Exserohilum turcicum, poses a grave threat to maize production worldwide. The resistance gene in A619Ht3, discovered decades ago, is an important genetic resource for NCLB control. By using a pair of near-isogenic lines (NILs) A619Ht3 and A619, together with the resistant and susceptible bulks derived from the cross of A619Ht3 and L3162 lines, we initially detected a Ht3-like (Ht3L) locus in bin 8.06 that was closely associated with NCLB resistance. We then performed five rounds of fine-mapping, which ultimately delimited the Ht3L locus to a 577-kb interval flanked by SNP markers KA002081 and KA002084. Plants homozygous for the Ht3L/Ht3L genotype exhibited an average reduction in diseased leaf area (DLA) by 16.5% compared to plants lacking Ht3L locus. The Ht3L locus showed extensive variation in genomic architecture among different maize lines and did not appear to contain any genes encoding canonical cell wall-associated kinases against NCLB. Moreover, the Ht3L locus was located ∼2.7 Mb away from the known Htn1 locus. We speculate that the Ht3L locus may contain a bona fide Ht3 gene or a novel NCLB resistance gene closely linked to Ht3. In practice, the Ht3L locus is a valuable resource for improving maize resistance to NCLB.Northern corn leaf blight (NCLB), caused by the fungal pathogen  Exserohilum turcicum (Zea mays L.) grown in areas with high humidity and moderate temperatures is more prone to NCLB outbreaks. During the grain-filling period, NCLB causes leaf necrosis and thus abolishes photosynthetic output, leading to lower grain yield (Northern corn leaf blight (NCLB), caused by the hemibiotrophic fungus turcicum , is one in yield . The devHelminthosporium turcium resistance l (Ht1), was identified in the inbred line ‘GE440’ and ‘Ladyfinger’ popcorn in 1959; this locus shows a partially dominant inheritance pattern and maps to chromosome 2L   show different symptoms, indicating that resistance mechanisms conferred by these genes are not equivalent  . Ht2 andtn1 gene . At the ormation . This obuivalent .qNLB1.06, was anchored to a 3.6-Mb region and narrowed down to two putative candidate genes by joint linkage and association mapping   at the Htn1 locus is the only resistance QTL against NCLB that has been cloned through map-based cloning (Previous studies have shown that quantitative trait loci (QTLs) for resistance to NCLB are dispersed over all 10 chromosomes in maize . Althoug mapping . The QTL (CSSLs) . Single- (CSSLs) . A total (CSSLs) . To date cloning . Further cloning .Htn1 locus is effective against most prevalent NCLB races, NCLB isolates with virulence in Htn1-bearing plants have been reported  (Triticum aestivum) (Ht3-like (Ht3L) locus in the resistant line A619Ht3 by combining NIL analysis, BSA and sequential QTL fine-mapping. Our results lay the foundation for the future map-based cloning of the causal gene at the Ht3L locus. The molecular markers on or adjacent to Ht3L described here can also be used for MABC to improve maize resistance to NCLB in breeding programs.Bulked-segregant analysis (BSA) is a rapid, technically simple method to identify markers linked to specific genes , which h sativa) , wheat (estivum)  and maizestivum) . Anotherestivum) . Many questivum) . In the Ht3 and A619 was obtained from the Eastern Cereal and Oilseed Research Centre of Agriculture and Agri-Food Canada. While A619Ht3 is highly resistant to NCLB, A619 is highly susceptible to NCLB, and both NILs share ∼98.84% genomic homozygosity (Ht3 (donor parent) and L3162 (recurrent parent), multiple backcross populations were developed for initial QTL detection and fine-mapping. In 2016, the two parental lines (A619Ht3 and L3162) and their BC2F1 backcross population comprising 362 individuals were grown at the Shenyang experimental station  in China for artificial inoculation. Twenty-nine highly susceptible and 29 highly resistant plants were selected to form the susceptible and resistant bulks, respectively, for initial mapping of the Ht3L locus. Subsequently, the BC3F1 progeny from recombination events in the mapped Ht3L region were identified and backcrossed to L3162  to produce BC4F1 progeny for fine-mapping. In the following five rounds of fine-mapping from 2017 to 2021, newly-occurring recombinants were screened in Shenyang and then backcrossed twice to L3162 in Shenyang and Hainan to generate progeny. All offspring were investigated for NCLB resistance at the Shenyang experimental station. A619Ht3 and L3162 were planted annually in each experimental block as resistant and susceptible controls, respectively.The pair of near-isogenic lines (NILs) A619zygosity . The eliExserohilum turcicum  was cultured on potato dextrose agar (PDA) medium for 15 days at 25°C and then stored in the dark at room temperature. Plant materials were artificially inoculated at the V8-V10 leaf stages with ∼5 mL of spore suspension (1 × 105-106 conidia per mL in 0.02% [v/v] Tween 20) per plant. Spray irrigation was then performed for 2 days following inoculation, three to four times a day, to maintain a humid environment. Four weeks after inoculation, NCLB lesions were prominent and suitable for first scoring, followed by two more scorings over the next 14 days. Diseased leaf area (DLA) was examined for three leaves (ear leaf and its upper and lower leaves) based on standards listed in Ht3 and A619) and the two parental lines (A619Ht3 and L3162). The Maize6K Chip was used to genotype each plant in the selected susceptible and resistant bulks. Kompetitive Allele-Specific PCR (KASP) markers, developed in the target Ht3L region, were subjected to automated genotyping with the QuantStudio 12 K Flex Real-Time PCR System (Applied Biosystems by Life Technologies).Fresh leaf tissue at the five-leaf seedling stage was harvested for high-throughput extraction of genomic DNA in 96-well plates. Plant genomic DNA was extracted according to the method described previously . The MaiHt3L. The average Δ(SNP-index) value of the SNPs located in each genomic interval was calculated using a sliding window with a window size of 10 Mb and 10-kb steps. The distribution of Δ(SNP-index) along the chromosomes was plotted using R software. SNP markers in the candidate Ht3L regions were also used to conduct regional association analyses. For each SNP marker, the significant difference in SNP-index between two bulks was determined by chi-squared test.The single-nucleotide polymorphism (SNP) index was calculated based on the heterozygosity at each SNP within a given bulk. For each SNP, the Δ(SNP-index) value was calculated using the following formula: Δ(SNP-index) = SNP-index in the resistant bulk – SNP-index in the susceptible bulk . The Δ by inoculating designated physiological races of Exserohilum turcicum  in growth chamber. The results showed that A619Ht3 exhibits susceptible to races 3 and 123N, while resistant to races 1 and 2 (Ht3 locus in OH43 and HZS genetic backgrounds (Ht3 as Ht3-like (Ht3L) to distinguish it from the recently reported Ht3 allele that is identical to Ht2 and allelic to Htn1  potentially covering the Ht3L locus. We detected three segments with stretches of polymorphisms between the two NILs in bins 3.08 (6 SNPs), 7.03/7.04 (12 SNPs) and 8.06 (12 SNPs) (Ht3L regions.The NILs A619 and A619esistant . We geno12 SNPs) , which wHt3L locus, we selected the susceptible line L3162 that is genetically distinct from A619Ht3 to generate mapping populations. We genotyped A619Ht3 and L3162 with the Maize3K Chip, which returned 493 SNPs distributed over all maize chromosomes, numbers sufficient for an initial mapping of Ht3L  for each SNP in the resistant and susceptible bulks. This analysis revealed a prominent peak on chromosome 8 .From a BCmosome 8 . We also.03/7.04 . By contbin 8.06 . Taken tHt3L region to saturate the target region. Accordingly, we retrieved the SNP markers in the Ht3L region between A619Ht3 and L3162 from the genotype data of the Maize6K Chip. We then converted all SNPs located in the Ht3L region into KASP markers and tested them against A619Ht3 and L3162. Totally, we obtained 18 effective KASP markers for fine-mapping  to detect recombination events in the 11.23-Mb Ht3L region from the BC2F1 population obtained in 2016. We identified seven BC2F1 recombinants, which we backcrossed twice to the susceptible parent L3162 to produce their corresponding BC4F1 populations. With an additional five newly-developed KASP markers , we determined the precise recombination breakpoint for each recombinant. To this end, we grew 1,678 plants from the BC4F1 progeny in the field and scored them for their extent of diseased leaf area (DLA) in Shenyang in 2017  from heterozygous (A619Ht3/L3162) BC4F1 plants. With the both genotypic and phenotypic data, an average DLA value can be estimated for both homozygous and heterozygous genotypes in each BC4F1 progeny. Significant difference in DLA between two genotypes indicated the presence of the Ht3L locus in the A619Ht3 donor segment; otherwise, there is no Ht3L locus. As shown, recombinants I to V showed a significant difference (P < 0.05) in their DLA values between homozygous and heterozygous genotypes in their BC4F1 progeny, indicating that they carried the Ht3L locus in the A619Ht3 donor segment (P > 0.05), and thus lacked the Ht3L locus. Recombinants V (with Ht3L) and VII (without Ht3L) defined the left boundary of the mapping interval to marker A007452, while recombinants I (with Ht3L) and VI (without Ht3L) marked the right boundary with marker A007455. The resulting interval spanned a region of 2.17 Mb (AGPv5)  .Ht3L locus with high-density markers. Thus, we backcrossed recombinants I and V to L3162 twice to produce their BC6F1 progeny. We also identified another three new recombinants within the 2.17-Mb region from the BC4F1 progeny, which, together with a heterozygous plant (as a positive control), were backcrossed twice to L3162 to produce BC6F1 progeny. We genotyped the five resulting BC6F1 progeny consisting of 847 individuals, grown in the field for NCLB testing in 2018, with 11 markers spanning the 2.17-Mb region, including seven newly-developed markers. We also tested the markers against the heterozygous Ht3L region to distinguish homozygous from heterozygous BC6F1 progeny. We detected a significant difference (P < 0.05) in DLA for recombinants I, II and III, together with the positive control .From the BCnt IV in  shows a e region . The oth10F1 plants corresponding to seven recombinants in Shenyang for further fine-mapping. Of them, two new recombinants (IV and VI in the P > 0.05) in DLA between the BC10F1 homozygous and heterozygous genotypes, indicating the A619Ht3 donor region lacked Ht3L. Based on these two recombinants, we moved the right boundary from marker KA002085 to marker KA002084. Phenotypic scorings for NCLB severity in the field indicated that the progeny from recombinants I-III showed a significant difference (P < 0.05) in DLA between the homozygotes and heterozygotes in their BC10F1 progeny, whereas the progeny from recombinants IV–VII did not show this difference. Thus, we ultimately anchored Ht3L to a 577-kb interval flanked by markers KA002081 and KA002084 (AGPv5).In the summer of 2020, we planted 3,200 BC10F1 progeny, we selfed heterozygous recombinants to develop three pairs of NILs, which were planted in the summer of 2021 to evaluate their DLA values. The first two pairs of NILs .From the BC did not . These rHt3L/ht3l genotype as the difference in the DLA values between heterozygous and homozygous genotypes for each of the BC4F1, BC6F1, BC8F1 and BC10F1 progeny. For the ht3l/ht3l and Ht3L/ht3l genotypes, we estimated the average DLA values to be 20.3% and 16.5% in BC4F1, 29.4% and 21.6% in BC6F1, 40.5% and 32.1% in BC8F1 and 42.2% and 35.9% in BC10F1, respectively  and resistant NILs (with Ht3L/Ht3L) were 40.4% and 23.9%, respectively. Thus, the genetic effect of the homozygous Ht3L/Ht3L genotype was 16.5% , a zinc knuckle (CCHC-type) protein (Zm00001eb361470), two violaxanthin de-epoxidases (Zm00001eb361480 and Zm00001eb361490), a brassinosteroid-insensitive protein (Zm00001eb361520), a potassium channel KAT protein (Zm00001eb361550) and a phosphatidylinositol kinase (Zm00001eb361560).Bin 8.06 is associated with several tructure . Within t report . Three oHt3L region from 25 sequenced founders of the NAM population (see text footnote 1). Of these 25 lines, seven were highly susceptible and another ten were highly resistant to NCLB, based on a previous report  in bin 8.06, flanked by the SNP markers PZE-108095959 and PZE-108110343. Notably, this 11.23-Mb segment overlapped with the known location of the Htn1 locus. After five rounds of fine-mapping, we delineated the Ht3L locus to a 577-kb interval. To our surprise, the Ht3L locus was located ∼2.7 Mb away from Htn1  with two loci (Ht3L and Htn1) did not display a lower DLA score than those BC2F1 recombinants (No. V) only harboring the Ht3L locus. The lack of phenotypic variation associated with the Htn1 locus between A619Ht3 and L3162 thus indicated that both lines have the same susceptible Htn1 allele. The other possibility is that Htn1 may lose its NCLB-resistance function due to the presence of an unidentified suppressor or the absence of a co-receptor in the L3162 background. For example, a dominant suppressor inhibiting the expression of Ht2 was found in lines related to ‘B14’  the 577-kb Ht3L locus interval may contain a bona fide Ht3 gene that is located ∼2.7 Mb away from the known Ht2/Ht3/Htn1 locus; or 2) A619Ht3 harbors a Ht3 gene that is allelic to Htn1, as claimed by Ht3L locus must contain a novel NCLB resistance gene. In this alternative scenario, we speculate that A619Ht3 is unlikely to harbor another resistant allele at the reported Ht2/Ht3 or Htn1 loci, as only one resistance QTL was detected in our mapping populations. Since the detailed genomic sequence of the parental line A619Ht3 is not currently available, we are still uncertain about the relationship between the currently mapped Ht3L locus and the reported Ht2/Ht3/Htn1 locus. In addition, the genetic backgrounds clearly have pronounced effects on Ht3L-conferred NCLB resistance, as we observed wide variation in DLA scores between homozygotes and heterozygotes in the fine-mapping progeny of different parental recombinants.We detected extensive genomic variation within the 577-kb n1 locus . In fielExserohilum turcicum shows clear physiological differentiation as a function of the resistance performance of inbred lines and differences of climatic conditions. Moreover, the distribution of physiological races is also complex. Twelve physiological races have been identified in China, of which races 0 and 1 are dominant  haplotypes into seven elite maize inbred lines exhibited enhanced resistance to maize stalk rot (Ht3L locus for NCLB resistance. Considering the substantial annual yield loss caused by NCLB, the Ht3L locus will be valuable in future breeding programs of NCLB-resistant maize.dominant . Becausetalk rot . The curHt3L to an interval of 577kb via five rounds of sequential fine-mapping. The homozygous Ht3L/Ht3L genotype reduced DLA by 16.5% compared to that in lines without Ht3L. Our results will facilitate the cloning of the causative gene underlying the Ht3L locus and accelerate application of Ht3L in the breeding of NCLB-resistant maize varieties.Mining and utilizing resistant loci/genes can greatly promote the development of resistant varieties, which will decrease yield losses and improve grain quality. With the availability of high-density SNP markers, we delimited The original contributions presented in this study are included in the article/MJ and YW designed the experiments. JM, XL, XQ, XG, and MJ were responsible for the field tests. YG, YZ, and XL conducted genotyping. MZ and YG analyzed the data. MZ, JM, and MJ wrote the manuscript. MJ and YW supervised the project. MZ, JM, and MJ revised the manuscript in response to the reviewers. All authors read and approved the manuscript.


The public health problems caused by schizophrenia are becoming increasingly prominent and can place a huge economic burden on society. This study takes Gansu Province as an example to analyze the level and changing trend of the economic burden of schizophrenia inpatients in economically underdeveloped areas of China. Using a multi-stage stratified cluster sampling method, 39,054 schizophrenics from 197 medical and health institutions in Gansu Province were selected as the research objects, and their medical expenses and related medical records were obtained from the medical information system. The rank sum test and Spearman rank correlation were used for univariate analysis. Quantile regression and random forest were used to analyze the influencing factors. The results show that the average length of stay of schizophrenics in Gansu Province of China was 52.01 days, and the average hospitalization cost was USD1653.96 from 2014 to 2019. During the six years, the average hospitalization costs per time decreased from USD2136.85 to USD1401.33. The average out-of-pocket costs per time decreased from USD1238.78 to USD267.68. And the average daily hospitalization costs increased from USD38.18 to USD41.25. The main factors influencing hospitalization costs are length of stay, proportion of medications, and schizophrenic subtype. The hospitalization costs per time of schizophrenics in Gansu Province have decreased but remain at a high level compared to some other chronic non-communicable diseases. In the future, attention should be paid to improving the efficiency of medical institutions, enhancing community management, and promoting the transformation of the management model of schizophrenia. And it remains one of the most expensive mental illnesses in human society2. The disease occurs in late adolescence and early adulthood, and has a poor prognosis3. The mortality rate is 3.12 times higher in the patient population than in the general population4. It is reported that the number of schizophrenics worldwide increased from 13.1 million in 1990 to 20.9 million in 20163. The global age-standardized prevalence of schizophrenia is 287.4 per 100,000 in 20195. And the lifetime prevalence in China is 0.6%6. The public health problems caused by schizophrenia have become increasingly prominent because of the concentration of the disease in young adults, the high unemployment rate of patients7, the susceptibility to co-morbidity with other somatic diseases8, and the high incidence of aggression and violence9. And they also bring huge economic burden to schizophrenic families and the society.Schizophrenia is a complex psychiatric disorder, often accompanied by obstacles in perception, thinking, emotion, and behavior10. It mainly includes direct economic burden, indirect economic burden, and intangible economic burden. The direct economic burden reflects the medical resources used in the course of disease treatment11, and the hospitalization expense is the largest contributor to the total direct medical costs of schizophrenia12. The existing research shows that the social costs due to schizophrenia vary widely globally13. The global lifetime social cost of schizophrenia per capita ranges from USD5818 in Thailand to USD94,587 in Norway14. The level of economic development and medical care varies greatly among different regions in China, therefore the level of the economic burden of this disease may also vary greatly. However, only small sample data or medical insurance data have been reported in China to analyze the economic burden of the illness17, and they are mostly concentrated in more economically developed regions. Few relevant studies using large sample treatment data in less economically developed regions have been seen.The economic burden of illness is measured and analyzed in terms of the economic costs or losses incurred by the population due to the disease, including the costs of treatment and care for the patients, as well as the loss of productivity due to injury, disability, caregiver assistance or the patient’s violent behavior18. At present, there is a shortage of full-time prevention and control personnel of mental health in the province. In 2018, the standardized management rate of registered patients with severe mental disorders was 71.78%, of which the regular medication-taking rate of schizophrenia was only 27.49%19. Therefore, this study using Gansu Province as an example has more research significance in analyzing the level of economic burden of schizophrenia in economically underdeveloped regions of China. This study used the treatment data of schizophrenia inpatients to analyze the composition and changing trend of hospitalization costs in Gansu Province of China from 2014 to 2019. At the same time, it explored the factors influencing the average hospitalization cost per time and the average daily hospitalization cost of schizophrenics and their significance. Thus, it provides a theoretical basis for policy-makers to formulate relevant health policies and reduce the economic burden of schizophrenia.Gansu Province is located in an economically underdeveloped region in northwestern China, with a resident population of 26.47 million at the end of 2019. The GDP of the province this year was 871.83 billion Chinese yuan, with 12 prefecture level cities and 2 autonomous prefectures under its jurisdictionA total of 39,054 inpatients with schizophrenia were enrolled in this study from 2014 to 2019, including 21,272 males (54.47%) and 17,782 females (45.53%). The average age was 37.28 ± 12.32 years old. The average length of stay was 52.01 ± 41.87 days. And the percentage of patients using the medical insurance reimbursement payment method was 59.17%. In terms of institutional level (graded by administrative subordination), 98.25% of patients were treated in provincial or municipal medical and health institutions. In terms of the type of institution, 94.32% of the patients were seen in specialized hospitals. Among schizophrenia inpatients with defined subtypes, paranoid schizophrenia had the highest percentage of 34.06%.P < 0.05). See Table The results of the univariate analysis showed statistically significant differences in the average costs of hospitalization per time between patients with different gender, ages, payment methods, hospital nature, hospital levels, hospital types, number of comorbidities, length of stay, proportion of medications, and schizophrenic subtypes . Among them, the treatment expenses were CNY4,568.64 , which accounted for the highest percentage. This was followed by bed and nursing fees of CNY1938.65 , medication costs of CNY1521.65 , and examination costs of CNY1301.55 . The proportion of consultation costs accounted for the lowest, at CNY286.00 . See Fig. rs = −0.161, P < 0.001). The average growth rate over the 6 years was −8.09%. Average inpatient out-of-pocket costs have also been decreasing from CNY8545.74 (USD1238.78) in 2014 to CNY1846.56 (USD267.68) in 2019 . The average growth rate was −26.39%. However, during the same period, the average daily hospitalization costs for patients showed an overall increasing trend from CNY263.41 (USD38.18) to CNY284.56 (USD41.25), with an average growth rate of 1.56%. See Table From 2014 to 2019, the average hospitalization costs of schizophrenics per time in Gansu Province showed an overall downward trend, from CNY14,741.08 (USD2136.85) to CNY9667.07.Natural log-transformed hospitalization costs per time and average daily hospitalization costs of schizophrenia patients were used as dependent variables. Variables that were statistically significant for univariate analysis, such as gender, age, payment method, hospital nature, hospital level, hospital type, number of comorbidities, length of stay, proportion of medications, schizophrenic subtype, and year of hospitalization, were used as independent variables. Incorporating each of the above variables into the quantile regression model simultaneously. The Pseudo P < 0.05) on all three quantile points of hospitalization costs per time, and the intensity of the effect was higher at the lower quantile point than at the higher point. Length of stay was positively associated with hospitalization costs, with the strength of its effect being slightly higher at the higher quartile point than at the lower. Among payment methods, full out-of-pocket patients had lower hospitalization costs at the 10th percentile and higher at the remaining two quartiles than medical insurance reimbursement patients . For the number of comorbidities, patients with one, two, and three or more comorbidities had higher hospitalization costs at the 50th and 90th percentiles than patients without comorbidities .Age, hospital level, and proportion of medications had a significant negative effect (P < 0.05). In contrast, payment method and hospital level had a significant positive impact on the three quantiles . For the number of comorbidities, patients with one, two, and three or more comorbidities had higher average daily hospitalization costs at the 50th and 90th percentiles than patients without comorbidities . See Table Hospital nature, length of stay, and proportion of medications had a significant negative effect at all three quartiles of average daily hospitalization costs  to 500, and the number of variables randomly sampled as candidates at each split (mtry) to 3. At this point, the standardized mean squared error (RMSE) of the random forest regression tree model for hospitalization costs per time was 0.266 with Finally, the importance of the influencing factors was analyzed according to the mean decrease accuracy (%IncMSE). That is, the larger the %IncMSE, the higher the importance. The results showed that the top five influencing factors for hospitalization costs per time were length of stay, proportion of medications, schizophrenic subtype, number of comorbidities, and year of hospitalization, respectively. The top five factors influencing average daily hospitalization costs were proportion of medications, length of stay, schizophrenic subtype, hospital type, and age. See Fig. 20. The median length of stay for schizophrenia inpatients in Portugal was 18.0 days, and the mean age of the patients was 41.68 ± 12.93 years21. In comparison, Chinese patients with schizophrenia were hospitalized at a younger age and for a longer length of stay. This may be because countries such as the United States have implemented integrated community-based rehabilitation interventions, which have shifted from an inpatient model to a community-based model for schizophrenia services, thereby the length of stay has been significantly reduced22. Medical resources are scarce in underdeveloped areas in northwest China. And there are few full-time mental health prevention and control personnel in grassroots institutions. It is difficult for patients to obtain high-quality community nursing services after discharge. The above together lead to the longer length of stay for schizophrenia in China. The average hospitalization costs per visit of schizophrenia patients in Gansu Province from 2014 to 2019 was CNY 11,409.83 (USD 1653.96). A higher percentage of these are for treatment costs, bed and nursing fees, and medication costs, with the three accounting for more than 80% of the total costs. The average hospitalization costs per time for schizophrenia is lower than that of gastric cancer 23, but much higher than some other chronic non-communicable diseases such as diabetes (CNY 5131.34 in 2018) and coronary heart disease (CNY 6791.38 in 2015)25. Unlike the medication-based cost model for non-psychiatric disorders, treatment costs, which measure the value of the medical workforce, account for the highest proportion of schizophrenia hospitalization costs. This is consistent with the findings of other similar schizophrenia studies15.This study analyzed the hospitalization costs of schizophrenia patients in economically underdeveloped areas of China, using a large sample of consecutive 6 years of treatment data in Gansu Province. The results showed that the average age of schizophrenia inpatients in Gansu Province from 2014 to 2019 was 37.28 ± 12.32 years, and the average length of stay was 52.01 ± 41.87 days. Previous studies have shown that the average length of stay for patients with schizophrenia in Central and Eastern Europe (CEE) and the United States was 25.3 and 9.08 days, respectively26. Average out-of-pocket costs per time declined obviously faster than hospitalization costs. This may be related to the comprehensive promotion of rescue and treatment assistance for severe mental disorders and the gradual improvement of medical coverage level for the patients in the region. The average daily hospitalization costs showed an overall increasing trend over the 6-year period, with an average rate of increase of 1.56%. This was driven primarily by the decrease in length of stay.The results showed that the average hospitalization costs and out-of-pocket costs per time of schizophrenia patients in Gansu Province showed a decreasing trend from 2014 to 2019, with an average growth rate of −8.09% and −26.39% respectively. The decrease in hospitalization costs may be related to the promotion of graded treatment in the region and the improvement of service capacity of primary medical and health institutions. The region has implemented the service model of “treating schizophrenia in the hospital and managing rehabilitation in the community”. For patients in the acute stage and unstable condition, the primary institutions promptly refer them to professional mental institutions for standardized treatment. After stabilization, they return to the community to receive maintenance treatment with basic psychiatric medicines28. This study used random forest regression tree model to obtain the importance ranking of independent variables on the degree of impact of hospitalization costs. The results showed that length of stay, medication proportion, and schizophrenic subtype are the main factors influencing the hospitalization costs. Combined with the quantile regression results, the average hospitalization cost per time was negatively correlated with age and medication proportion, and positively correlated with length of stay and number of comorbidities. A previous Chinese study showed that the optimal length of stay for functional recovery in schizophrenics was between 20 and 50 days29. Therefore, the length of stay for schizophrenia patients should be reasonably reduced and other physical illnesses of the patients should be actively prevented. The efficiency of treatment in medical institutions should also be continuously improving. This will, on the one hand, help to increase bed turnover and bring more economic benefits to medical institutions, and on the other hand, help to reduce the patient’s medical costs. The average cost of a hospital stay at district and county level is lower than at provincial and municipal level. And the cost of specialized hospitals is higher than that of general hospitals. The number of inpatients with schizophrenia attending provincial and municipal level medical institutions is much higher than that of district and county level. This may be due to the fact that higher-grading institutions are better equipped and have a higher level of diagnosis and treatment. Therefore, patients are more likely to visit such facilities, and the costs are correspondingly higher. The hospitalization costs for full out-of-pocket patients are higher than for medical insurance reimbursement patients. This suggests that medical insurance has had a cost-controlling effect on schizophrenia inpatients. It has reduced unnecessary expenditure in medical costs, thus having a positive effect on reducing the economic burden of the disease.Random forest is an integrated learning algorithm based on decision trees, which has better tolerance for outliers and noise and is less prone to overfittingThis study also has some limitations. Firstly, the data for the study were obtained from the billing and settlement systems of healthcare institutions. Hence the variables included were somewhat limited and could not include other variables such as disease severity that may affect hospitalization costs. Second, for reasons such as the poor implementation of schizophrenia typing in Chinese clinical practice, some of the available statistics do not contain detailed information on disease subtypes. This may limit the further mining of data information.In conclusion, the average costs per time and the length of stay of schizophrenia inpatients in Gansu Province, China, decreased from 2014 to 2019, but currently remain at a high level. Therefore, in order to further reduce the level of the patient’s economic burden, the length of stay should be reasonably reduced. At the same time, the treatment efficiency of medical institutions should be improved, and the capacity of primary medical and health institutions for prevention and control of mental illness should be strengthened. Thus, the community management level of schizophrenia can be further improved and the rational distribution of medical resources can be continuously promoted.In this study, a multi-stage stratified cluster sampling method was used. In stage 1, among the provincial institutions, half of the number of general hospitals and Traditional Chinese Medicine hospitals  and one of each different type of specialized hospitals were selected as sample institutions using simple random sampling method. In stage 2, based on the economic level, geographical condition, and population size of the 14 cities and prefectures in Gansu Province, five cities, namely Pingliang, Dingxi, Zhangye, Wuwei, and Tianshui, were finally selected as sample cities. The cities follow the same sampling principle as the provincial medical and health institutions for municipal institutions. In stage 3, according to the geographical location and the urban and rural characteristics, one district and two counties were selected in each sample city, totaling 15 counties (districts). And then according to the provincial institutions sampling principle, the county (district) level medical and health institutions shall be selected. In stage 4, among the selected counties (districts), 5–8 township health centers or community health service centers were selected by simple random sampling method. Eventually, a total of 197 medical and health institutions were acquired.According to the code F20 of the International Classification of Diseases 10th Revision (ICD-10) issued by WHO, the medical records of inpatients with a primary diagnosis of schizophrenia were collected in each sample institution from 2014 to 2019. Among them, the ICD-10 code F20.0 refers to paranoid schizophrenia. F20.1 refers to hebephrenic schizophrenia. F20.2 refers to catatonic schizophrenia. F20.3 refers to unidentified schizophrenia. F20.4 refers to post-schizophrenic depression. F20.5 refers to residual schizophrenia. F20.6 refers to simple schizophrenia. F20.8 refers to other schizophrenia. F20.9 refers to schizophrenia, unspecified. We obtain the patient’s case information, including general demographic information  and hospitalization cost information . Finally, a database of hospitalization costs of schizophrenia patients was formed.Hospitalization costs are the costs incurred by the patient for the diagnosis and treatment of the disease during hospitalization. Among them, medication costs include TCM costs and western medicine costs. Examination costs include check fees, test fees, and radiate fees. Bed and nursing fees include bed fees and nursing fees. Other costs include all expenses except treatment costs, medication costs, examination costs, consultation costs, and bed and nursing fees. Proportion of medications refers to the proportion of medication costs to the total hospitalization costs.After data entry, the integrity and authenticity of the data were verified and the logical error correction was performed promptly. Subsequently, the information with incorrect ICD-10 coding should be corrected. Reject the unqualified data, including: (1) Primary diagnosis of non-schizophrenia. 2) Missing and poorly documented major items. (3) Logical relationships between costs that were clearly incorrect and could not be logically corrected. (4) The presence of outlier data. A total of 39,054 hospitalization data for patients with schizophrenia were eventually collected in this study. To ensure comparability between cost data across years, the costs were converted comparably using the GDP deflator for 2019 as the base year. The relevant calculation formula is shown below Eqs. ] were used to describe their distribution. Univariate analysis of average hospitalization costs per time of each classification of patients using Mann–Whitney U test and Kruskal–Wallis H test. Spearman’s rank correlation was used to analyze the correlation between non-normally distributed hospitalization costs and age, length of stay, proportion of medications, and year. Transforming the hospitalization costs into a natural logarithm so that it follows an approximately normal distribution. The factors influencing the hospitalization costs and their importance were analyzed using quantile regression model and random forest regression tree model. In this study, based on the experience of previous similar studies30, the 10th, 50th, and 90th percentiles of hospitalization costs were selected for fitting in the quantile regression model. This provides a comprehensive picture of the effect of independent variables on low, medium, and high levels of hospitalization costs. Variable assignment is shown in Table α = 0.05.Excel 2016 was used to standardize the data obtained from the field survey. Stata 14.0 and R 4.1.3 software were used to build the database and perform data cleaning and statistical analysis. And GraphPad Prism 8.3.0 and R 4.1.3 software were used to make graphs. Qualitative data were expressed as number of cases (composition ratio) [n(q%)], and measurement data were expressed as mean ± standard deviation [mean(SE)]. Since hospitalization costs are positively skewed, the median and quartiles [M(P


Insensitivity is an MHC-independent strategy for controlling antiviral T cells activation and inflammatory cytokines production. It is likely a critical aspect of virus spread in the infected host. The ability of monkeypox virus to spread efficiently as cell-associated viremia may be explained by the evasion strategies employed by the virus to subvert immunological surveillance by virus-specific T cells.The mpox (disease caused by the monkeypox virus) epidemic in 2022 provides a good opportunity to study the immune response to mpox. Vaccinia virus-infected monocytes could be recognized by monkeypox virus-specific CD4+ and CD8+ T cells, which produce inflammatory cytokines including IFNγ and TNFα. However, these cells are mostly unable to react to monkeypox virus-infected cells. The monkeypox virus also has no effect on the expression of MHC classes. Cells infected with monkeypox virus can prevent T cells from being activated  Many viruses evade the immune system through various mechanisms. Poxviruses can specifically avoid being detected by antiviral chemokines, cytokines, and antigen presentation. Monkeypox virus (MPXV), an orthopoxvirus belonging to the poxviridae family, was first identified as a human pathogen in the Congo region of Africa in 1972. Previous mpox outbreaks  before 2In 2003, 37 confirmed cases in the Midwestern United States of the first mpox outbreak in the Western Hemisphere. However, this outbreak did not result in any fatalities. The monkeypox virus isolates from the United States were found to be more closely related to the less virulent West African monkeypox virus strains than to the more virulent Central African strains by DNA sequencing . This maWe claimed in our earlier paper  that theAll verified MPXV and HIV cases had bacterial infections, vaginal ulcers, or skin rashes. As a result, this may be the cause of the evolutionary dynamics that aid in the development and reemergence of viruses caused by human activity and behavior .Considering that the current human population has little or no immunity to smallpox, this is very problematic. Although the vaccinia virus  is 85% effective in preventing disease, it is highly unlikely that it will be used as a pre-exposure vaccine because of the adverse side effects of vaccination . For thein vitro, wild-type virus pathogenesis depends on these two domains. The PKR pathway can be completely inhibited only when the N-and C-terminal domains of the VACV-E3 protein are present  and VACV-E3L37N , have been shown in  culture . However culture .Genomic studies revealed sequence similarities between MPXV and VACV. With 92% nucleotide and 88% protein sequence similarity, the F3 protein of MPXV and E3 protein of VACV are homologous to each other . AccordiIn addition, cowpox virus (CPV), another orthopoxvirus, has been shown to inhibit intracellular transport of MHC class I, a process associated with CPV evasion of the CD8+ T-cell antiviral responses . CPV ORFImmunity to mpox is typically determined by studying interactions between various orthopoxviruses like the vaccinia virus (VACV). It has been proposed that the poxvirus antigen on neutrophils and monocytes significantly determines mpox mortality. It was also found that primary human M2-like macrophages support VACV replication and transmission. After becoming infected, these primary macrophages produced actin tails, cell junctions, lamellipodia, and branching structures related to VACV virions, indicating that these cells may aid in the spread of the MPXV . A similSince the end of smallpox vaccination, the prevalence of mpox has increased. As a member of the poxvirus family, monkeypox virus has the ability to manipulate both the innate and adaptive immune systems. It is important for the development of vaccines and antiviral therapeutics to know the strategies of monkeypox virus to evade immune.MZ conceived the idea and developed concepts, and wrote and edited the paper. MS and FH wrote, edited and revised the paper. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Mass vaccination of dogs as important rabies reservoir is proposed to most effectively reduce and eliminate rabies also in humans. However, a minimum coverage of 70% needs to be achieved for control of the disease in zoonotic regions. In numerous developing countries, dog vaccination rate is still dangerously low because of economic constraints and due to a high turnover in dog populations. Improved vaccine production processes may help to alleviate cost and supply limitations. In this work, we studied and optimized the replication and vaccine potency of PV rabies virus strain in the muscovy-duck derived AGE1.CR and AGE1.CR.pIX suspension cell lines.8 FFU/ml were obtained. The potency of inactivated rabies virus harvest according to the NIH test was 3.5 IU/ml. Perfusion with the chemically defined medium during the virus replication phase improved the potency of the vaccine twofold, and increased the number of doses 9.6 fold.The BHK-21-adapted PV rabies virus strain replicated efficiently in the avian cell lines without requirement for prior passaging. CR.pIX was previously shown to augment heat shock responses and supported slightly higher infectious titers compared to the parental CR cell line. Both cell lines allowed replication of rabies virus also in absence of recombinant IGF, the only complex component of the chemically defined medium that was developed for the two cell lines. After scale-up from optimization experiments in shake flask to production in 7-l bioreactors peak virus titers of 2.4 × 10This study demonstrates that a rabies vaccine for animal vaccination can be produced efficiently in the AGE1.CR.pIX suspension cell line in a scalable process in chemically defined medium. Lyssavirus genus. While all mammals appear to be susceptible to the disease, carnivora and chiroptera are considered to be the most important reservoirs. Human rabies mortality exceeds 60,000 deaths every year worldwide and 99% of the cases occur in Africa and Asia  .The protective activity of inactivated rabies virus harvests was determined according to the NIH test . Swiss m


Haemaphysalis longicornis  (Acari: Ixodidae), the Asian longhorned tick, is an invasive tick species present in the USA since at least 2017 and has been detected in one-third of Virginia counties. While this species is associated with the transmission of multiple pathogens in its native geographical range of eastern Asia, little is known about its ability to acquire and transmit pathogens in the USA, specifically those that are transmissible to humans, although from an animal health perspective, it has already been shown to vector Theileria orientalis Ikeda strains. Emerging tick-borne viruses such as Bourbon virus (genus: Thogotovirus) are of concern, as these newly discovered pathogenic agents have caused fatal clinical cases, and little is known about their distribution or enzootic maintenance. This study examined H. longicornis collected within Virginia (from ten counties) for Bourbon and Heartland viruses using PCR methods. All ticks tested negative for Heartland virus via qRT-PCR (S segment target). Bourbon-virus-positive samples were confirmed on two different gene targets and with Sanger sequencing of the PB2 (segment 1) gene. Bourbon virus RNA was detected in one nymphal stage H. longicornis from Patrick County, one nymph from Staunton City, and one larval pool and one adult female tick from Wythe County, Virginia. An additional 100 Amblyomma americanum  collected at the same Patrick County site revealed one positive nymphal pool, suggesting that Bourbon virus may have spilled over from the native vector, potentially by co-feeding on a shared Bourbon-virus-infected vertebrate host. Blood tested from local harvested deer revealed a 11.1% antibody seroprevalence against Bourbon virus, exposure which further corroborates that this tick-borne virus is circulating in the southwest Virginia region. Through these results, it can be concluded that H. longicornis can carry Bourbon virus and that pathogen spillover may occur from native to invasive tick species. Thogotovirus, family Orthomyxoviridae, associated with clinical disease cases that were initially identified in Kansas, Oklahoma, and Missouri in 2014, 2015, and 2017, respectively [Hyalomma dromedarii  in 1973 [Bourbon virus (BRBV) is a vector-borne agent from the genus ectively . Thogoto in 1973  and was i Koch, 144 in 197 in 1973 . The disPhenuviridae, genus Bandavirus, first identified in 2009 when it was isolated from two clinical cases in Missouri [Haemaphysalis longicornis in its native range [Another emerging arbovirus of medical importance in the USA is Heartland virus (HRTV). Heartland virus is a tick-borne phlebovirus from the family Missouri . Closelyve range ,13,14. Cve range . Heartlave range ,17,18.Amblyomma americanum L. tick [A. americanum [The most probable route of transmission for BRBV and HRTV to humans is from the bite of an infected  L. tick ,20,21. A L. tick ,23,24,25ericanum ,21,26.Ixodes scapularis , Dermacentor variabilis , A. americanum, and, more recently, H. longicornis, the Asian longhorned tick. Haemaphysalis longicornis is an invasive tick species in Virginia and across the eastern USA, following first reports of the species on a sheep in New Jersey in 2017 [H. longicornis have since been reported in 17 states, including 38 counties in Virginia as of March 2022 [H. longicornis is a vector for multiple bacterial, viral, and protozoan pathogens, and thus, its arrival in the USA represents a huge concern for public and animal health. Research efforts are focused on understanding the threat it poses as a vector of existing or novel tick-borne pathogens within the USA. In western Virginia, incidence of vector-borne disease is increasing [I. scapularis, A. americanum, and D. variabilis with H. longicornis creates an opportunity for pathogen spillover through co-feeding and sharing of hosts. Haemaphysalis longicornis is already responsible for the transmission of Theileria orientalis Ikeda strain to cattle in Virginia [H. longicornis can acquire and transmit Rickettsia rickettsii, the causative agent of Rocky Mountain spotted fever [H. longicornis can harbor Anaplasma phagocytophilum, the causative agent of human granulocytic anaplasmosis [The Commonwealth State of Virginia, USA, is home to a variety of tick species, including ticks of medical and veterinary importance— in 2017 . Populatrch 2022 . In its creasing ,30, inclcreasing . The preVirginia ,33. Addied fever . More relasmosis .H. longicornis populations for emerging tick-borne viruses in Virginia, specifically BRBV and HRTV. Although there have been neither reports of human cases of BRBV or HRTV infection in Virginia nor previous detections of the virus in ticks in the region, it was hypothesized that BRBV and HRTV were circulating in Virginia. Supportive of that notion are preliminary serological studies conducted in our laboratory revealing the presence of BRBV and HRTV antibodies in wildlife of northern Virginia, including striped skunks, white-tailed deer, and northern raccoons, among others [H. longicornis ticks in the USA as well as the first detection of BRBV-infected ticks in Virginia.The purpose of this study was to screen local g others . In thisH. longicornis  were collected from sites across western Virginia in 2021  were tested for BRBV and HRTV using real-time RT-PCR. No pools were positive for HRTV via the probe-based RT-PCR assay. Of the 34 tick pools, 4 (11.8%) were found potentially positive for BRBV using the SYBR Green RT-PCR assay amplifying a portion of the PB1 (segment 2) gene  and one adult female from a pool of 8 from two separate sites in Wythe County. Across all 10 counties, the minimum infection rate (MIR) was calculated based on the number of positive pools divided by the total number of ticks yielding a minimum infection percentage of 0.62%. Only one nymph was collected in Patrick County and 15 ticks in Staunton City, and thus, no MIR was calculated for these counties, but a minimum infection percentage for Wythe County was calculated as 1.04% of ticks tested as being positive for BRBV. By subsequently examining individual ticks that had comprised each positive pool, one female from the pool of 8 adults from Wythe County was found to be BRBV-positive, along with one nymph from the pools of 10 nymphs from Staunton.A total of 34 tick pools ( 2) gene . We deteA. americanum (n = 100) collected at the same site as the BRBV-positive H. longicornis nymph in Patrick County were also tested for BRBV and HRTV. Seventy-one nymphal A. americanum ticks were tested as 12 nymphal pools (up to nine ticks per pool), and 29 adult ticks were tested individually. Evidence of BRBV was detected in 1 nymphal pool with a C(q) value of 34.98. Unfortunately, we were unable to obtain a sequence amplicon from this pool.A subset of n = 10), the larval pool from Wythe County, and a single female tick from the Wythe County pool (n = 8) were successfully sequenced in both directions for the larger PB2 (segment 1) gene fragment . The amplicons that were purified and sequenced were the direct products of the real-time RT-PCR assays. Amplicons from the single nymph in the pool from Patrick County, a single nymph from the Staunton City nymphal pool  collected in 2021 were screened for antibody evidence of exposure to BRBV. Nine white-tailed deer (of eighty-one tested), one raccoon (of four tested), and three groundhogs (of five tested) showed neutralizing antibodies against BRBV  [We corroborate evidence that BRBV is now circulating in multiple counties in western Virginia through detection of the virus in locally collected ticks and BRBV-seropositive white-tailed deer and groundhogs. Although seropositivity does not indicate a reservoir host role, it does indicate exposure to an infectious agent; serosurveys of wild and domestic animal species as well as continued vector surveillance can demonstrate the introduction and expansion of novel arboviral pathogens. The three counties from which BRBV-seropositive wildlife were detected  border or are close to Wythe and Patrick County, where BRBV-positive ticks were identified in the current study. Patrick County is at the southern border of Virginia adjacent to North Carolina, a state where harvested deer in two counties have shown BRBV-seroprevalence albeit at a higher reported rate than in this study  . Even ifA. americanum ticks, highlighting the potential for pathogen spillover through co-feeding or sharing of hosts as a likely transmission route for BRBV to the nymph. If BRBV is circulating in Virginia  in wildlife hosts or A. americanum ticks, these interactions are the suggested means of which H. longicornis in Patrick, Wythe, and Staunton counties are acquiring BRBV. Each of these counties has reported established A. americanum and white-tailed deer (the key vertebrate host of adult A. americanum and a species upon which H. longicornis have been reported) populations [Potentially, the BRBV-infected ticks detected in this study could have fed on an infected vertebrate host or co-fed with infected ticks on the same host. Our site in Patrick County, for example, is inundated with ulations ,40. FutuA. americanum, for both Heartland and Bourbon virus [H. longicornis ticks may acquire BRBV through transovarial transmission. Thus far, no field-collected larvae in the USA have been detected with BRBV in environmental surveillance sampling. Experimentally infected A. americanum larval ticks showed transstadial transmission of BRBV and a low rate of vertical transmission from infected female ticks [Rhipicephalus appendiculatus, Thogoto virus was reported to be transmitted transstadially but not transovarially [Transovarial transmission can be ecologically significant for the persistence of several arboviruses, e.g., La Crosse virus ; howeveron virus ,42. It ile ticks . In a lavarially .A. americanum is described as 3.1% for nymphs and 3.2% for adult males in Missouri [H. longicornis sampled .The prevalence rate of BRBV in field-collected Missouri  based onH. longicornis. Unfortunately, culturing of molecularly positive tick homogenates on Vero-76 cells in this study did not establish a novel isolate of BRBV. Potentially the strains of BRBV that we detected in H. longicornis ticks have a different phenotype, or potentially, culturing and sequencing was challenged due to low concentrations of viral particles in the tick specimens. It is possible that H. longicornis is not a competent vector or amplifying host for BRBV transmission as compared to A. americanum.Being able to isolate the strain(s) circulating in local populations and characterize their disease dynamics will provide a better idea of the risk this poses to local residents, domestic animals, and wildlife in counties with infected A. americanum and H. longicornis would be beneficial in understanding the disease ecology of this emerging pathogen in the USA.Nevertheless, these findings influence future studies of BRBV surveillance by highlighting an additional tick species that could be playing a role in the enzootic cycle of BRBV. Future studies of the transmission dynamics of BRBV in both A. americanum [H. longicornis, which also exists in the northeastern USA, has yet to be seen.While no evidence of HRTV was detected during the current study, future research is needed to establish whether this virus is circulating with an underlying presence in the Virginia region and if any risk of pathogen spillover to different tick vectors exists in general. Although previous studies did not find serological evidence of HRTV in Virginia, this phlebovirus is suggested to be widespread in the eastern USA . Indeed,ericanum . The potH. longicornis and an ability to acquire BRBV and other pathogens of human disease within the USA [H. longicornis is a competent vector of viral pathogens, then people living in areas with large populations of this tick species would be at an increased risk for encountering an infected tick, as H. longicornis has been reported to bite humans [A. americanum, future spillover events are not impossible.The rapidly expanding distribution of  the USA ,35 raisee humans . Given tBourbon virus is a recently identified human-pathogenic thogotovirus which has been exclusively detected in the USA. Although human disease cases are rare and have so far been restricted in occurrence in the Midwest , seroprevalence in new regions and detection of BRBV in novel tick species warrant vigilance of this arbovirus as a public health concern.Haemaphysalis longicornis ticks were collected during routine tick surveillance at 13 sites across 10 counties in the western region of Virginia throughout the year of 2021  and 16S−1 (5′-CCG GTC TGA ACT CAG ATC AAG T-3′), described in Black and Piesman [Ticks were collected and transported alive to the laboratory for immediate storage at −80 °C. Ticks were identified morphologically using keys  and a co Piesman  and de l Piesman , in a 25 Piesman .Both DNA and RNA were extracted from tick specimens using a whole nucleic acid extraction system developed by Crowder et al. , with DNNucleic acid from each pool was initially screened for BRBV using a SYBR Green RT-PCR assay to amplify a portion of the PB1 (segment 2) gene using primers developed for this study . The 20 Sample pools were also screened for HRTV using a probe-based RT-PCR assay developed by Savage et al. . Brieflywww.ncbi.nlm.nih.gov/genbank (accessed on 19 January 2022)) using the basic local alignment search tool (BLAST) [Virus-positive PCR amplicons were subjected to further confirmation using Sanger sequencing and compared to reported sequences of BRBV in NCBI GenBank ( (BLAST) . We atte2 environment for 1 h, rocked every 10 min. Following adsorption, liquid overlay of 1 mL of Dulbecco’s Modified Eagles Medium (DMEM), supplemented with 2% FBS and 1% P/S, was added into each well. Samples were incubated at 37 °C and 5% CO2 for eight days and monitored daily for cytopathic effect (CPE) using an EVOS M5000 imaging system  microscope. Following this incubation, a 50 µL aliquot of each sample supernatant was transferred to a fresh Vero monolayer in a blind passage (even if not displaying CPE) and monitored for a further seven days. At the end of a second passage, RNA was extracted from the culture supernatant (regardless of whether CPE had been displayed) and tested as above.With the objective of viral isolation, 50 µL of a homogenate supernatant from each virus-positive tick sample were inoculated onto a confluent monolayer of Vero-76 cells  in 12-well culture plates . Briefly, cryofrozen tick halves from each virus-positive tick pool were first dry-homogenized as described above, then suspended in 200 µL of tick diluent consisting of Dulbecco’s phosphate-buffered saline supplemented with 20% fetal bovine serum (FBS), 1% 100X solution of Penicillin-Streptomycin (P/S), and 1% amphotericin B solution at 250 µg/mL final concentration. Samples were incubated at 37°C with a 5% COSample collection. Local wildlife sera, collected as part of a separate project, were tested for evidence of exposure to Bourbon virus. Deer blood was collected in November 2021 (under Virginia Department of Wildlife Resources (DWR) permit #069946, VT IACUC#20-197), from the body cavity of 81 harvested deer brought to check stations for chronic wasting disease monitoring. Samples were obtained from Floyd (n = 33), Montgomery (n = 30), Giles (n = 1), and Pulaski (n = 17) counties, Virginia; these counties are adjacent to each other in southwest Virginia. In addition, blood was collected from n = 12 individuals from fresh roadkill and hunter donations in 2021 , including 4 northern raccoons (Procyon lotor), 5 groundhogs (Marmota monox), 2 striped skunks (Mephitis mephitis), and 1 eastern cottontail (Sylvilagus floridanus), from Roanoke, Floyd, and Montgomery Counties. Blood was centrifuged at 5000 rpm for 6 min and subsequent sera transferred to a storage vial and heat inactivated using a bead bath (56 °C for 1 h) prior to analysis.Serology testing. All sera were tested, initially at 1:20 dilution, by plaque reduction neutralization tests (PRNT), as described previously by Eastwood et al. [80) titers against BRBV. Serum was diluted in DMEM supplemented with 2% FBS and 1% P/S. Bourbon virus Original strain  was used in these challenge assays at a working concentration of 800 PFU/mL and as a virus-positive control (further diluted two-fold from 1:2 to 1:64); DMEM was used as a negative control; BRBV-positive rabbit antisera (provided by CDC) was used as a positive control. Briefly, 60 µL of serum and 60 µL of virus were challenged during 1 h incubation at 37 °C, and then, a volume of 50 µL was inoculated in duplicate into wells of a twelve-well culture plate. The modified method applied an overlay of 1.2% bactoagar  and 2X MEM  following the initial adsorption. This same overlay, later containing 1.5% Neutral Red solution , was applied after 48 h incubation at 37 °C, then plaques were visualized after a further 24 h of incubation using a light box. Testing was performed at a BSL3 facility of Virginia Tech since BRBV is a risk group-3 pathogen [d et al. , with sl


After a period where many countries have let the SARS-CoV-2 virus spread more or less freely, individuals and communities are now grappling with the many negative health effects and economic ramifications from high levels of illness over long periods. As evidence of the detrimental long-term effects of the virus mount, it is increasingly clear that the policy vacuum comes at an unacceptable price both in the short and long term; its only justification would be if there was no other alternative that did not come at an even greater cost. Entering the cold season, the number of infections will most likely increase significantly in Europe (≈ one – two order of magnitude in 2021). While the world awaits and hopes for new and more effective vaccines, we need tools in the toolbox that can effectively control transmission of rapidly spreading new variants, especially if more pathogenic. Otherwise, we may face significant disruptions and enormous costs due to repeated waves of illness, with each wave increasing the numbers of workers thrown out of the workforce from long term health effects. Lockdowns, due to their social restrictions and high short-term economic costs, are no longer the best available option. We here point out that mass testing  is an alternative approach that can dramatically reduce cases and quickly restore economic and social activity.R is possible  is lower and individuals may be infectious while still testing negative. However, given their easy accessibility, LFTs can remain a useful tool of surveillance, for instance as a supplement for mass testing, where more accurate tests (PCR or LAMP) are unavailable. If we assume R = 10 without testing (or significant other measures), we might expect daily testing to reduce the number of new cases by greater than a factor of 9 each week  and daily testing of cross border travelers for two weeks. Non-intrusive HEPA purification in restaurants, retail and workplaces and masking where needed, should continue for robustness in preventing new outbreaks.11 per person can and are being realized using pooling strategies, simple test collection, or new testing methods such as LAMP tests. LAMP tests start at 5 euros per test and pooling five individuals per test can reduce the costs by an additional factor of 5.Mass testing impacts the economy far less than a lockdown, and results in far less social disruption. Calculations by Institut économique Molinari based on OECD quarterly accounts for instance show, that if France, Germany, the EU, or the US were to implement mass testing for every individual every day for a month at 10 dollars a test, the cost for one month of testing would respectively be 12 times, 8 times, 9 and 7 times lower than the loss of GDP per inhabitant measured by comparing the loss of GDP over a year (between 2020 and 2019). Test costs on the order of 10 dollars or even less This strategy will not only reduce the amount of illness and suffering in the population but likely also still be financially profitable; although society has opened up, the accelerating costs of long COVID, and the increase in prevalence of many other medical conditions after COVID-19, continue to mount with the continuous increase in new cases. The cost of long COVID with today's knowledge was recently estimated to equal the cost of the great recession.,,Np, the size resulting in the minimal number of tests needed to identify a positive case, increases as the number of infections goes down . At 100 infections per Million people, the pool size could therefore be increased to ∼100 people per pool. At such low infection rates, the continuous surveillance of an entire German elementary school would therefore produce the costs equivalent to 3 PCR tests a few times a week and even a soccer game, with 40.000 visitors, could be monitored with just a few 100 PCR tests allowing to keep schools, social gatherings as well as the economy open and safe. But there are more examples demonstrating the feasibility of the concept, such as the efforts of the University of Davis and others.The feasibility has not only been described repeatedly on theoretical grounds,Sufficiently high rates of mass testing can replace lockdowns as the main tool to reduce transmission. When combined with other measures it helps to achieve the shortest pandemic intervention that can stop social and health related collateral damage and keep the economy open. It is a less expensive, less interfering but highly effective and complementary risk mitigation tool compared to measures such as stay-at-home orders, while ensuring that most people stay healthy. If implemented on a larger scale, mass testing may propel the world towards a real end to this pandemic without the need of lockdowns.Here we have discussed the opportunity to stop transmission using alternatives to lockdowns. Given the ongoing level of disease and long COVID accumulating in the population, the consequences for health, social stability, and economy even with vaccination are significant and require a reconsideration of the currently prevailing strategy.All authors contributed to the concept and writing of the manuscript.The authors declare no competing interests.


Medicago sativa L.) is one of the most extensively cultivated forage legumes in the world. It is currently the third most valuable field crop in the United States with an estimated value of over $9.3 billion. Alfalfa productivity is limited by various infectious diseases that can reduce forage yield and quality and shorten stand life. The crop can frequently be infected with a diverse array of pathogens and other organisms that have distinct life cycles, biology, and mode of action. Among them are many coinfecting viruses, that greatly contribute to the heterogeneity of within-host pathogenic communities, representing a ubiquitous and abundant background for all other host–pathogen interactions. Regrettably, the impact of viral diseases, their role in alfalfa health and involvement in the severity of multi-pathogen infections are often underestimated and not well understood. As high-throughput sequencing approaches have been developed, opportunities to delve into these complex interactions can be realized. In this work, we have characterized a diversity of viral populations in several commercial alfalfa production fields located in the U.S. Pacific Northwest. At least 45 distinct viruses have been identified in all alfalfa samples. Among them some were known to infect the crop prior to this study, and others were designated as emerging, novel and viruses integrated into the alfalfa genome. Known viruses included alfalfa mosaic virus, pea streak virus and bean leafroll virus, while among emerging and novel agents were alfalfa virus S, cherry virus Trakiya, several rhabdoviruses and others. Additional biological and impact studies will be needed to determine if newly identified viruses, especially those that have not been reported from alfalfa before, should be considered pathogens of this crop.Alfalfa ( Medicago sativa L.) is a major forage crop worldwide and the third most valuable field crop in the United States with an estimated value of over $9.3 billion1. Viral infections of alfalfa are widespread in major cultivation areas. Alfalfa may also serve as a natural reservoir for dissemination of viruses to other agriculturally important crops. Although several economically important viral diseases infecting alfalfa have been previously described, novel viruses continue to be discovered at accelerated rates thanks to the advent of high-throughput sequencing (HTS) technologies9. In the United States, among the major alfalfa-infecting viruses are alfalfa mosaic virus (AMV), bean leaf roll virus (BLRV), pea streak virus (PeSV) and others10. The makeup of the alfalfa virome continues to expand to include new and emergent species, different geographic locations, and previously unknown adaptations of viruses to alfalfa as a new host1.Alfalfa , thus confirming a complexity of co-infections Table . A totalCommon viruses known in alfalfa in the U.S. included alfalfa mosaic virus (AMV), pea streak virus (PeSV), bean leafroll virus (BLRV), Medicago sativa alphapartitiviruses 1 and 2 (MsAPV1 and MsAPV1), Medicago sativa deltapartitivirus 1 (MsDPV1) and Medicago sativa amalgavirus 1 (MsAV1). These viruses were detected in many samples, some of them  infecting more than 90% of all the plant samples evaluated Table , they ap8 was found in 21% of the samples collected in 2019 , which is believed to be an emerging and possibly seed-transmitted virus13 was not reported to infect alfalfa as a host prior to this work. CVT-alfalfa strain (CVT-A) had 93% of complete genome identity to the reported cherry strain . Since assembled contigs of the CVT-A covered a significant portion of the putative viral genome me Table , we haveme Table . The comThe total length of CVT-A genome was found to be 8706 nt, excluding a poly(A) tail. Like the cherry strain, the genome of CVT-A encoded two open reading frames (ORFs) and contained non-coding regions (NCRs) at the 5′-end (1–314 nt), 3′- end  and an intergenic NCR (2697–2937 nt). ORF1 encoded the putative coat protein (CP) consisting of 794 deduced amino acids (aa) with a predicted MW of 87.8 kDa. It was only 60.9% identical to the CP of CVT cherry strain . However, ORF2 of CVT-A, encoding the putative replicase protein and comprised of 1892 aa with MW 215 kDa, was 94% identical to the reference strain .Picornavirales, positioned CVT-A isolate in the same cluster with the cherry isolate  and potato virus X (PVX). Sequenced portions of their genomes were highly identical to the reference viruses. Alfalfa-derived HLV contigs had on average 97–100% identity; while those that matched to the PVX contigs were 95–100% identical. Assembled HLV reads formed several short contigs; the longest of them was 926 nt and corresponded to the viral CP. The RT-PCR with several infected samples and primers specific for the nucleotide sequence of this HLV contig .Despite the positive RT-PCR detection of these two viruses in alfalfa samples, an incidental contamination of the alfalfa plants rather than a true infection remains a strong possibility. When indicator species 14 and Medicago sativa deltapartitivirus 1 (MsDPV 1)15. Although LTSV strain STN1 has been recently reported in the U.S.16, it was isolated from soybean thrips (Neohydatothrips variabilis) and not from naturally infected alfalfa plants. It thus cannot be considered an alfalfa isolate since LTSV naturally infects at least 18 species in four plant families18.Viruses not known to occur in the US, but reported from other countries, included lucerne transient streak virus (LTSV)16.LTSV was found in 12% of tested samples originating from eight different alfalfa fields. The longest assembled LTSV contig (4257 nt) apparently represented a coding-complete genome of the US alfalfa isolate of LTSV (LTSVa-US). The top BLAST hits for this nucleotide sequence  were genomic sequences of LTSV US isolate from thrips (MT224146.1) and LTSV-Can, a Canadian isolate of the virus (JQ782213.1). LTSVa-US encoded four ORFs, presumably corresponding to the movement protein (ORF1); viral polyprotein translating via − 1 ribosomal frameshifting into replicase-associated proteins (ORF2a and ORF2b); and CP (ORF4). The translated ORFs were 96–99% identical to the US isolate of the virusSouthern bean mosaic virus, a type species of the genus Sobemovirus, was evident, relatively low bootstrap values indicated its divergent status within the group. The nucleotide sequence of the LTSVa-US was submitted to GenBank under accession number OK181163.Phylogenetic analysis, deduced from the alignment of the LTSVa-US with available complete nucleotide sequences of LTSV, placed LTSVa-US in the same cluster with the virus isolate from thrips Fig. A. While RdRp of the MsDPV1 isolates was 99–100% identical at both nucleotide and amino acid levels to the previously reported isolates of the virus indicating a low genetic diversity among American isolates of MsDPV1 Table .Potentially novel species included rhabdo-, toti-, partiti, and picorna-like viruses. Top BLASTX hits were recorded for maize yellow striate cytorhabdovirus ; datura yellow vein nucleorhabdovirus ; maize sterile stunt cytorhabdovirus ; maize-associated cytorhabdovirus ; zhuye pepper nucleorhabdovirus , maize-associated picornavirus ; and horseradish latent virus . Low identity and coverage levels indicated that these viruses are likely new species or at the very least new strains of the known pathogens (Table 7), the percent identity was 34.5%, indicating unlikely relationship between these two viruses. Provisionally, we named this virus alfalfa nucleorhabdovirus 1 (ANRV1). The longest contig homologous to ZPNRV  was 98% identical to the ANRV1 and therefore most likely depicted the same virus. Using PCR primers designed according to the nucleotide sequence of the ANRV1 contig_23296 . The largest translated ORF of the DYVV-like virus was 449 aa-long and only 65.7% identical to the nucleocapsid protein of the DYVV (YP_009176972.1). When this ORF was compared to the nucleocapsid protein of recently discovered alfalfa associated nucleorhabdovirus .The second longest contig had a top BLAST hit with MYSV (2444 nt) and contained one complete ORF 437 aa-long that was 31% identical  to the N protein of MYSV (YP_010086409.1) and 33%  identical to the N protein of barley yellow striate mosaic cytorhabdovirus. At the nucleotide level, however, no significant similarity with other viruses was found, not counting short stretches of the sequence with a low coverage (2%). Despite the low levels of identity, these were sufficient to establish affiliation with the family The third longest contig among those potentially belonging to the new viruses, consisted of 1903 nt and encoded a 260 aa-long fragment 38%, 37% and 35% identical to the nucleocapsid proteins of MSSV (QBJ27588.1), MYSV (YP_010086409.1), and MaCV (ARS22490.1), respectively. This virus was provisionally named alfalfa cytorhabdovirus 2 (ACRV2). Nucleotide sequence identities between partial segments of ACRV1 and ACRV2 was ~ 52%. Amino acid sequence identities between their translated ORFs, though, was ~ 84%, suggesting these could be different strains of the same virus.Rhabdoviridae  with a hypothetical protein of uncategorized Hubei toti-like virus 2 (YP_009336495.1). Nucleotide sequence search has not resulted in any hits. Although totiviruses are associated with latent infections of fungal or protozoan hosts19, among the top BLAST hits were also CP of several unclassified toti-like viruses apparently infecting higher plants: peach-associated virus 2 (QSV39137.1), black raspberry virus F (YP_001497150.1), and maize-associated totivirus 2 (YP_009259485.2). Phylogenetic analysis of the contig_30828 along with complete nucleotide sequences of the representative species and unclassified members of the family Totiviridae positioned it, albeit with poor bootstrap support, in the same cluster with Giardia lamblia virus (GLV), the prototype virus of the genus Giardiavirus that infects the parasitic protozoan G. lamblia , although a possibility exists that this is a contaminant virus originating from a non-plant host.Contig_30828 ORF Table  was the en masse by Shi et al.20. The longest contig of 3387 nt translated into two proteins of 639 and 452 aa-long, with an intergenic region of 65 nt. The largest ORF had a low identity (31%) BLAST hit with a hypothetical protein of Ryboviria spp. (QKN89031.1), while the second ORF was paired with uncategorized Arma picornavirus GZ . Nucleotide sequence search has not resulted in any significant hits with viral genomes. This virus was tentatively named alfalfa-associated picorna-like virus (AAPLV 1).Several contigs, according to BLAST analysis, were associated with a group of different unclassified Hubei picorna-like viruses, reported 21 and the latter was recently found in larvae of alfalfa weevils (Hypera postica), a herbivorous pest feeding on alfalfa and other legumes22. As of today, the only known plant-infecting ifla-like virus has been identified from asymptomatic tomato plants23. However, the percent identity of the unknown picorna-like virus with the polyproteins of both Watson virus and HPAV was rather low , indicating that it could be an undescribed species originated from alfalfa or an ambiguous host. Phylogenetic analysis, encompassing the picorna-like virus, its top BLAST matches, and selected species from the order Picornavirales indicated its relationship with iflaviruses. The virus appeared only remotely connected to the AAPLV1 that clustered with Solenopsis invicta virus 2 from the family Polycipiviridae  of this virus produced a 7277 nt-long sequence that translated into one polyprotein of 2412 aa. The ORF had best BLAST hits with Watson virus (QED21508.1) and Hypera postica-associated iflavirus 2 . The former is an unclassified picornavirus reported from Australian fleasDeltapartitivirus, family Partitiviridae. The virus was first detected in Japanese pear24. Translated ORF (445 aa) of the longest contig  had only 36% identity with PpCV , thus assuming that this could be a fragment of the new partitivirus infecting alfalfa. This contig presumably represented a complete nucleotide sequence of the RNA 2 segment of the virus, including poly (A) tail. Indeed, phylogenetic analysis clustered contig_67368 with PpCV, suggesting their relationship, as well as association with other deltapartitiviruses , which was also reflected in their distant phylogenetic grouping within the family.More than half of the samples collected in 2019 contained nucleotide sequences that had BLAST hits with capsid protein of Pyrus pyrifolia cryptic virus (PpCV), a tentative member of the genus 25 and diploid , carry endogenous viral elements (EVEs) resembling SbCMV and FMV26 and that SbCMV EVEs may represent a new virus, provisionally named alfalfa endogenous pararetrovirus (AePV). In the phylogenetic tree, deduced from the alignment of the 6563 nt-long SbCMVL sequence with AePV and the complete nucleotide sequences of the representative species from the family Caulimoviridae, SbCMVL grouped in the same cluster with SbCMV and AePV  had sequences homologous to soybean chlorotic mottle virus  and 47% of the samples contained sequences homologous to figwort mosaic virus . While the SbCMV-like (SbCMVL) contigs derived from different samples assembled into nearly complete (~ 80%) genome of 6553 nt in length, FMV-like sequences were relatively short and assembled into a partial genomic fragment Table . We haveTo further confirm that SbCMVL segments identified in this study represent EVEs rather than an exogenous infection, the 6553 nt-long SbCMVL fragment was BLAST searched against the tetraploid alfalfa genome. Table 26.When we mapped the longest FMV fragment (892 nt) from the transcriptome assembly against the tetraploid alfalfa genome, up to 228 bp matched the genome Table , assuminContaminant viruses, likely originating from different hosts in the same ecosystem—mostly insects—represented a substantial portion of the findings Table . They we28. According to this analysis, many alfalfa samples infected with viruses, also contained described bacterial pathogens, including Clavibacter michiganensis, a causal agent of bacterial wilt; Xanthomonas campestris, a causal agent of bacterial leaf spot; Xylella fastidosa, a causal agent of alfalfa dwarf disease; Agrobacterium tumefaciens, a causal agent of crown gall disease. In addition, several species in the genus Pseudomonas, namely P. viridiflava, P. syringae, and P. sevastanoi were identified, which have been associated with bacterial stem blight of alfalfa  and alfalfa in Illinois, according to the plant disease report from the University of Illinois31.Alfalfa may potentially be a host for viruses that were not previously reported to infect the plant, such as CVT-A. While detection of CVT-A strain is most likely due to the presence of authentic infection, occurrence of HLV and PVX, despite RT-PCR evidence, could be the result of external contamination of alfalfa plants via aphids (HLV) or contaminated equipment (PVX). Negative results of 32. However, it is not known whether these viruses are new to the U.S. The fact that they have been identified in this study may indicate that they are widespread in the surveyed regions and possibly have always been present but gone unnoticed. More information is needed to determine the biological significance of these described viruses.Several conceivably novel viruses were identified in the survey, including three different rhabdoviruses, a toti-like virus, two picorna-like viruses, and a deltapartitivirus Table . Their e33.Each tested sample was co-infected with a cluster of different viruses. Occurrence of multiple viral infections can affect within-plant virulence, disease dynamics, host–pathogen interactions, and as a result, trigger epidemiological implications35. Consequently, host fitness may be altered triggering susceptibility, expanding disease damage to the plant and, potentially, disease epidemiology  adapted to growing in the areas.37. High-throughput sequencing of paired-end reads (2 × 150) was performed on a HiSeq Illumina Platform by Novogene .Ten samples collected from 10 different plants in each of the 24 fields were split in two to three groups, 3–5 plants in the group. In each group, three to five leaves were taken from each plant, pooled together, and used for total RNA extraction. Total RNA was extracted with TRIzol Reagent . cDNA libraries were prepared from total RNA using poly (A) selection protocol for higher exonic coverage and better accuracy of gene quantificationhttps://jgi.doe.gov/data-and-tools/software-tools/bbtools/) to remove low-quality reads and adapters sequences. The cleaned reads were mapped to the alfalfa genome and the unmapped reads were used for further analysis. Clean reads were assembled de novo using CLC Genomics Workbench  and mapped to the reference viral genomes, when available, using CLS’s maximal exact match algorithm or Bowtie 2 aligner38. Other assemblies were created from the complete set of reads for each sample after trimming with Trimmomatic39 and then running BLASTX against a custom viral detection database containing all viral sequences in NCBI refseq (https://www.ncbi.nlm.nih.gov/genome/viruses/), viruses that have not been deposited in refseq yet, plus Arabidopsis lyrata and A. thaliana proteins added from the reference proteome of UniProt for an in-BLAST subtraction.The reads were cleaned using BBMAP  hit-region and expect-value.Contig matches and relatives to known viruses were then mapped in CLC Genomics Workbench (minimum 80% identity for 50% of read length); contigs were extracted and read counts and coverages were generated for each sample. For identification of the endogenous viruses, the 6.5 kb putative soybean chlorotic mottle virus-like (SbCMVL) and 892 nt figwort mosaic virus-like (FMVL) transcripts identified in the alfalfa transcriptome were searched using the BLASTN program against the tetraploid alfalfa genome28. Paired-end reads were merged using BBmerge40 and both merged and unmerged single-end reads were concatenated, followed by BLASTX screen against the nr database using the DIAMOND BLAST program (v2.0.6.144). The BLAST results were meganized27 and inputted into MEGAN6 for taxonomic identification. In addition, paired-end reads were inputted into the Kracken2 (v2.1.1) pipeline using the standard Kracken database for taxonomic identification. Relevant taxa were then checked manually using the BLASTN program against nr at the NCBI.Contigs derived from bacteria and fungi were identified by using the MEGAN/Diamond BLAST and Kracken2 programsAdditional sequence information on a select group of genomes was obtained by SMARTer 5′/3′ RACE , cloning and sequencing of the multiple RT-PCR products. RT-PCRs were performed with total RNA employing the SuperScript RT-PCR system per the manufacturer’s directions . Gel-purified RT-PCR products were either sequenced directly or cloned into the pCRII-TOPO vector with dual promoter  for an automated Sanger sequencing.41 with Maximum Likelihood method based on the Tamura-Nei model and bootstrap analysis of 1000 replicates.Phylogenetic trees were deduced from CLUSTALW alignments and built using MEGA 7 softwareSupplementary Information 1.Supplementary Information 2.Supplementary Information 3.Supplementary Information 4.Supplementary Information 5.Supplementary Information 6.Supplementary Information 7.


There are three major and many minor global problems today: the coronavirus pandemic and other global health threats, global climate change, and the Russian invasion of Ukraine being among them. The first is a long-term and continuing problem, the second is a devastating problem, and the last is an acute problem. There are others as well; there always are.This is the major current problem. Although it has been reported by the World Health Organization that the global human cases total more than 530 million and world death toll from this disease probably has been upwards of 18 million people . Clinicians are hard at work to characterize and understand the disease it causes and the peculiarities of its various manifestations. Other researchers are trying to understand and identify the relationships and functions of wild vertebrates that may serve as reservoir hosts of the virus, the relationship between the size of coronavirus genomes and the mutation rate, the prevention  of mutations in different geographic areas and among different human populations, and even more, as we recognize each day. Thus far, understanding has not led to solutions.Many of the suggested solutions to current problems are unlikely to be carried out. National representatives and some others assigned to the United Nations have suggested nothing truly useful; they are just counting numbers. (“A committee is a cul-de-sac down which ideas are lured then quietly strangled.” – Sir Barnett Cocks)Orthopoxvirus genus in the family Poxviridae. The disease that monkeypox virus causes was first identified in laboratory cynomolgus monkeys in Denmark by Preben von Magnus in 1958, when two outbreaks of a smallpox-like disease occurred in colonies of monkeys captured in Malaysia, and transported via Singapore . Almost 50 cases were reported between 1970 and 1979, with more than two thirds of these being from the DRC. It is likely that many more cases have occurred but not been diagnosed as such. Other cases originated from Liberia, Nigeria, Ivory Coast, and Sierra Leone. By 1986, more than 400 cases in humans had been reported. Small viral outbreaks with a death rate in the range of 10% and a secondary human-to-human infection rate of about the same percent occur routinely in equatorial Central and West Africa.Cricetomys gambianus) may have mild symptoms. Some people keep these animals as pets, for reasons unknown to me. During the 2003 outbreak in the U.S.A., affected (pet!) prairie dogs (Cynomys species) presented with fever, cough, sore eyes, poor feeding, and rash. Non-human primates present similarly. They may have breathing problems, facial swelling, mouth ulcers, and swollen glands. In cynomolgus monkeys, the time from exposure to symptoms was noted to be about a week. Rabbits and rodents typically present with fever, cough, runny nose, sore eyes, and swollen glands. They develop small vesicles that fill with yellow fluid and may have patches of hair loss and may develop pneumonia. The disease has also been reported in dormice (Glis species), tree squirrels (family Sciuridae), and rope squirrels  , well more than 35000 human infections with this virus have been recognized globally in the current outbreak, although numbers of case reports appear to be declining. Transmission of this virus between humans has been attributed to close contacts, perhaps even very close contact; however, this virus has been shown to be aerosol-transmitted between humans. Fewer human personal interactions likely would decrease the transmission rate, as would vaccination. There is one virus-specific vaccine approved for prevention of monkeypox, as is the vaccinia virus vaccine used to eradicate the closely related, but much more pathogenic, smallpox virus (variola virus). It is unlikely that monkeypox virus will be responsible for a pandemic, although an increasing number of cases might very well be found world-wide. Still, until the spread of this virus is stopped and awareness of it enlarged, it will not be stopped.Whether God or another unknown source has provided us with the abundance of resources and natural beauty, we have not taken care of our environment, as obviously is required for maintenance and, ultimately, survival of life. No need to discuss this aspect of our slovenly attempts – everyone knows what they are, even those who have become wealthy by closing their eyes to the conditions under which their children and grandchildren will live – or die.The immediate solution to this problem is to change our energy sources from fossil fuels to highly available renewable resources. Immediately. Full stop. This will not be done, of course. When we had the opportunity to do this, we did not do it. Now we are far behind where we need to be. The causes of this disastrous hesitancy are obvious.Russian armed forces most recently began attacking Ukraine again on February 24, 2022. Russia is suffering tremendous losses of their fighting forces and their military equipment. Sooner or later, peace will be declared but that will require concessions, which are unlikely. A recent rumor is that Russia has offered to generously accept one-third of Ukraine territory in return for its withdrawal of Russian troops and equipment. If true, and it probably is not, this may be a reasonable request. In response to this proposal, it seems only fair that Ukraine would receive one-third of Russian territory in return.Having all that territory in a downsized Russia would allow some breathing room (дихання номер) for the Ukrainians. They could expand their human population from about 44 million to, say, 80 million. That would still leave Russia with 30 times as much land as Ukraine. As for language differences, both speak Slavonic languages, but there is an asymmetry between them such that Ukrainians who do not speak Russian fluently can understand Russian much better than Russians, who don't speak Ukrainian fluently, can understand Ukrainian. Does not much matter now, as most Ukrainians do not want to speak with Russians anyway.As for the patently false assertion/propaganda that Russia has attacked Ukraine to remove Nazis from power there, there probably are only four or five Nazis in Ukraine and they are all in their 90s, eating breakfast cereal three times a day in nursing homes. The former comedian-actor-lawyer Volodymyr Zelenskyy, now Ukraine’s President, has been a strong and heroic leader against Russia’s genocidal attacks. He is Jewish and part of his family was killed by Nazis during the Holocaust so, if he is a Nazi, he is not a very competent one. As for Russia, they will now have to keep working to reform their military for the next 100 years, which should keep them busy and out of trouble. In sum, as there is no valid reason for this war, the simplest solution is to simply stop fighting. Stopping doing stupid things should be easy to do.Middle East conflicts: These seem to continue ad nauseum. Except for the way they dress, due to my own ignorance I cannot seem to tell the difference between them. Both sides should be given one, and only one, more opportunity to make peace. Will Rogers said, “Diplomacy is the art of saying ‘Nice doggie!’ until you can find a rock.” The two sides have had more than enough time to negotiate a settlement, if both sides were serious about finding a solution to the interminable hostility between them. Enough is enough, no matter who is right and who is wrong.Inevitably, they will not agree to peace because they see each other with their clothes on. All we (the rest of the world) need to do is make them take off their clothes in the summer and move them all to the states of North and South Dakota in North America; one group to North Dakota, the other to South Dakota; it does not matter which. Residents of the New Dakotas would have to spend the next few years producing warm clothes and foods. The land of Israel and surrounding controversial territories could be turned into an International Religious Territory, a sort of museum, with visitors limited to a few each year who win a lottery. The New Dakotans would surely complain but would be allowed to fight with each other in a sort of isolation.North Dakotans and South Dakotans: Current residents of the Dakotas could be moved to the mid-Saharan desert or some other isolated and woebegone location. Most of these many places were only producing nice sweaters, oil, and camels anyway and the world already has an excess of these and certainly do not need any more.Dictators: We are done with Adolf Hitler, Mao Zedong, Joseph Stalin, Pol Pot, Leopold II, Kim Il-Sung, Idi Amin, and similar if not equal tyrants, but we still have Kim Jong-un, Vladimir Putin, and more than one hundred military dictatorships in charge of various governments. Once again, the rest of the world could get together and move these people and their families to, say, an uninhabited island. After all the airports and shipping docks are destroyed, so that no one can leave or enter, the dictators can rule whatever part of the island they have dominated (that is what they do) and leave the rest of us alone. Specifically, killing women and children would remain illegal, as it already is in most civilized countries, but not in these new dictatorships.Problems recognized; some solutions proposed.


Experts in infectious diseases have long cautioned that climate change and the destruction of nature will raise the likelihood of ‘zoonotic spillovers’, or the transmission of viruses from animals to people Santana . RecentlHenipavirus genus in the family Paramyxoviridae, which has a single-stranded RNA genome with a negative orientation. Prior to LayV, the genus Henipavirus included Hendra, Nipah, Cedar, Mojiang, and the Ghanaian bat virus. Fruit bats are the natural host for Hendra and Nipah virus  in China (Wu et al. This letter aims to highlight the possible impact of the LayV, assess the existent risk, and delineate the possible containment measures to mitigate the same. LayV virus belongs to the While the LayV was first identified in the north-eastern provinces of Shandong and Henan in late 2018 , it was Unsurprisingly, there aren’t any approved treatments or specific vaccines for henipaviruses, including the Langya virus, to date. Ribavirin may be a good option when there no other treatments are available for viral infections. Ribavirin is effective against the Hendra and Nipah viruses. These two might also be treated with chloroquine, a medication for malaria. Therefore, if necessary, these two treatments may also aid in controlling the Langya virus.Scientists have estimated that 70% of newly emerging infectious diseases have been transmitted to humans through interaction with animals, a trend that has accelerated as the expanding human populations have a greater impact on wildlife habitats, especially viral pathogens (Saied et al.