercial techniques routinely used in our laboratory is excellent. Our laboratory is certified according to COFRAC NF EN ISO 15189.We used commercial kits to detect viral genomes. For pooled collections, we used Allplex Seegene  . At eachWe initially designed this study as a proof-of-concept study; our objective was to describe the circulation of respiratory viruses in communities of children. The results led us to consider a more individual approach, and we were able to gather enough data despite the COVID-19 pandemic. In the first part of the study, using pooled material, we gathered a large amount of data. Collecting used tissues from both institutions, the daycare center and the preschool, did not raise concerns from the professionals or the parents; both groups seemed interested in the study and awaited the weekly results. One objective of the regular monitoring of virus circulation would be to communicate the results on a weekly basis throughout the year to limit the unnecessary use of antimicrobial drugs when a viral infection is identified in a community, to inform the parents about what to do in such circumstances, and to possibly limit general practitioner visits. Parents who are informed about the circulation of a specific virus in a group of children may be less worried and could share their concerns with others. Collecting used tissues is noninvasive, well accepted by children and parents, and provides valuable information about the epidemiology of viruses in a specific community.Despite proximity, older and younger children were not affected by the same viruses at the same time, which indicates that hygienic rules were probably well applied, particularly in the daycare center, but also that children of different ages are not susceptible to the same infections. That information reflects development of immunity during growth, but collecting data throughout the year demonstrates the susceptibility of children in each age group to viral infection.Constant circulation of rhinovirus was not unexpected and may indicate rather weak immunity to the virus and highlights the relatively mild signs/symptoms in young children. Detecting parainfluenza virus in autumn in both communities of children fits well with the recent work from Horemheb-Rubio et al.  to the population. One may imagine a surveillance model similar to what has been done with sewage water for SARS-CoV-2  of cases . The medBlowing in a tissue is an individual action, and its effectiveness largely depends on the person. Nasopharyngeal swabbing also depends on the skill of a professional but may be less subject to variability. One possible way to assess the sampling quality is to quantify a cellular gene, yet this strategy is not routinely applied and lacks consensus among scientists .Of note, This study reflects major changes in virology that have occurred in recent years. Although growing the virus in cell culture was the reference for diagnosis, we slightly moved to molecular detection of viral nucleic acids. The information gained by each strategy is different: in the first instance, we identified live virus, probably infectious; in the second, we found only the presence of viral components. Yet the overall sensitivity of nucleic acid amplification methods now offers improved clinical sensitivity despite loss of information regarding the viability of the virus and possibly its infectiousness. Detecting viral nucleic acids in used tissues is feasible and possibly as sensitive as classical sampling methods, if not more so. That possibility provides the option for virus testing of pooled samples in communities to provide information on the dissemination of any respiratory-transmitted virus and to propose a noninvasive method for the general population. Situations in which persons need to be repeatedly tested, such as athletes during a several-day event, may also largely benefit from this strategy. Being able to send tissues by regular mail for remote diagnosis is also a real asset with potential application for gaining information about the diffusion of an infection in a population. Although this study should be considered as a proof-of-concept work, we believe that the accumulation of data justifies considering the use of our methods on a larger scale.


African swine fever (ASF) is causing a pandemic affecting swine in a large geographical area of the Eastern Hemisphere, from Central Europe to East and Southeast Asia, and recently in the Americas, the Dominican Republic and Haiti. The etiological agent, ASF virus (ASFV), infects both domestic and wild swine and produces a variety of clinical presentations depending on the virus strain and the genetics of the pigs infected. No commercial vaccines are currently available, although experimental recombinant live attenuated vaccine candidates have been shown to be efficacious in protecting animals against disease when challenged with homologous virulent strains. This review attempts to systematically provide an overview of all the live attenuated strains that have been shown to be experimental vaccine candidates. Moreover, it aims to analyze the development of these vaccine candidates, obtained by deleting specific genes or group of genes, and their efficacy in preventing virus infection and clinical disease after being challenged with virulent isolates. This report summarizes all the experimental vaccine strains that have shown promise against the contemporary pandemic strain of African swine fever. African swine fever virus (ASFV) is causing a disease pandemic that affects swine industries from Central Europe to Southeast Asia . RecentlThe most promising vaccine candidates have been derived from live attenuated strains of ASFV. Pigs infected with live attenuated virus strains are usually protected from infection or disease caused by virus isolates that are genetically and antigenically related (homologous) to the immunizing virus. ASF live attenuated virus strains have been obtained following different approaches. Initially, naturally attenuated virus strains were isolated from infected animals in the field ,7,8. TheAn alternative approach in the development of ASF attenuated virus strains is the progressive adaptation of virulent field isolates to replicate in a variety of primary swine cell cultures or established cell lines. The process of adaptation is usually accompanied by a progressive loss of virulence when inoculated into domestic pigs. This has been reported during the attenuation of several field isolates ,13,17. AThe last methodological approach used to generate attenuated strains of ASFV is the genetic manipulation of highly virulent field isolates. This approach requires, as a prerequisite, basic knowledge of virulence genes, followed by the removal of these genes from the virus genome to achieve attenuation. This systematic review summarizes all the published information regarding the development of attenuated ASFV strains by genetic manipulation, and the status of experimental vaccine development. 2 or 103 tissue-culture infectious dose (TCID50) of ASFV∆NL-S remained clinically normal, presenting a long viremia (2 to 3 weeks). These animals survived a 30-day post-vaccination (pv) challenge with 102 TCID50 of the homologous virulent E70 isolate, without presenting any clinical signs associated with ASF.The involvement of specific ASF virus genes, or groups of genes, in the process of virus virulence and disease production in the natural hosts remained largely unknown until the 1990s. The development of a method to delete specific genes from virulent ASFV isolates made possible the identification of virus genes involved in virulence during the infection of domestic pigs; these results are summarized in This report constituted the first identification of a genetic determinant of virulence in ASFV and presented the possibility of developing a recombinant ASF attenuated virus strain using genetic manipulation. This pioneering work facilitated the rational development of live attenuated vaccine candidates for ASF. The identification and subsequent manipulation of a genetic determinant of virulence has become a standard method for the development of experimental live attenuated vaccine strains.2 TCID50 IM of an ASFV E70 lacking the UK gene (∆UK) survived infection, but presented with a transient period of fever and lethargy right after inoculation. Similarly to observations after deletion of the NL-S gene, deletion of the UK gene does not affect the ability of the ∆UK virus to replicate in swine macrophages. Much like the in vivo studies with the ∆NL-S virus, animals infected with the ∆UK virus had prolonged viremias (4 to 6 weeks) and survived the IM challenge (by day 42 pv) with 104 TCID50 of the homologous virulent E70 isolate, remaining clinically normal.The next ASFV gene to be discovered as a determinant of virulence was the UK (DP96R) gene . This ge4 TCID50 of the Malawi isolate, all the animals remained clinically normal.Deletion of the thymidine kinase (TK) gene was demonstrated in the highly virulent Malawi isolate. Since deletion of the TK gene impedes the replication of ASFV in swine macrophages, a TK deletion mutant virus (∆TK) was developed using a Malawi strain partially adapted to grow in Vero cells, although still fully virulent in domestic pigs . Althoug2 to 106 TCID50), with animals displaying a transient mild rise in body temperature and a reduced viremia lasting 3 to 4 weeks. The ASFV challenge was performed at 42 days pv using 104 TCID50 of parental highly virulent Malawi Lil-20/1 administrated IM; all animals were completely protected, with the exception of a transient rise in body temperature in animals inoculated with the lower ∆9GL dose. The contribution of this research group to the development of attenuated ASF viruses was closed with the identification of a novel determinant of virulence, the 9GL gene . This prTo summarize, these four genes  were the first to be identified as determinants of virulence; this is because, when deleted from the respective ASFV isolate, it resulted in a drastic reduction in the virulence of the parental virus in domestic swine. Importantly, all four attenuated strains  efficiently protected pigs from disease produced after the challenge with the corresponding parental virus. Interestingly, although these genes were conserved across isolates, deletion of the same gene from the genome of different ASFV strains did not always produce the same phenotype in terms of virulence attenuation and protection efficacy, as described in the next section.3 HAD50 of the BeninΔDP148R virus only developed a transient rise in body temperature without additional signs related to ASF. The virus remained in the blood for approximately 3 weeks after infection. After receiving a booster 21 days after the first dose, animals were challenged IM with 104 HAD50 of the parental virulent virus 3 weeks after the booster. The challenged animals survived with only a transitory period of fever. Interestingly, deletion of the DP148R gene from the genome of the highly virulent field Chinese isolate HLJ/18 did not reduce virulence in domestic swine [3 HAD50, showed a virulent phenotype indistinguishable from that of the parental virus. Similarly, deletion of the DP148R gene from the genome of the Georgia 2007 isolate did not attenuate the virus [3 HAD50 of Georgia∆DP148R developed a fatal disease with severity and kinetics similar to those of the parental Georgia strain. These reports constitute an example of how deletion of the same gene in different ASFV strains may produce recombinant viruses with very different virulent phenotypes. The next single gene described that was involved in ASFV virulence was a previously uncharacterized gene mapped on the right end of the virus genome DP148R . Deletioic swine . The reche virus . Animals4 or 106 PFU of BA71ΔCD2 remained clinically normal with just a transient rise in body temperature and, importantly, were protected when IM challenged 24 days later with 103 HAD50 of the virulent BA71 virus. Remarkably, all pigs immunized with BA71ΔCD2 also survived the IM challenge with 20 LD of the heterologous Georgia 2007/1 isolate. Interestingly, deletion of the CD2 gene from other virus isolates has not been associated with a reduction in virulence as in the case of BA71ΔCD2. Deletion of the CD2 gene from isolates such as Malawi Lil-20/1 [Deletion of the viral CD2 (EP402R) gene produced complete attenuation of the virulent strain BA71 in swine . BA71ΔCDLil-20/1 , GeorgiaLil-20/1  and CN/GLil-20/1  did not Lil-20/1 . As lateLil-20/1 ,31.The 2007 outbreak of the disease in the Republic of Georgia re-energized research in the development of ASF vaccines. The genetic determinants of virulence  initially described above were used as the first gene candidates for deletion from the parental ASFV Georgia isolate . 4 HAD50 either died from acute ASF disease or developed a delayed and subclinical form of the disease. Similarly, deletion of the UK gene from the Georgia2010 isolate did not result in reduced virulence in domestic pigs [4 HAD50 of ASFV-G-∆UK developed a fatal and acute disease indistinguishable from that caused by the parental Georgia2010 virus. The deletion of the NL-S gene from the highly virulent ASFV Georgia2010 isolate (genotype II) did not produce a similar result  to when tic pigs  as it ditic pigs . Pigs re6 TCID50, did not induce ASF disease; however, interestingly, these animals were not protected when IM challenged with 103 TCID50 of the virulent parental Georgia strain [Deletion of the TK gene in a virulent strain of ASFV Georgia adapted to replicate in Vero cells produced a virus (ASFV-G/V-∆TK) that, when IM inoculated into pigs up to 10a strain . 3 HAD50) of ASFV-G-∆9GL remained clinically normal , pigs receiving 104 HAD developed a fatal acute disease. These results indicate that ASFV-G-∆9GL retains residual virulence that is not seen in Malawi Lil-20/1, which remained attenuated even when IM administered at high doses (106 HAD) [Although deletion of the 9GL gene from the ASFV isolate Pretoriuskop/96/4 (Pret4) produced similar attenuation results  as in th106 HAD) .These results highlight phenotypic differences when evaluating attenuation and protective efficacy induced in diverse ASFV isolates through the deletion of the same gene, even in the case of those genes being highly conserved across strains. The molecular basis of these differences is not understood at this time, but needs to be considered in the development of attenuated virus strains using field isolates.3 HAD50), but produced residual virulence at a higher dose. Regardless of its residual virulence, when used at a sublethal dose (102 or 103 HAD50), ASFV-G-∆9GL induced protection against the IM challenge with 103 HAD50 of the Georgia2007 isolate, both at 21 and 28 days pv. The challenged animals remained clinically normal with an absence of replication of the challenge virus (sterile immunity). ASFV-G-∆9GL was one of the first recombinant attenuated viruses reported to induce protection against the epidemiologically important Georgia isolate.The first successful attempt to develop a live attenuated vaccine candidate based on the Georgia 2007 isolate was deletion of the 9GL gene, as described above . Deletio6 HAD50 IM remained clinically normal, including an absence of fever. Importantly, animals receiving a wide range of ASFV-G-∆I177L doses were completely protected against the IM challenge with 102 HAD50 of Georgia2007 virus at 28 days pv, even those receiving as little as 102 HAD50 of ASFV-G-∆I177L. Remarkably, the animals vaccinated with doses of 104 HAD50 or higher of ASFV-G-∆I177L developed sterile immunity against the challenge virus. Another recently identified determinant of virulence is the previously uncharacterized I177L gene . DeletioThe efficacy of ASFV-G-∆I177L was further tested, using, as the challenge virus, a highly virulent field isolate from Vietnam, TTKN/ASFV/DN/2019 . TTKN/AS6 HAD50 of ASFV-G-∆I177L and IM challenged 28 days later with 102 HAD50 of the virulent Georgia isolate were protected, with an absence of clinical disease associated with ASF [The effect of the administrative route was also assessed using ASFV-G-∆I177L, to determine its potential use as an oral vaccine. Animals oronasally inoculated with 10with ASF .A limitation of most of the experimental LAVs is the ability of the vaccine viruses to be produced in stable cell cultures. The exception is BA71∆CD2, which replicates in Cos-1 cells ; all oth1 HAD50 of ASFV-∆9L survived the infection, with only a transient fever and low viremia titers. No data were presented regarding the ability of ASFV-∆9L to protect against the challenge with the virulent parental virus. The ASFV gene MGF-110-9L, a previously uncharacterized gene located on the left side of the virus genome, was shown to be involved in disease production . A recom1 HA50 of the recombinant virus had no clinical sings associated with the disease during the 3-week observation period. ASFV-∆7R replication in the blood and tissues of inoculated pigs was decreased in comparison with the parental ASFV.A report analyzed the role of another gene member of the MGF cluster, MGF-505-7R, in ASFV virulence in pigs . The man50 presented with a fatal disease, another 20% had a transient clinical disease, and the rest remained clinically normal. Viremias in all the ASFV-∆360-9L infected pigs were present for more than 2 weeks. Another gene involved in disease production is MGF360-9L . It was 50 of ASFVΔI267L, a recombinant virus obtained by deleting I267L from the genome of the virulent Chinese field strain CN/GS/2018. Eighty percent of the pigs inoculated with the ASFVΔI267L virus exhibited mild clinical signs, surviving during the 3-week observation period; the remaining 20% of animals developed a protracted and fatal disease. All these animals experienced prolonged viremias in comparison with pigs infected with the parental virus. Interestingly, another group showed that deletion of I267L from the virulent Chinese field isolate SY18 genome produced a virus (SY18ΔI267L) that, when IM inoculated at doses of 102 TCID50, caused a severe and lethal disease in all the animals [50 showed either just a transient rise in body temperature or a complete absence of clinical signs associated with ASF [Recently, the ASFV gene I267L has been shown to inhibit RNA Pol-III-RIG-I-mediated innate antiviral responses  and has  animals . Perhapswith ASF . In theswith ASF , no data7 TCID50 remained clinically normal and had reduced viremia. After the IM challenge with 104 TCID50 of the parental strain at 21 days pv, all the challenged pigs survived with no development of ASF disease.It was recently reported that I226R, a previously uncharacterized ASFV gene located on the right end of the virus genome, was shown to be involved in disease production . Deletio2 HAD50 of ASFV-G-∆A137R remained clinically healthy during the 28-day observational period, with medium-to-high viremias. Importantly, all the ASFV-G-∆A137R-inoculated animals were protected when IM challenged with 102 HAD50 of the virulent parental strain ASFV-G. No evidence of replication of the challenge virus was observed in the ASFV-G-∆A137R-inoculated animals.The ASFV gene A137R has also been reported to be involved in virus virulence . A137R i2 HAD50 of a recombinant virus lacking the E184L gene (ASFV-G∆E184L) survived the infection with only a transient fever, while the remaining animals experienced a significantly delayed presentation of fatal disease. All the animals surviving ASFV-G∆E184L infection were protected when IM challenged with 102 HAD50 of the parental Georgia isolate.Deletion of the ASFV gene E184L, located on the right end of the virus genome, is also involved in disease production by the Georgia2010 isolate . Sixty pA summary of the determinants of virulence that attenuate ASFV-Georgia-derived viruses is in Several groups have attempted the development of recombinant viruses including the deletion of more than one gene. Multiple-gene deletion has been used to produce an attenuation when there is no clear identification of individual target genes, or to enhance the level of attenuation of a virus already harboring deletions affecting virulence. The obtained results are not always as expected, since combining different gene deletions does not produce a predictable linear effect.4 HAD50 remained clinically healthy. Importantly, when animals receiving as little as 102 HAD50 were challenged with the virulent parental Georgia2007 strain, no signs of the disease were observed, despite detection of the challenge virus. A recombinant virus (HLJ/18-6GD) harboring similar gene deletion was developed using the highly virulent Chinese field isolate HLJ/18 [4 HAD50 remained clinically normal and were protected against the challenge with 200 Pig Lethal Dose 50 (PLD50). Reversion to virulence studies performed with the recombinant virus HLJ/18-6GD demonstrated the presence of clinical signs associated with the disease in one of the animals included in the passage 5 and 6 groups, prompting questions about the genetic stability of the recombinant virus [An example of attenuation of virulent ASFV through deletion of a group of genes is the production of an attenuated strain, ASFV-G-∆MGF, with removal of six genes belonging to the MGF360 and MGF505 groups  from the genome of the highly virulent Georgia2007 isolate . These ge HLJ/18 , confirmnt virus . No infont virus . The samnt virus . 4 HAD50 and challenge experiments performed with Georgia 2007. The co-deletion of MGF360-12L, MGF360-13L, MGF360-14L and MGF505-1R reduced virus replication in macrophages and resulted in complete attenuation in pigs; 64% of animals were protected after the challenge. Simultaneous deletion of MGF360-13L, MGF360-14L, MGF505-2R and MGF505-3R did not reduce virus replication in macrophages, and produced a delayed but lethal disease in all the inoculated animals. Lastly, co-deletion of genes MGF360-12L and MGF505-1R reduced virus replication in macrophages and eliminated virulence in pigs; 34% of the pigs were protected when challenged with the Georgia isolate. These results suggest that the deletion of genes MGF360-12L and MGF505-1R attenuates Georgia2007, but this recombinant virus is unable to efficaciously protect against the challenge. None of the recombinant viruses developed in this report [2 HAD50 and 104 HAD50, respectively, only developed a transient fever. These animals were protected from the IM challenge at 6 weeks pv with 104 HAD50 of the parental virus, with a complete absence of clinical signs associated with the disease. It should be mentioned that the only available study on long-lasting immunity induced by a recombinant vaccine candidate was performed with the Benin∆MGF virus [4 HAD50 of the Benin∆MGF virus were not protected when IM challenged 130 days later with 104 HAD50 of the virulent parental Benin 97/1 strain.A recent report  further s report  were abls report  and HLJ/s report , includis report . Animals4 HAD50) in swine [2 HAD50 of the virulent parental virus.A novel recombinant virus, ASFV-∆QP509L/QP383R—produced by the deletion of two previously uncharacterized virus genes, QP509L and QP383R, from the genome of a highly virulent ASF virus CN/GS/2018 strain—was highly attenuated when IM inoculated  located on the right end of the virus genome of the virulent strain SY18, demonstrated a partially attenuated phenotype in pigs . SY18∆L74 TCID50 did not develop clinical signs associated with ASF, except for a transient fever. After the IM challenge at 28 days pv with 104 TCID50 of the parental virus, all the ASFV-SY18-∆CD2v/UK-inoculated pigs remained healthy. A similar recombinant virus, HLJ/18-9GL&UK, harboring the same double gene deletion in the same parental virus retained residual virulence, producing a lethal disease in 50% of the animals IM inoculated with 104 TCID50 [An excellent example of unexpected synergistic activity between genes individually unable to induce attenuation by themselves is the recombinant virus ASFV-SY18-∆CD2v/UK . This vi4 TCID50 . This isAs mentioned earlier, the effect of multiple-gene deletions from the genome of a virus to enhance attenuation is difficult to predict. When attempting to improve a vaccine candidate’s safety profile, these additional deletions often do not increase attenuation, and/or there is a loss in vaccine efficacy.3 HAD50) into domestic pigs. Importantly, both failed to protect against the challenge with the parental virulent ASFV-Georgia. Therefore, the deletion of CD2-like and C-type lectin-like genes significantly decreased the protective potential of ASFV-G-∆9GL as a vaccine candidate.This scenario played out during an attempt to increase the attenuation of a vaccine candidate through the deletion of two virus genes involved in pathogenesis: CD2v (EP402R) and the C-type lectin-like (EP153R) genes from the genome of the vaccine candidate ASFV-G-∆9GL . ASFV-G-The effects of the individual or combined deletion of the same genes, CD2 and EP153R, from the Benin∆DP148R vaccine candidate produced different effects  than whaSimilarly, to improve the safety profile of BA71∆CD2, two double recombinant viruses were developed, deleting either the EP153R gene or the UK gene (DP96R), BA71∆CD2EP153R and BA71∆CD2DP96R, respectively . Compara6 HAD50. These animals did not present detectable levels of virus in blood at any time post-infection, and they were not protected against the challenge IM with 106 HAD50 of the virulent parental ASFV-G isolate [ASFV-G-∆9GL represents another example of the unpredictability of genetic manipulation involving deletions of multiple genes . The 9GL isolate .4 HAD50 of ASFV-G-∆9GL developed a fatal form of ASF, those IM inoculated with up to 106 HAD50 of ASFV-G-∆9GL/∆UK remained clinically normal. ASFV-G-∆9GL/∆UK grows in swine macrophages with a decreased ability compared with the parental ASFV-G-∆9GL, but effectively protected pigs IM inoculated with either 104 or 106 HAD50 of the virus against the IM challenge with 103 HAD50 of Georgia isolate.In summary, most attempts to increase the safety profile of ASFV-G-∆9GL by combining the deletion of the 9GL gene with additional deletions have been unsuccessful, as evidenced by the significantly decreased protective efficacy ,51. One These many studies show that the deletion of similar genes in different vaccine candidates may produce a variety of phenotypic changes, and this unpredictability can be extended to the addition of multiple-gene deletions to an already attenuated strain. This observation is not surprising, considering that much remains unknown about the role of the deleted genes in virus virulence. Developing and testing recombinant viruses harboring each particular combination of gene deletions appears to be the only accurate approach to assessing the potential effect of those genetic manipulations on virulence attenuation and protective efficacy.More than 40 recombinant virus strains have been developed by deleting genes from ASFV isolates, many of which were single deletions resulting in no observable phenotype to virus virulence ; of thesThe majority of the published results support the use of genetic manipulation to produce recombinant viruses with potential use as vaccine candidates. The lack of extensive experimental data and results for many of these recombinant viruses impedes further consideration of their potential as vaccine candidates. Basic information is needed before a recombinant virus can be seriously evaluated as a vaccine candidate, including: the onset and duration of protective efficacy; genetic stability; the stability of the attenuation phenotype; minimal protection doses; the possibility of being used with alternative routes of inoculation , etc.6 HAD50), using pigs of different ages, and challenge procedures are using different doses (from 10 to 105 HAD50) performed at different times post-immunization. Theses variables make it difficult to conduct a comparative analysis of all the published vaccine candidates. The very low doses used to test attenuation by some laboratories raise questions about the presence of residual virulence of those viruses if they were tested at higher doses. Residual virulence at higher doses is a common outcome during these types of studies and is a major objectionable factor in a vaccine strain. Differences in the challenge dose used is another consideration when comparing vaccine efficacy between studies. Standardized ASF vaccine development protocols would make for more robust evaluation of virus attenuation and assessment of protective efficacy.The analysis of the published information makes it apparent that there is heterogeneity in the methods employed to evaluate the attenuation and protective efficacy of each of the vaccine candidates . VaccineThe residual virulence and stability of the attenuated phenotype are two concerns when utilizing live attenuated viruses as vaccine strains in field conditions. Many recent reports have described the development of novel vaccine strains, but most of these are not accompanied by additional studies evaluating their safety profiles. Basic safety considerations include an absence of general and local toxicity and detection of reversion to virulence. The recombinant viruses HLJ/18-6GD and HLJ/18-7GD are an exception, with safety data available for both viruses .Another topic that needs more attention is the development of DIVA-capable vaccine candidates. Currently, only two reports discuss the identification of potential target genes to support the development of accompanying DIVA tests ,47. As hHeterogeneous phenotypes produced by the deletion of a specific gene or group of genes from different virus isolates is another research area that requires further investigation ,32,33,51Finally, it is important to continue the effort to systematically discover and characterize virus genes associated with virulence. The significant work performed by the scientific community in discovering novel determinants of virulence has resulted in the development of a relatively large set of attenuated virus strains, and in the characterization of many virus genes with previously unknown functions in virus virulence. This knowledge is the essential initial step required for the development of potential novel vaccine candidates.


Malaria in pregnancy remains a major health threat in sub-Saharan Africa to both expectant mothers and their unborn children. To date, there have been very few studies focused on the out of pocket costs associated with seeking treatment for malaria during pregnancy.A cross-sectional survey was undertaken in Burkina Faso and The Gambia to estimate the direct and indirect costs associated with outpatient consultations (OP) and inpatient admissions (IP). Direct costs were broken down into medical , and non-medical (transportation and food). Indirect costs reflected time lost due to illness. In total, 220 pregnant women in Burkina Faso and 263 in The Gambia were interviewed about their treatment seeking decisions, expenditure, time use and financial support associated with each malaria episode.In Burkina Faso 6.7% sought treatment elsewhere before their OP visits, and 27.1% before their IP visits. This compares to 1.3% for OP and 25.92% for IP in The Gambia. Once at the facility, the average direct costs (out of pocket) were 3.91US$ for an OP visit and 15.38US$ of an IP visit in Burkina Faso, and 0.80US$ for an OP visit and 9.19US$ for an IP visit in The Gambia. Inpatient direct costs were driven by drug costs (9.27US$) and transportation costs (2.72US$) in Burkina Faso and drug costs (3.44 US$) and food costs (3.44 US$) in The Gambia. Indirect costs of IP visits, valued as the opportunity cost of time lost due to the illness, were estimated at 11.85US$ in Burkina Faso and 4.07US$ in The Gambia. The difference across the two countries was mainly due to the longer time of hospitalization in Burkina Faso compared to The Gambia. In The Gambia, the vast majority of pregnant women reported receiving financial support from family members living abroad, most commonly siblings (65%).High malaria treatment costs are incurred by pregnant women in Burkina Faso and The Gambia. Beyond the medical costs of fees and drugs, costs in terms of transport, food and time are significant drivers. The role of remittances, particularly their effect on accessing health care, needs further investigation. Malaria in pregnancy (MiP) is a major public health problem in sub-Saharan Africa (SSA) . It alsoThe success of IPTp delivery depends partly on access to, and use of, antenatal care (ANC) by pregnant women. In 2016 about 26% of women did not attend ANC facilities during their pregnancy . It is eA range of geographic, economic, social and cultural factors contribute to the low use of antenatal clinics. These factors include, among others, distance and poor road infrastructure, low level of education, poverty, limited access to information or traditional values , 11. At Costing studies associated with MiP are few in number compared to other malaria interventions , 14 and While our focus is on the costs of seeking treatment for malaria while pregnant, we also asked some explorative questions about financial support the respondents may have received. We were particularly interested in the frequency and sources of remittances. Evidence shows that remittances improve various health outcomes –28. The The costing study was ancillary to the cluster randomized trial called “Community-based scheduled screening and treatment of malaria in pregnancy for improved maternal and infant health: a cluster-randomized trial in The Gambia, Burkina Faso and Benin” (commonly referred to as COSMIC study). The study aimed to determine whether adding community-based scheduled screening and treatment of malaria (CSST) by community health workers (CHWs) to standard IPTp-SP would further reduce placental malaria compared to IPTp-SP alone.  In practBetween November 2013 and November 2015, a total of 4731 pregnant women across the three countries took part in the 2-arm cluster-randomized, controlled trials to assess the clinical outcomes. The cross-sectional costing study presented here was conducted between August 2014 and December 2015 and designed to assess the direct and indirect costs associated with a single malaria episode from the pregnant women’s perspective in all three countries. However, data from Benin was excluded because the clinical trial became subject to rumours and accusations of placenta being sold for mystical and financial gain by staff despite community sensitization meetings and standardized informed consent procedures. After discussions with the data and safety monitoring board and the local ethics committee, it was decided to stop the data collection in this study site .In The Gambia, the study was conducted in the eastern part of the country on the southern bank of the Upper River Region , around the Basse Health Centre and satellite health facilities. Although malaria in The Gambia has declined significantly over the last 10–15 years, there is still moderate and highly seasonal (July–December) transmission in its eastern region. Despite reductions in malaria, about 15% of pregnant women are still shown to have placental malaria at delivery . In The Prior to starting the COSMIC study, community sensitization was carried out at each study site. Following cultural norms in both settings, consent was sought at the community level through discussions with village elders. Where appropriate further community meetings were then held to explain the study objectives to all community members. Experienced data collectors explained in the local language the written information and consent forms to potential participants. This conversation outlined study goals, the topic and type of questions respondents could expect and highlighted their right to decline participation, to interrupt or withdraw from the questionnaire at any time without giving a reason. If an individual agreed to answer the costing questionnaire, they signed the informed consent (by thumb print and the signature of an independent witness in case of illiteracy). The economic protocol followed the same inclusion and exclusion criteria as that used in the clinical trial.  PregnantFor those who consented, structured questionnaires were administered. The questionnaires comprised of five sections: 1. socio-demographic characteristics of the study participants such as age, level of education and receipt of money from anyone living abroad; 2. transportation costs to the health facility; 3. treatment seeking behavior and previous treatments for symptoms associated with the same malaria episode; 4. direct costs of treatment which was confirmed with data abstracted from the prescription orders; and 5. time lost because of the illness. See Annex 1–4 for English and French versions of the questionnaire.Costs were broken down into both direct and indirect costs. Direct costs (out-of-pocket expenses) were further broken down into medical , and non-medical (such as transportation and food). Indirect costs reflected time lost because of the illness. Indirect costs were collected for inpatient visits only. They were calculated by multiplying reported time lost by the nominal value of the median monthly permanent income per capita of households in Burkina Faso and The Gambia, represented by country specific estimates by the International Labour Organisation , 24, 34.All data were collected through standardized questionnaires, double entered into a specially designed database and verified. Analysis was conducted using Microsoft Excel and Stata software . All questionnaires were labelled with the pregnant woman’s unique identity number and date of collection, thus ensuring anonymity. Costs were collected in local currencies.Missing values were rare, never beyond the 3% of a variable, and reflected information that participants did not want to disclose or could not remember. Our values can, therefore, be interpreted as a lower bound of the estimated categories.Costs were then converted to US dollars (US$) using an average exchange rate for 2016 . In mostThree hundred fifty-five outpatients (150 in Burkina Faso and 155 in The Gambia) and 178 inpatients (70 in Burkina Faso and 108 in The Gambia) were interviewed  than in The Gambia (64.5%), with only 10.6% of women having completed secondary education in Burkina Faso and 13.5% in The Gambia. In both countries, the main activity was farming, for the women (54.6% in Burkina Faso and 54.1% in The Gambia) and their partners (68.0% in Burkina Faso and 66.9% in The Gambia). About half (52.7%) of the women in Burkina Faso were Catholics while most (97.4%) Gambian women were Muslims.Inpatient characteristics were similar to outpatient ones. Women in The Gambia were older (27.02 years) and had a higher number of children (1.75) than those in Burkina Faso (23.87 years and 1.47). Similarly, most women were farmers and had no formal education.Table Table Table Before their IP visit, 27.1% and 25.9% of women sought treatment in Burkina Faso and The Gambia, respectively. Inpatients reported being ill on average 3.21 (range 1–20) days in Burkina Faso and 1.83 (range 1–4) days in The Gambia before being hospitalized. The mean cost of the first treatment for IP was higher in The Gambia (2.74US$) than in Burkina Faso (2.21US$).The mean total cost for an OP consultation, including any previous treatment, was 2.72US$ in Burkina Faso and 0.86US$ in The Gambia. For hospitalization, the cost was 4.27US$ in Burkina Faso and 2.74US$ in The Gambia for an admission. For inpatients, costs for first treatment sought and total were statistically different between the two countries . For outpatients, costs for first, second treatment sought and total were not statistically different between the two countries .Table Table This study represented a rare opportunity to cost malaria treatment in two different settings, in Burkina Faso and The Gambia, from the perspective of pregnant women. We present the unit costs and cost breakdowns of both inpatient and outpatient, data that has not been available to date. We are able to show in Gambia treatment costs are lower, largely due to lower drug costs. Our data suggests that approximately 27% of pregnant women in both settings had sought care elsewhere before their inpatient visit, notably from home or the pharmacy. The vast majority of women in The Gambia reported financial support directed at their household, our study fell short of confirming if these remittances were used by the pregnant women towards health care.The direct cost of inpatient treatment for malaria in Burkina Faso was US$15.38, the equivalent of 55% of the monthly female agricultural wage (estimated at US$28.31 per month ). In TheIn 2016, a few months after the end of our data collection, the government of Burkina Faso established a free health care policy for women. The benefit package now covers a wide range of services including antenatal care, the prevention of anaemia and malaria. Consultation fees, prescriptions fees, laboratory tests, hospitalization expenses and the expenses of ambulance transportation between health facilities are also covered. A recent study showed that while the policy has provided effective financial protection, a significant proportion of women continue to pay for services and consumables that should be free of charge . Our stuIn Nigeria, the average cost of treating an episode of malaria during pregnancy was reported as US$11.86  and US$18.97  . While tThis is the first study to explore the frequency of financial support, more specifically remittances to households with pregnant women in both settings. While there appears little or no health insurance, in The Gambia most women received remittance from family members living abroad, mainly from a few European countries  and the United States. Estimates of the global Gambian diaspora range between 118,485 to 200,000, one of the highest rates of emigration in Africa, at approximately 9.2% of the population . Our finThere are limitations to this study. The costs data were obtained from a cross-sectional survey. Ideally, a longitudinal data would allow us to estimate the economic burden of malaria over the course of the entire malaria season and/or individual pregnancies to better reflect the impact of multiple episodes. We were only able to capture the costs of malaria episodes from women who visited the health facilities included in the study. There is always the risk of the Hawthorne effect whereby the pregnant women might have modified aspects of their behaviour in response to their awareness of being part of a trial, but this is unlikely, given the trial offered no additional benefits in terms of access to care or finances in either trial arm. Finally, the costs in this study are associated with the immediate malaria infections in pregnant women and do not consider the long-term costs of treating the consequences of maternal infection on the infant. For instance, the consequences of low birth weight have been studied previously –43.A recent review of the status of MiP highlights three main economic issues than need further attention. First, MiP accounts for a notable proportion of the total health-care budget; second, direct and indirect costs incurred by pregnant women for malaria prevention and treatment are high and; third, strategies to decrease costs incurred  are effective, but they need to be scaled up . In thisThe key message to policy makers emanating from this study is the same in both countries; reaching pregnant women in rural areas and encouraging them to engage with ANC services early during their pregnancy is essential for malaria prevention and control as direct and indirect treatment costs associated with malaria episodes amongst this vulnerable group are high even if many services are officially provided for ‘free’ to pregnant women.Our results revealed the high costs incurred by pregnant women in Burkina Faso and in The Gambia. Even when accounting for the removal of user fees, the indirect costs of transport and food can be significant. The costs incurred by pregnant women may have a large impact on the budgets of households, and consequences on the allocation of their limited resources. Our findings also suggest that the role of remittances, particularly in The Gambia, needs further investigation with a focus on the effect they may have on accessing health care and improving health outcomes.