Phoma matteucciicola strain LG915-1. The genome of PmOLV1 was 2603 nucleotides long and contained a single open reading frame (ORF), which could be translated into a product of RNA-dependent RNA polymerase (RdRp) by both standard and mitochondrial genetic codons. Cellular fractionation assay indicated that PmOLV1 RNAs are likely more enriched in mitochondria than in cytoplasm. Phylogenetic analysis indicated that PmOLV1 is a new member of the genus Penoulivirus (recently proposed) within the family Botourmiaviridae.Here, we report a novel (+) ssRNA mycovirus, Phoma matteucciicola ourmia-like virus 1 (PmOLV1), isolated from  Cryphonectria parasitica and Sclerotiorum sclerotiorum, respectively [Mycoviruses  infect and replicate in all major filamentous fungal groups, yeasts, and oomycetes ,2,3. In ectively ,8.Alphaflexiviridae, Gammaflexiviridae, Barnaviridae, Hypoviridae, Mymonaviridae, Narnaviridae, and Botourmiaviridae [So far, more than 300 mycoviral sequences have been reported in the National Center of Biotechnology Information (NCBI) database. The identification of newly isolated mycoviruses may contribute to our understanding of the diversity, evolution, and ecology of viruses ,10. Mostaviridae ,12,13.Narnaviridae are the simplest viruses and have been discovered in filamentous fungi, yeast, and oomycetes [Ourmiavirus represents a link between fungal viruses and viruses of other higher organisms [Botourmiaviridae currently comprised four approved genera  and one recently proposed genus (Penoulivirus), among which Botoulivirus, Penoulivirus, and Magoulivirus infect only fungi; Scleroulivirus infects both fungi and plants; and Ourmiavirus comprises only viruses isolated from a plant host .Viruses in the family omycetes . The genomycetes . Phylogeomycetes . Thereforganisms . The famPhoma matteuccicola is the causal agent of leaf blight disease in Curcuma wenyujin, and can generate huge economic losses [P. matteuccicola [P. matteuccicola and provisionally named it Phoma matteucciicola ourmia-like virus 1 (PmOLV1). We showed here that PmOLV1 is a new member of the recent proposed genus Penoulivirus within the family Botourmiaviridae, and is likely enriched in mitochondria.c losses . So far,uccicola . In the P. matteuccicola strain LG915-1 was isolated from C. wenyujin, which showed symptoms of leaf blight disease in Hainan province, China, in 2016. It was cultured on potato dextrose agar (PDA) at 25 °C. For dsRNA extraction, mycelium was cultured on a PDA plate covered with cellophane membranes at 25 °C for 10 days.DsRNA was extracted from 3.0 g of frozen fungal mycelium using the method described previously by Morris and Dodds (1979) with minor modifications, and by selective absorption to columns of cellulose powder CF-11  in the presence of 16% ethanol . To impr®UltraTM RNA Library Prep Kit  according to the methods described previously [The purified dsRNA sample was used as a template for cDNA synthesis. The cDNA library was constructed using the NEBNexteviously . To get eviously . Each bahttp://www.ncbi.nlm.nih.gov). Motifs searches were conducted in the PROSITE database (http://www.expasy.ch/). Multiple sequence alignments were performed using the CLUSTALX 1.81 program [Open reading frames (ORFs) and conserved domain(s) were determined using ORF Finder program and CD-search in the National Center for Biotechnology Information (NCBI) ( program . Phyloge program . Potenti program .W/V), the membranes were immunoblotted with the two antibodies: cytosolic Pgk1  and mitochondrial Porin . Then, the membranes were further incubated with peroxidase conjugated secondary antibodies, followed by the protein bands detected using enhanced chemiluminescence HRP substrate and images taken using Chemiluminescence instrument.To determine the subcellular distribution of PmOLV1 mRNAs in fungal cells, an enriched mitochondria fraction was extracted using the mitochondria isolation kit  according to the manufacturer’s recommendation. Then, the protein concentration of supernatant and mitochondrial enriched precipitation fraction was quantified via bicinchoninic acid (BCA). The proteins were separated using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto polyvinylidene fluoride (PVDF) membranes. After blocked with 5% dry milk in TBST . The RNA concentration was detected and the cDNA was prepared from 1 μL RNA using the RevertAid First Strand cDNA Synthesis Kit for qPCR . Phoma matteuciicola partitivirus 1 (PmPV1), a co-infecting and cytoplasmic virus, was used as a control in this experiment . The speMitovirus [The full-length cDNA sequence of Phoma matteucciicola ourmia-like virus 1 (PmOLV1) was 2603 bp in length, with a G+C content of 53.67%. The cDNA sequence of PmOLV1 was deposited in GenBank under the accession number MN473199. It contains a single ORF being 2258 bp long that initiated at the nucleotide position 263 and terminated at position 2521, potentially encoding 751 amino acid (aa) residues with a calculated molecular weight of 83.5 kDa between the initiation AUG triplet and the termination UGA triplet A. In addA homology search with BLASTP showed that this 83.5 kDa protein was most closely related to the RNA dependent RNA polymerases (RdRps) of Epicoccum nigrum ourmia-like virus 2, Penicillium sumatrense ourmia-like virus 1, and Pyricularia oryzae ourmia-like virus 1 . FurtherPenoulivirus within the family Botourmiaviridae [To analyze the relationship between PmOLV1 and other mycoviruses, phylogenetic tree was constructed based on the RdRp aa sequence of PmOLV1 and other related viral sequences, including those of the ourmia-like viruses, mitoviruses, narnaviruses, and ourmiaviruses. In the obtained phylogenetic tree, PmOLV1 was grouped with penouliviruses, such as the Phomopsis longicolla RNA virus 1, Epicoccum nigrum ourmia-like virus 2, Penicillium sumatrense ourmia-like virus 1, and Pyricularia oryzae ourmia-like virus 1 . Therefoaviridae .As the PmOLV1 genome can encode putative RdRp protein with both standard and mitochondrial codons, we further tested whether PmOLV1 could localize in mitochondria, for which we performed an enrichment experiment with the strain containing both PmPV1 (a putative cytoplasmic virus) and PmOLV1. With a commercial mitochondria isolation kit  demonstrated to be efficient for isolating yeast mitochondrial fraction , we obta7 times, as the threshold cycle (CT) was 16.20 ± 0.44 in the cytoplasmic fraction and 22.87 ± 0.89 in the mitochondrial (from three independent experiments)  B. HoweveP. matteucciicola, the causal agent of leaf blight disease in Curcuma wenyujin. The genome of PmOLV1 contained a single open reading frame (ORF), which encoded a single protein sharing amino acid sequence identity with the RdRp sequences of ourmia-like mycoviruses. Interestingly, the ORF of PmOLV1 could be translated by both standard and mitochondrial genetic codons into a product of RdRp with UGA and UAA as termination condons, respectively. Similar translation machinery has been reported with Phomopsis longicolla RNA virus 1 (PlRV1) and Tuber aestivum mitovirus (TaMV), but their termination codons are UAG and UAA. It is worth noting that the termination code of PmOLV1 is UGA, not UAG, when standard genetic code was invoked. However, the mitoviruses always have the UAA termination codon, such as Cryphonectria parasitica mitovirus 1 (CpMV1), Sclerotinia sclerotiorum mitovirus 1 (SsMV1), and Botrytis cinereal mitovirus 1 (BcMV1). In addition, the narnaviruses always have the UGA termination codon.In this study, we identified a novel mycovirus, PmOLV1, being isolated from the plant pathogenic fungus Although the cellular fractionation assay indicated that PmOLV1 mRNA is likely more enriched in mitochondria than in cytoplasm, in this study, we cannot rule out the potential contamination of the mitochondrial fraction by vesicular bodies. This is only a biochemical enrichment experiment. Therefore, in the near future, additional studies are necessary in vivo to confirm whether the virus PmOLV1 is associated with mitochondria themselves or other vesicular bodies potentially also enriched in the fraction.P. matteucciicola strain LG915-1. 24 strains were obtained by protoplast regeneration, 44 strains were obtained through single spore isolation, and 56 isolates were obtained through hyphal tip sub-culturing following ribavirin and cycloheximide treatment, as reported previously [PmOLV1 infection is very stable in the mycelia of eviously . HoweverPenoulivirus within the family Botourmiaviridae.Phylogenetic analysis with RdRp sequence placed PmOLV1 in a distinctive clade with closely related to the ourmiaviruses rather than to any of the narnaviruses . It is r


This cohort study analyzes the implications of pandemic-related stay-at-home restrictions for the positivity rates of influenza and other seasonal respiratory viruses in Northern California. Was the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)–related shelter-in-place order associated with lower rates of common respiratory viruses in a community?In this cohort study using interrupted time analysis of 46 128 tests for viral respiratory pathogens, statistically significant lower rates of influenza and rhinovirus or enterovirus infections were found after the institution of a shelter-in-place order to prevent SARS-CoV-2 transmission.Results of this study showed that public health initiatives, such as shelter-in-place orders during a pandemic, appeared to be associated with lower transmission rates of other respiratory viruses in a community. Public health initiatives that include shelter-in-place orders are expensive and unpopular. Demonstrating the success of these initiatives is essential to justify their systemic or individual cost.To examine the association of a shelter-in-place order with lower rates of seasonal respiratory viral activity.This cohort study with interrupted time series analysis obtained monthly counts of respiratory virus testing results at UC Davis Health from August 1, 2014, to July 31, 2020. Patients of all ages underwent testing conducted by the laboratory at UC Davis Health, a referral center for a 65 000-square-mile area that includes 33 counties and more than 6 million Northern California residents.A statewide shelter-in-place order was instituted on March 19, 2020, restricting residents to their homes except for traveling for essential activities. Large social gatherings were prohibited, schools were closed, and nonessential personnel worked remotely. Those who had to leave their homes were mandated to wear face masks, engage in frequent handwashing, and maintain physical distancing.Positivity rates of common respiratory viruses within the community served by UC Davis Health.A total of 46 128 tests for viral respiratory pathogens over a 6-year period were included in the analysis. For the postexposure period (March 25-July 31), approximately 168 positive test results occurred for the studied organisms in the 2020 virus year, a positivity rate of 9.88 positive results per 100 tests that was much lower than the positivity rate of 29.90 positive results per 100 tests observed for this date range in the previous 5 virus years. In contrast, the positivity rates were similar for the preexposure time frame (August 1-March 24) in the 2020 virus year and for the same time periods in the 5 previous years (30.40 vs 33.68 positive results per 100 tests). In the regression analyses, statistically significant decreases in viral activity were observed in the postexposure period for influenza  and for rhinovirus or enterovirus  infections. Lower rates of postexposure viral activity were seen for respiratory syncytial virus, parainfluenzavirus, coronaviruses, and adenoviruses; however, these associations were not statistically significant.Using interrupted time series analysis of testing for viral respiratory pathogens, this study found that statistically significant lower rates of common community respiratory viruses appeared to be associated with a shelter-in-place order during the coronavirus disease 2019 pandemic. As part of this directive, nonessential personnel worked remotely, large social gatherings and worship services were canceled, and California schools were closed. Essential workers and consumers leaving their homes were mandated to wear face masks, engage in frequent handwashing, and maintain a 6-foot distance from people outside their household.1 The March 19 stay-at-home order remained in effect through the summer and fall of 2020, with travel permissible only for health care needs; food procurement; outdoor exercise or recreation; outdoor museum visits; and activities at open, low-risk businesses and workplaces for which telework was not possible, including retail, manufacturing, and offices.The 2019-2020 respiratory virus season was like no other in human history, with the emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and its circulation alongside common respiratory viruses. In response to the coronavirus disease 2019 (COVID-19) pandemic, federal, state, and local governments placed restrictions on travel and instituted physical distancing protocols to curb the spread of this novel virus. On March 19, 2020, California governor Gavin Newsom issued a statewide shelter-in-place order.The end of the 2019-2020 respiratory virus season presented a unique opportunity to evaluate the outcome of public health interventions in response to SARS-CoV-2 for the epidemiologic characteristics of other circulating respiratory viruses. In this cohort study, we examined the association of the shelter-in-place order with lower rates of seasonal respiratory viral activity using the incidence of common respiratory viruses in a Northern California health system during the statewide shelter-in-place order. Public health initiatives that include shelter-in-place orders are expensive and unpopular. Demonstrating their success is essential to justify their systemic or individual cost.TREND) reporting guideline.All data used in this study were deidentified, so the study did not qualify as human research according to the policies of the US Department of Health and Human Services or the US Food and Drug Administration; therefore, institutional review board approval was not required. We followed the Transparent Reporting of Evaluations With Nonrandomized Designs , which tests for influenza A subtypes H1, H1 2019, and H3; influenza B; respiratory syncytial virus (RSV) A and B; parainfluenzavirus 1 to 4; human coronaviruses 229E, HKU1, NL63, and OC43; human metapneumovirus; rhinovirus or enterovirus; and adenovirus. Respiratory syncytial virus and coronaviruses were added to UC Davis Health’s current respiratory pathogen panel in July 2016, limiting our evaluation of these 2 viruses to 5 rather than 7 years. Human metapneumovirus rates during the study period were substantially lower than the rates of other viruses; thus, this organism was not included in the analysis.Results of point-of-care real-time polymerase chain reaction test for influenza A and B and RSV  were also included in this study. Point-of-care RSV testing was available at UC Davis Health starting in 2018. Respiratory pathogen panel and point-of-care testing data were extracted from clinical encounters in multiple settings, including the hospital, emergency department, and more than 40 outpatient clinics.Our interest was in ascertaining whether the seasonally adjusted incidence rate of respiratory virus detection during the shelter-in-place period differed from the expected rates based on previous years. The units of analysis were monthly counts of positive and negative test results. In this study, the shelter-in-place order divided the current virus season into preexposure  and postexposure  periods. This calendar time division was used for the 5 previous virus years to permit the comparison of similar calendar periods between virus years while accounting for between-year variation. In the unadjusted analysis of virus incidence, we proportionally allocated the March data in a 24:7 ratio to the preexposure and postexposure periods.2 (SAS Institute Inc) to fit marginal regression models for overdispersed Poisson data with autocorrelated errors for each organism’s monthly time series of positive test result counts. This model included a variable coded 0 for all months before or 1 for all months after March 2020, and coded with the value 7/31 for March 2020, to adjust for the shelter-in-place order affecting the last week of March and fully affecting all weeks subsequent to that. The estimated regression coefficient for this variable was used to estimate the seasonally adjusted incidence rate ratio (IRR) associated with the shelter-in-place order, which was also expressed as a percentage reduction (100 × [1 – IRR]). The Wald test for this regression coefficient was evaluated for statistical significance with a 2-sided α = 5%. All models included additional covariates to adjust for year-to-year differences and cosinor analysis terms to account for sinusoidal seasonal effects.3 For each organism, we fit and compared 6 models that varied in how they controlled for historical patterns  and how they controlled for seasonal effects ,4 and we used the Akaike information criterion to select the best model.5For analyses adjusting for between-year and seasonal effects, we used the GLIMMIX procedure in SAS/STAT softwareOffset terms based on the monthly number of definitive test results were included in the model so that that the incidence rates refer to incidence proportions. The small number of indeterminate test results (<0.14%) were excluded from the analysis. In sensitivity analyses, we changed the offset terms to refer to number of days in the month so that incidence rates refer to number of positive test results per day , approximately 168 positive test results occurred in the 2020 virus year, a positivity rate of 9.88 positive results per 100 tests, which was much lower than the positivity rate of 29.90 positive results per 100 tests observed for this date range in the previous 5 virus years. In contrast, the positivity rates were similar at the preexposure time frame (August 1-March 24) for the 2020 virus year and for the previous 5 virus years (30.40 vs 33.68 positive results per 100 tests) .We observed lower rates of all incident viral respiratory infections after the institution of the statewide shelter-in-place order compared with the previous 5 to 7 years (2014-2019). Specifically, a statistically significant decrease of 93%  was found in 2020 for laboratory-confirmed cases of influenza and 81%  for laboratory-confirmed cases of rhinovirus or enterovirus. Lower rates of viral activity ranging from 63% to 91%  were also observed in all other viral pathogens, but these rates were not statistically significant because of smaller incidence rates, higher seasonal variability, and thus wider CIs .6 indicated that in Japan, seasonal influenza activity was lower in the 2019-2020 respiratory season compared with the 5 previous years. Studies from Taiwan, Singapore, Korea, and Thailand have also shown decreased influenza incidence after countrywide public health measures were instituted to reduce the transmission of COVID-19.10 To our knowledge, this study is the first in the United States to examine the implications of SARS-CoV-2 public health measures for influenza rates as well as the first in the world to examine the implications of these public initiatives for other seasonal respiratory viruses.We hypothesized that the statewide shelter-in-place order that was instituted to curb the spread of SARS-CoV-2 would be associated with lower rates of common viral infections circulating in the community. In April 2020, a report by Sakamoto et alThis study has several limitations. First, although UC Davis Health serves as a referral center for more than 6 million Northern California residents, this is a study of a single health system. Second, although the statistical analysis rigorously estimated the seasonally adjusted outcome of a shelter-in-place order as an IRR, the order was issued toward the end of the respiratory viral season in California. The implication of this public health intervention for the 2020-2021 respiratory viral season is unknown and could be altered by favorable  or unfavorable  factors. Third, health care avoidance because of concerns about COVID-19, particularly by those with milder infections, was a possible confounder in the number of laboratory-confirmed cases. Increased use of telehealth and limits on testing capabilities are other possible confounders. We used the rate of positive cases to control for fewer tests attributed to changes in health-seeking behavior. In sensitivity analyses that included daily rates of positive cases, the estimated IRRs were similar, with IRRs for parainfluenzavirus and adenovirus becoming statistically significant (eTable in the 11 However, this cohort study shows that public health strategies have been associated with lower rates of respiratory viruses in the community served by UC Davis Health.The restrictions on travel, mandated mask wearing, and physical distancing that are embedded in the California shelter-in-place order have been controversial because of their substantial social and economic costs.


Drosophila melanogaster with no known characterized phenotype. In this study, geotaxis assays and longevity analyses were used to determine if Nora virus infection affects D. melanogaster's locomotor ability. In addition, Drosophila C virus (DCV), a well-characterized D. melanogaster virus, was used as a positive control, as it has previously shown a locomotor defect in infected flies. Stocks infected with NV (NV+) and DCV (DCV+) and virus-free (NV-/DCV-) stocks were established. Over a 3-year period, approximately 2,500 virgin female flies were tested for geotaxis and longevity using Kaplan–Meier analyses, as well as the Cox Proportional Hazards regression for survivorship. There was a significant decrease in the geotaxis when the D. melanogaster flies were infected with Nora virus compared to uninfected controls, but no difference was found between DCV+ and NV+ trials. There were not significant differences in longevity between the three groups. This is the first time that a phenotype has been recorded in association with Nora virus infection. Overall, the data demonstrate that geotaxis dysfunction may be a phenotypic hallmark of Nora virus infection.Nora virus (NV) is a picorna-like virus that contains a positive-sense, single-stranded RNA genome. The virus infects  Drosophila melanogaster is a model organism commonly used in biological studies to study virus effects because its DNA is similar enough to humans that they can be used as a model for human immunity and disease [D. melanogaster and humans is geotaxis [D. melanogaster, geotaxis is an innate behavior that causes the fly to climb the wall of a cylinder after being tapped to the bottom [D. melanogaster is comparatively short, which makes studying the effects of a virus on longevity relatively easy. D. melanogaster viruses have variable effects on longevity. There is genetic variation in survivorship with DCV infection, which is dependent upon a number of factors. One early study demonstrated that DCV infection caused a decrease in lifespan with 30–50% of the flies dying within a week [D. melanogaster makes these good metrics for characterizing a possible disease phenotype associated with a viral infection, especially one that has no known phenotype, such as Nora virus. disease , as wellgeotaxis . Geotaxie bottom . By quane bottom . Althouge bottom . Anothern a week . While on a week . TherefoD. melanogaster and is transmitted horizontally via the fecal-oral route [D. melanogaster flies that are chronically infected with Nora virus as the primary innate immune response using siRNA showed no effect on Nora virus. This is unlike most viral infections, as this is active against foreign RNA and is essential in defense of most RNA viral infections [D. melanogaster [D. melanogaster.Nora virus is a picorna-like virus, similar to poliovirus, rhinoviruses, and enteroviruses, that replicates in the gut of al route . The viral route . Interesfections . In addinogaster . TherefoD. melanogaster. For comparison, DCV, a well-characterized D. melanogaster virus, was used as a control because infected flies have been shown to have decreased locomotor function in terms of geotaxis and, in some studies, decreased longevity. We predicted that D. melanogaster infected with Nora virus will show reduced climbing ability and possibly a decrease in lifespan compared to virus-free flies.In this study, geotaxis assays and lifespan analysis were used to observe the effects of Nora virus infection on both locomotor function and longevity of RelE23 stocks  were maintained at 25°C on standard cornmeal, molasses, and torula yeast medium with diurnal light. Flies were infected fecal-orally to establish either Nora virus infected or DCV infected stocks for further analysis [Witi analysis . Once adNORF1 55–844  primers or DCV using DCV7/8  primers and Verso 1-Step RT-PCR Hot-Start Kit according to manufacturer's instructions . The positive controls were an RNA extraction that previously tested positive for Nora virus infection and DCV cDNA for DCV infection. Reactions were set up under the following conditions: Nora virus: 50°C for 30 min; 94°C for 2 min; 94°C for 30 s, 55°C for 30 s, and 68°C for 1 min for 30 cycles; 68°C for 5 min; and hold at 4°C. DCV: 50°C for 30 min; 94°C for 4 min; 94°C for 40 s, 52°C for 40 s, and 72°C for 1 min for 35 cycles; 72°C for 7 min; and hold at 4°C. Samples were analyzed on a 0.8% agarose gel in a TAE buffer solution at 80 V for 1 hour. A positive reaction yielded a product at approximately 790 bp for Nora virus and 524 bp for DCV.Total RNA extraction was performed using TRIzol® as per manufacturer's instructions . Each sample was quantified using a NanoDrop™ 2000c spectrophotometer (Thermo Fisher Scientific) to assess RNA purity and concentration. Samples were analyzed for the presence of Nora virus using Once stocks were confirmed to be either Nora virus or DCV infected or uninfected, bottles of Nora virus positive (NV+), Nora virus negative (NV-), and/or DCV positive (DCV+) stocks were established using 30 mating pairs in each and allowed to mate for 24 hours. After 24 hours of mating, the parental generation was transferred into new bottled medium and allowed to mate for another 24 hours. The F1 generation of virgin female flies were collected every 6 hours after first emergence. Experimental flies were kept in small cages, approximately a liter in volume , with ventilation and food vial extension tube. New vial food media were replaced every 3 days. Each cage contained 60 females, and at the end of 3 years there were 1140 NV+ females tested, 840 NV− females tested, and 600 DCV+ females tested. Overall, 20 cages were completed for NV+, 15 for NV−, and 10 for DCV+.D. melanogaster were frozen at −80°C for future RNA analysis for the presence or absence of Nora virus, as well as quantification. After the dead were removed from the cage, the food was removed, and the hole was blocked with an empty food vial. Each cage was gently tapped on the counter to get all flies into the bottom of the cage. A timer was set for 1 minute and the flies were allowed to climb to the top. At the end of the 1 minute, the number of flies that climbed to the threshold line, which was 2/3 of the way to the top (10.5 cm), was counted and recorded. The empty food vial was then replaced with a new food vial. The data was collected for three years and sent to the Bioinformatics and Systems Biology Core at the University of Nebraska Medical Center (UNMC) for statistical analysis. For both geotaxis and longevity assays, the statistical significance between treatment groups was determined using Kaplan–Meier analysis. In addition, the longevity data was subjected to Cox Proportional Hazards regression analysis. Both tests were done with an alpha of 0.05.The treatment groups were maintained in the liter sized cages for the entirety of their lifespan. Every 3 days, the dead were collected via aspiration and the number was tallied. This continued until all the flies died. Dead Ribosomal protein L32  and Nora virus (AIRSA9W). Reactions were carried out in triplicate using the following conditions: 48°C for 30 min, 95°C for 10 min  repeated for 40 cycles. PCR products were analyzed in the linear range for amplification using the 7500 Real Time PCR Sequence Detection System software and normalized to RpL32. The quantitative results were analyzed on a log 2 scale and with the ΔΔCT method to determine viral load [t-test with an alpha of 0.05.Total RNA was extracted as described in ral load . Statistp < 0.0001). The uninfected group had a greater climbing ability than the NV+ (p < 0.0001) with the NV+ having greater locomotor function than the DCV+ (p < 0.0001). The uninfected control flies had greater locomotor function than the DCV+ (data not shown).The geotaxis analysis showed a significant difference in the locomotor function between NV+ and uninfected flies ( the NV+ . When cothe DCV+ . In DCV+p < 0.0001). When comparing two treatment groups at a time, for NV+ versus uninfected, it was p < 0.0001 . When studying the survivorship curves, it is evident that they appear to cross each other at around 20 days of age . Quantitative RT-PCR was used to analyze Nora viral load at three time points . Average fold change in Nora virus load was determined relative to day 2 of infection. As infection progressed relative to day 2, the viral load fluctuated, decreasing at day 21 (1.5-fold decrease) and increasing again at day 40 (25.05-fold increase). The decrease between days 2 and 21 was not statistically different. The increase in Nora virus viral load at day 40 was statistically significant (p < 0.02) when compared to both days 2 and 21. The average fold change from days 21 to 40 was a 39.16-fold increase.All Nora virus infected samples demonstrated the expected RT-PCR amplification product for Nora virus D. melanogaster has been used to measure the pathogenicity of other human diseases, including Parkinson's disease [D. melanogaster. Indeed, the geotaxis analysis showed that there was a significant decline in the climbing ability of Nora virus infected flies as compared to the uninfected flies . Nora virus is a picorna-like virus , which i disease , 15 and  disease . Becauseed flies . This reed flies . This iswith DCV , 5. It ar skills , 17.D. melanogaster, recording deaths every three days in conjunction with the geotaxis assays. The Kaplan–Meier survivorship analysis using a log-rank Mantel–Cox test indicated a significant decline in longevity between the Nora virus infected and uninfected groups (D. melanogaster flies age, they may accumulate NV and die more rapidly (In toto, the data suggest that Nora virus infection may decrease the overall longevity of D. melanogaster, but this could be due to a number of factors including genetic variation and not only infection with Nora virus.To begin to understand this, we carried out longevity analyses in conjunction with the geotaxis assays. A previous study suggested that the survival rates were similar between Nora virus infected and uninfected flies. However, in that study, they did not perform the statistical analyses to determine if longevity was in fact impacted . In thisd groups , as welld groups , but no d groups  and DCV+d groups  flies, i rapidly . InteresD. melanogaster flies have an open circulatory system, meaning that the hemolymph is pumped by the heart into the tissues and is diffused back to the heart. In the case of DCV, infected hemolymph is circulated to the brain, via the open circulatory system, where it establishes infection [Overall, the data suggest that Nora virus infection leads to a decrease in geotaxis. The data demonstrates that Nora virus infection is similar to DCV infection with a decline in locomotor ability, and therefore the locomotor defect may result in the same way with both viruses. nfection , and qui


It provides a convincing answer to an important research question. We would like to highlight some points about the paper that may merit attention.We read with interest the paper by Esmaeilzadeh et al. titled “Economic Burden of Thalassemia Major in Iran, 2015” published in the Journal of Research in Health Sciences 6, more than 50% of total costs was associated with mortality costs. For example, a study by Daroudi et al.3 suggested that mortality costs accounted for 77% of total costs of breast cancer in Iran in 2010. We believe that the study underestimate the costs of thalassemia major in Iran. Therefore, it should have been mentioned as one of the study limitations.First, although the authors aimed to estimate the economic burden of thalassemia major from the society perspective, mortality cost associated with the disease was not considered as a 'cost' to society in the study. In fact, based on the current literature 7 showed that intangible costs were responsible for 17.27% of total costs of economic burden of road traffic crashes (RTC) in Iran in 2009.Second, the authors mentioned that they calculated direct, indirect and intangible costs when they calculated total costs of thalassemia major. However, there is no information on intangible costs in the results section. It seems that the authors did not consider intangible costs when they estimated total costs of the diseases even though these costs were one of the main components of total cost of the disease from societal perspective. For example, a study by Rezaei et al.Third, the method section of the paper states that the prevalence approach was used to estimate the economic burden of thalassemia major. Nevertheless, the use of this method in the study is not clear throughout the paper because the authors did not mention which part of the findings obtained from using this approach.Fourth, the authors estimated total costs associated with thalassemia major for 198 patients in 2015. Hence, the title of article should not refer to economic burden of thalassemia major in Iran as a whole. This could have been mentioned as one of the limitations of the study.8, it would be useful to present direct healthcare costs, direct non-healthcare costs, patient time costs and costs of premature death in separate table.Fifth, similar to previous studies a, Behzad Karami Matin (PhD)a, Mohammad Hajizadeh (PhD)b*Satar Rezaei (PhD)a Research Center for Environmental Determinants of Health, Kermanshah University of Medical Sciences, Kermanshah, Iran.b School of Health Administration, Faculty of Health Professions, Dalhousie University, Halifax, CanadaCorrespondence to:Mohammad Hajizadeh (PhD)E-mail:m.hajizadeh@dal.caThanks for writing the article "Economic Burden of Thalassemia Major in Iran, 2015". Hereby, the authors respond to the problems stated in Comment on: "Economic Burden of Thalassemia Major in Iran, 2015"In this study, the economic burden of thalassemia for using in planning and policy-making has been estimated both with and without taking into account the costs associated with premature deaths and disabilities. The results related to intangible costs have been presented below Table 2. In addition, the overall results in which the intangible costs as well as the costs of premature death are included can be seen in the second paragraph of the discussion section. Regarding the intangible costs of accidents pointed in this letter, it is important to note that intangible costs or premature death caused by diseases are not comparable in any way. For example, a disease such as rabies that causes the patient to die within a few days does not create much intangible costs. On the contrary, a disease such as cancer with which that patient may live several years might cause substantial intangible costs. Some parts of the article that contains the expressed results:“According to Naghavi, Years Lost due to Disability associated with thalassemia is 25%. Given the per capita GDP (Gross Domestic Product) of $ 5442 for Iran in 2014, it can be said that, in addition to the costs expressed in Table 2, almost all patients lose $1360.5 a year for costs associated with pain and suffering caused by the disease.Taking into consideration the life expectancy of 50 years for thalassemic patients 24, the annual cost of the lost opportunity due to premature death in these patients will be equal to $ 2558.1. By adding the lost welfare costs as well as the direct and indirect treatment costs, the annual and the lifetime expenses of a thalassemic patient (without discount fees) were estimated $ 12,240.4 and $612,020.0”1, referred to in the methodology section. In the cost estimation method, it has been clearly stated how the annual costs were collected (one strength of this study was to estimate the costs very accurately).In the method of estimating the costs based on the prevalence -based approach, the annual costs of the disease are estimated2According to the results, 85% of the costs of thalassemia is paid by the insurance companies and the government, much of which relate to drugs and transfusion that the patients receive. Given that the payers are the same in different regions, the payments by the government and insurance companies cannot vary too much. Regarding the costs that the patients pay, the highest share of costs relates to the lost time for blood collection and testing that can be somewhat similar for the patients. On the other hand, similar studies conducted in Thailand and Taiwan with sample sizes smaller than ours, the heading "Economic burden of thalassemia in Thailand and Taiwan" had been used,Another strength of this study was to present the costs in terms of age groups (Table 1), cost resources (Table 2) and cost payers  that can be really helpful in controlling the costs. On the other hand, it was pointed out in the discussion and conclusion section that how much of the costs related to intangible costs and premature deaths. .a, Batoul Ahmadi (PhD)a*Firooz Esmaeilzadeh (MSc)a Department of Health Management & Economics, School of Public Health, Tehran University of Medical Sciences, Tehran, IranE-mail:ahmadiba@sina.tums.ac.ir


Bemisia tabaci, is a vector of this group of viruses. This research studied the transmission biology of tomato yellow leaf curl Thailand virus (TYLCTHV) by B. tabaci, including virus-infected tissues, virus translocation, virus replication, and transovarial transmission . We discovered that the virus first infects the alimentary gut, then the hemolymph, and finally the salivary glands of the whitefly. The virus did not replicate in the whitefly during infection. In addition, TYLCTHV was detected in only 10% of infected females’ first-generation progeny, but the progeny was unable to cause viral infection of tomato plants; therefore, there was no evidence of transovarial transmission. When combined with the current literature, our results suggest that B. tabaci transmits TYLCTHV in a persistent-circulative mode.Tomato yellow leaf curl viruses cause disease epidemics in tomato crops in tropical and subtropical areas worldwide. The sweet potato whitefly, Begomovirus, family Geminiviridae. Tomato-infecting begomoviruses cause epidemics in tomato crops in tropical, subtropical, and Mediterranean climates, and they are exclusively transmitted by Bemisia tabaci in the field. The objective of the present study was to examine the transmission biology of the tomato yellow leaf curl Thailand virus (TYLCTHV) by B. tabaci, including virus-infected tissues, virus translocation, virus replication, and transovarial transmission. The results demonstrated that the virus translocates from the alimentary gut to the salivary glands via the hemolymph, without apparent replication when acquired by B. tabaci. Furthermore, the virus was detected in 10% of the first-generation progeny of viruliferous females, but the progeny was unable to cause the viral infection of host plants. There was no evidence of transovarial transmission of TYLCTHV in B. tabaci. When combined with the current literature, our results suggest that B. tabaci transmits TYLCTHV in a persistent-circulative mode. The present study enhances our understanding of virus–vector interaction and the transmission biology of TYLCTHV in B. tabaci.Viruses that cause tomato yellow leaf curl disease are part of a group of viruses of the genus  A majority of pathogenic plant viruses are known to have associations with insect vectors, which are responsible for virus transmission among plant hosts ,2. EspecBegomovirus in the family Geminiviridae [Bemisia tabaci [Tomato yellow leaf curl viruses comprise a group of viruses of the genus iviridae . They ara tabaci . Tomato-a tabaci ,10,11. Aa tabaci , whilst a tabaci , and it a tabaci ,15. TYLCa tabaci .B. tabaci transmits TYLCV in a persistent-circulative mode [B. tabaci. Some studies support this idea [B. tabaci [B. tabaci transmits tomato-infecting begomoviruses in a persistent-circulative or persistent-propagative manner is still a controversial issue.It has been suggested that ive mode , which mhis idea ,19, whil. tabaci ,21,22,23B. tabaci that had inherited TYLCV from a viruliferous mother could cause viral infection of host plants . So far, transovarial transmission has only been evidenced in TYLCV but not in other tomato begomoviruses.In addition to replicating in insect vectors, TYLCV has shown vertical transmission in viruliferous female whiteflies ,25,26,27B. tabaci, including virus-infected tissues, virus translocation, virus replication, and transovarial transmission. Our results provide important insights into the transmission mechanism of TYLCTHV by B. tabaci.Control programs for insect-transmitted plant diseases need to be based on a solid understanding of the causative pathogen’s transmission biology in order to be effective. However, at present, the virus–vector interaction and transmission biology of TYLCTHV remain unclear. The objective of the present study was to examine the transmission biology of TYLCTHV in B. tabaci colony and TYLCTHV are described in Weng et al. [B. tabaci Middle East–Asia Minor 1 (MEAM1) putative species was reared on Chinese kale (Brassica oleracea cv. Alboglabra Group) in whitefly-proof net cages , at 28 °C, under a photoperiod of L:D 16:8 h. Chinese kale is a non-host of TYLCTHV; therefore, whiteflies reared on it are non-viruliferous.The sources of our g et al. . The labSolanum lycopersicum cv. ANT22), by vector transmission, using B. tabaci. TYLCTHV-infected and healthy tomato plants were prepared and maintained as previously described [TYLCTHV was maintained in tomato plants  were transferred to a detached leaf of a TYLCTHV-infected tomato plant and placed in a plastic cup (250 mL) for 2, 3, 4, 14, or 24 h of feeding. The petiole of the detached leaf was inserted into a glass vial of water, to prevent wilting. After the acquisition period, the whiteflies were transferred to a Chinese kale leaf for 2 h, to clear possible gut residue from the infected phloem sap. At each time point, 10 whiteflies were collected, and their midgut, PSGs, and legs (a surrogate for hemolymph) were removed. DNA was extracted from the recovered organs, using QuickExtraction DNA Extraction Solution  as previously described [Taq DNA polymerase (Invitrogen) and the TYLCTHV-specific primer set ThCP-F (5’-ACGCCCGTCTCGAAAGTA-3’) and ThCP-R (5’-CCAGCTTCCTGGTGATTGTA-3’) [To investigate virus translocation after escribed . PCR wasTGTA-3’) . PCR conB. tabaci, the viral genome in viruliferous whitefly was quantified by real-time PCR, after the whitefly had fed on Chinese kale. Approximately 300 adult whiteflies (0–5 days old) were transferred by mouth aspirator to a net cage enclosed with a TYLCTHV-infected tomato plant for 24 h of feeding. Subsequently, the whiteflies were transferred to another net cage enclosed with a Chinese kale. Ten females and 10 males were then randomly collected by mouth aspirator every 24 h until day 10. Each pool of 10 whiteflies was stored in a 1.7 mL microcentrifuge tube, at −80 °C, until DNA extraction.To determine whether TYLCTHV replicates in heat shock protein 90 (HSP90) gene of B. tabaci was amplified, using the primer set HSP90-F (5’-ATCGCCAAATCTGGAACTAAAGC-3’) and HSP90-R (5’-GTGTTTTGAGACGACT GTGACGGTG-3’) [HSP90 gene was calculated, using the 2−∆Ct method.The DNA of each whitefly pool was extracted, using DNeasy Blood and Tissue kit , following the manufacturer’s instructions. The TYLCTHV genome was quantified by real-time PCR, as previously described , using tGGTG-3’) , to servRelative quantities of TYLCTHV genome between days were analyzed, using the Kruskal–Wallis test. All data analyses were performed, using SPSS 22.0.2, 109 mesh/inch, Megaview) for an inoculation access period (IAP) of 48 h. Twenty-five test plants were inoculated by the F1 progeny. After the IAP, inoculated plants were treated with a systemic insecticide (acetamiprid), to kill all remaining insects on the plants. PCR assays were conducted to assess the infection of TYLCTHV in the inoculated plants three weeks after the IAP. DNA extraction from tomato leaves and whiteflies, as well as the PCR procedure, was performed as described previously [B. tabaci, and were kept in the same environmental growth chamber as the inoculated test plants, until the PCR assay.Twenty female and 20 male adult whiteflies (0–2 days old) were given an AAP of 48 h on a TYLCTHV-infected tomato plant in the aforementioned net cage. After the AAP, all viruliferous whiteflies were transferred, by mouth aspirator, to another net cage and enclosed with a Chinese kale for an additional 5 days for oviposition. During this period, females had the opportunity to lay eggs on the Chinese kale. All adults were removed from the Chinese kale on the sixth day, with only the eggs and nymphs of first-generation progeny (F1) remaining on the plant. In this setting, the progeny of the viruliferous whiteflies hatched and developed on the Chinese kale, without contact of TYLCTHV. Twenty-five of the F1 progeny were individually subjected to DNA extraction and PCR assay when they emerged as adults, to examine whether they had inherited TYLCTHV from their viruliferous mothers. Additionally, to evaluate whether the transovarially transmitted TYLCTHV in the F1 progeny was infectious, 5 adults of the F1 progeny were used to inoculate healthy tomato plants by enclosing them with a tomato leaf in a gauze bag . Regression analysis showed that the relationship between viral load and time was negatively correlated with a slope of −2.47, suggesting that viral load decreased with time. However, in males, the viral load did not significantly alter over the course of 10 days (p = 0.56). The results therefore suggest that TYLCTHV does not replicate in viruliferous B. tabaci.A time-series real-time PCR assay was conducted, to investigate whether TYLCTHV replicates in o day 10 ; howeverB. tabaci, the F1 progeny of viruliferous females were examined by PCR and a transmission assay. Ten F1 individuals tested positive for TYLCTHV from a total of 100. However, none of the tomato plants (n = 100) inoculated with F1 progenies tested positive for TYLCTHV. None of the negative controls tested positive for TYLCTHV. The results imply that TYLCTHV is inherited from viruliferous mother to F1 progeny, with 10% of F1 adults carrying the virus; however, the F1 progeny is unable to cause viral infection of host plants.To determine whether TYLCTHV is transovarially transmitted by B. tabaci alimentary gut. This is similar to virus accumulation in B. tabaci reported for TYLCV, tomato yellow leaf curl China virus (TYLCCNV), and tomato yellow leaf curl Sardinia virus (TYLCSV) [B. tabaci MEAM1 species but not Mediterranean (MED) species, whereas TYLCV can be detected in the secretory section of PSGs of both MEAM1 and MED species [The confocal microscopy results demonstrate that TYLCTHV accumulates in the PSGs, gastric caeca, midgut, and filter chamber of the (TYLCSV) ,33,34,35B. tabaci, through confocal microscopy. A fluorescence signal was only detected in follicle cells surrounding the oocyte. However, 10% of the F1 progeny harbored the virus, as confirmed by PCR. Therefore, it is likely that the F1 progeny of viruliferous females inherit TYLCTHV, but the amount of virus in most F1 progeny is below the PCR detection threshold. Their viral loads were also too low to be detected by the immunofluorescence assay.Furthermore, we found that TYLCTHV accumulated in the ovarioles but not oocytes of B. tabaci MEAM1 pathosystem, but the transovarial transmission of TYLCTHV was not supported by our data. Our results are similar to those reported in the transmission of TYLCSV by B. tabaci [B. tabaci MEAM1 species to transmit the virus to plants. So far, transovarial transmission has only been evidenced in TYLCV, but not in other tomato begomoviruses [B. tabaci complex transmit TYLCV transovarially. Guo et al. [B. tabaci species and discovered that this only occurred in MEAM1 species.The F1 progeny of viruliferous females cannot cause viral infection of host plant; however, some progeny inherit TYLCTHV from viruliferous females. Transovarial passage is possible in the TYLCTHV-. tabaci , where toviruses ,27. Howeo et al.  tested tB. tabaci increases with time after viruliferous whiteflies are transferred to a non-host plant of the virus [B. tabaci. In the present study, the amount of TYLCTHV in female whiteflies gradually decreased with time, whilst remaining constant in males.Whether tomato begomoviruses replicate in whitefly vectors is still controversial. Some studies have reported that the amount of TYLCV in he virus ,21; othehe virus ,23. Howehe virus ,19,34 nohe virus  replicatB. tabaci MEAM1 species were previously examined by our group [The present TYLCTHV translocation study demonstrated that the virus infected the midgut and hemolymph within 3 h, and then the PSGs within 14 h of feeding on an infected plant. The time needed for TYLCTHV to infect the PSGs is useful for disease control, since the virus cannot be transmitted to other host plants until it reaches the PSGs. The transmission characteristics of TYLCTHV by ur group , and illur group . AccordiB. tabaci MEAM1 species, but not the hindgut and oocyte. When acquired by B. tabaci, the virus translocates from the alimentary gut to the salivary glands via the hemolymph, without apparent replication. The virus was detected in a small proportion of the F1 progeny of viruliferous females, but the F1 progeny was unable to cause viral infection of test plants. Therefore, transovarial transmission is not evidenced for TYLCTHV.TYLCTHV infects and accumulates in the PSGs, gastric caeca, midgut, and filter chamber of


As is well known the novel coronavirus (COVID-19) is a zoonotic virus and our model is concerned with the effect of the zoonotic source of the coronavirus during the outbreak in China. We present a SEIS complex network epidemic model for the novel coronavirus. Our model is presented in fractional form and with varying population. The steady states and the basic reproductive number are calculated. We also present some numerical examples and the sensitivity analysis of the basic reproductive number for the parameters. The corona virus family continues to clone new strains that threaten human life. The world is witnessing these days the emergence of a new strain of corona virus in the Chinese city of Wuhan. This novel corona virus strain (COVID-19) is the seventh of the corona family, which includes, for example, influenza, SARS, and Middle East respiratory syndrome (MERS). The family of coronaviruses is characterized by being common among different types of animal species, such as bats, cats, camels, and cattle.In December 31, 2019, the Chinese city of Wuhan announced the outbreak of a new strain of coronavirus. And this new strain is considered among the zoonotic viruses that are transmitted from animal to human and then transmitted from human to human .On February 27, 2020 (at the time of writing), there were 82,294 infected cases of this virus worldwide, according to the World Health Organization’s report No. 38 on the epidemiological situation of the virus, including 2747 deaths in China and 57 death cases outside China [The Chinese city of Wuhan in Hubei Province is the origin of this virus, which quickly spread to many Chinese cities (34 cities so far). Then it moved out of the Chinese territory until it reached 46 countries worldwide . NowadayThe Chinese city of Wuhan is among the most important cities in China in terms of combining many transportation lines between Chinese cities as well as internal transportation outside China. Wuhan also contains a large market for seafood and animals, which is the source of the emergence and spread of COVID-19.And when looking at how COVID-19 spreads from person to person, we find that the pattern of spread is not known yet, but most of the current information about the method of spread is based on previous information on corona viruses. Also, the spread of COVID-19 from a person infected with the virus to a healthy person needs close communication with the infected person where there will be an effect of cough and sneezing droplets. It turned out from the current cases of infection, whether simple or severe, that symptoms of this disease (COVID-19) appear in the form of fever, shortness of breath and cough. To date, there is no vaccine for this virus, so general prevention instructions such as avoiding direct contact with infected people and using gloves and face masks should be adhered to.The study focused in this model on the zoonotic nature of the virus because of its continuous effect on the spread of the virus, especially at the beginning of the spread. In addition, the model was placed in a fractional form, with the community being represented by a heterogeneous network, in order for the model to be more realistic.In the following section we present a heterogeneous network epidemic model for COVID-19 in a fractional form –10 usingIn this model we divided the population into three compartments susceptible, exposed and infected. The susceptible individuals can be exposed because of being in close contact with infected one. Also, the infection could be transmitted to a susceptible individual from a zoonotic source of COVID-19 . This interaction between susceptible and the zoonotic source happened in a homogeneous pattern during buying and walking in the seafood market. Also, the number of zoonotic sources is considered to be constant in the seafood market . The exposed individual become infected after the incubation period. The infected individual became susceptible again after the infectious period. The city’s population (Wuhan city) is changing as a result of traveling continuously to and from the city. In this model we ignored the births and the deaths.k is the degree of the node, n is the maximum degree of a node. According to the above system dynamic description, the model is defined as k. mentclass2pt{minimLet r system .We will find the equilibrium points of system  by puttit system  has a unThe value of the endemic point changes with respect to the existence of By substituting with the value of We can put it in the following form: Now, we need to get a solution for Therefore, we have two cases.Case 1: If Case 2: If F given by V represents the rate of transferring out of and into the two compartments F are given by V take the form Only the exposed and infected compartments will be used to find the basic reproductive value . The ratmatrices . The foln eigenvalues λ of matrix The characteristic equation for the 2Define the basic reproductive numberas follows: Ifandthen system  with the definition r by v% increases (decreases) the value of r by v% decreases (increases) the value of γ is the most sensitive parameter i.e. if this parameter increased by 10%, the value of Sensitivity analysis shows us which of the parameters used in our mathematical model is the most effective in spreading the infection . In the entclass1pt{minimam is a constant satisfies In this section, we used an Adams-type predictor-corrector method , 20 for k with fractional order In the absence of the zoonotic effect  prevalence pattern using a fractional-order system. Taking into account the effect of the zoonotic source origin of the disease, as well as the continuous transport movement in Wuhan, the mainland city of the virus. We calculated the basic reproduction number, which significantly depends on traveling and movement rates from and outside the city. In addition we calculated the equilibrium positions for this system, as well as showing the local stability of the disease-free situation if the value of the


HoBiPeV should be considered among the bovine pathogens that impact on reproduction, but there is a need for more specific and sensitive diagnostic methods, while the cross-protection elicited by commercially available BVDV vaccines should be better investigated.HoBi-like pestivirus (HoBiPeV) is an emerging group of pestiviruses that has been detected in cattle and other ruminants in South America, Europe, and Asia. Analogous to other bovine pestiviruses, namely bovine viral diarrhea (BVDV) 1 and 2, HoBiPeV is able to cause a variety of clinical forms that range from asymptomatic infections to fatal disease, having a great impact on cattle productions and causing substantial economic losses, mainly as a consequence of the occurrence of reproductive failures. The manuscript aims to provide an updated review of the currently available literature about the impact of HoBiPeV infection on cattle reproduction. The reproductive disorders observed in cattle due to natural and experimental infections caused by this virus are reported along with the few available  Flaviviridae, genus Pestivirus) are single-strand, positive-sense RNA viruses, whose genome, 12.3 Kb in size, encodes for a polyprotein that is cleaved by viral and cellular proteases in structural  and nonstructural  proteins , genus Pestivirus is composed of 11 recognized species, namely Pestivirus A , Pestivirus B , Pestivirus C , Pestivirus D , Pestivirus E (pronghorn pestivirus), Pestivirus F (Bungowannah virus), Pestivirus G (giraffe pestivirus), Pestivirus H , Pestivirus I (Aydin-like pestivirus), Pestivirus J (rat pestivirus) and Pestivirus K   animals –3. Pestitivirus) . HoBiPeVtivirus)  and latetivirus) . Furthertivirus) –20. Howetivirus) , 19–30. The first report of HoBiPeV sequences detected in bovine fetuses dates back to 2006, when a study was published aiming to investigate the genetic variability of BVDV strains from archival samples . Two fet2 (kidney of one fetus) to 5.78 × 104 (lung of the other fetus) RNA copies per μl of template, by a specific real-time RT-PCR assay. No additional pathogens were detected by molecular methods for abortogenic agents of cattle. By immunofluorescence assay using an NS3 monoclonal antibody, a cytoplasmic fluorescence was evident in cryostat sections obtained from the lungs and spleen of the fetuses, whereas successful isolation of a HoBiPeV ncp strain was obtained from the same fetuses. Sequence and phylogenetic analyses of pestivirus's most informative genomic regions  revealed that the abortogenic HoBiPeV strain was closely related to the virus which had been responsible for the respiratory outbreak a few months before , stillbirth (n = 4) or generation of PI lambs (n = 4). Aborted fetuses, stillborn and dead lambs displayed extensive histopathological changes, including hemorrhages, congestion and mononuclear infiltration in internal organs, and immunohistochemical detection of pestiviral antigens in affected tissues. PI lambs were constantly viremic, shed the virus through the nasal secretions and feces and, with one exception, were HoBiPeV seronegative. The single seropositive infected lamb showed low-titer viremia and viral shedding that ceased only several weeks after the 3-month observation period. Therefore, the sheep model reproduced the reproductive disorders observed in cattle due to natural or experimental infection with HoBiPeV and represents a suitable model for the evaluation of the fetal protection induced by pestivirus vaccines.A comprehensive study was carried out in pregnant sheep to evaluate their suitability as a model for vaccination trials of HoBiPeV experimental formulation for cattle . In factBos taurus taurus), indicine (Bos taurus indicus), and mixed taurine and indicine (Bos taurus taurus × Bos taurus indicus) breeds (A study was carried out to evaluate the growth of different bovine pestivirus species in testicle cell cultures obtained from taurine  breeds . In thisin-vitro maturation. After in-vitro fertilization, zygotes were cultured for seven days, and the developed embryos were evaluated according to the guidelines of the International Embryo Transfer Society and submitted to pestivirus detection by real-time RT-PCR. The obtained results showed that in-vitro produced embryos from BVDV-1 and BVDV-2 infected oocytes developed normally, but were infected, thus carrying the virus. In contrast, HoBiPeV infected oocytes had reduced cleavage and cause pre-implantation embryo loss, but viable embryos did not carry the virus, suggesting that HoBiPeV infection may cause embryo loss before blastocyst development. Therefore, the study demonstrated that oocyte exposure to HoBiPeV resulted in reduced embryo development, having the potential to impact more severely on cattle reproduction than other bovine pestiviruses.Another study aimed to evaluate the effect of pestivirus infection on embryo development as a consequence of exposure of bovine oocytes to different pestivirus species . Bovine in utero fetal exposure is critical in the perpetuation of the virus in an infected herd or spread to other susceptible herds (Pestivirus infections cause economically significant diseases affecting the cattle industry, with losses stemming from decreased production and reproductive performance, and control costs. Economic losses associated with reproduction are relevant since these viruses may have negative effects on all phases of bovine reproduction. Reduced conception rates, early embryonic death, abortion, congenital defects, and weak calves have all been associated with pestivirus infection of susceptible cows. Besides, the birth of PI calves as a consequence of le herds , 40. Allle herds , 26, 35.Consequently, this group of emerging viruses should be considered among bovine pathogens that impact reproduction and be included in the diagnostic algorithms of reproductive disorders in cattle. A major issue in HoBiPeV detection is that antigenic, molecular, and serological tests commercially available for BVDV-1 and BVDV-2 may not react or react at a lower extent with this emerging virus , 41. AlsND conceived and wrote the article.The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


In the last decades, Italy as well as other developed countries have registered a decrease in the population size of many local horse breeds. The continuous crossbreeding has determined the dilution of genetic heritage of several native breeds. The Italian Heavy Draught Horse (IHD) is the only autochthonous Italian coldblooded horse among these breeds; therefore, it represents a resource to be preserved. In 1927, the first generation of this breed was officially created by crossing different Heavy Draught horses with local mares and recorded in a Studbook.N = 568) to have a more complete scenario and to understand the relationships with other European Heavy Draught horse breeds.To provide the first comprehensive overview of the genetic diversity of Italian Heavy Draught horses from Central Italy, we produced and phylogenetically analysed 52 mitochondrial DNA (mtDNA) control-region sequences. Furthermore, we evaluated data available from GenBank  found in our samples reflected the multiple maternal origins of the horses. Our results highlighted a considerable percentage of haplotypes shared especially with Bardigiano and Hungarian Heavy Draught breeds. Furthermore, both the presence of four unique haplotypes detected in our samples and their absence among all equine mitochondrial published data demonstrate a mitochondrial peculiarity that needs to be further investigated and preserved with careful breeding practices. Equus caballus is certainly one of the most investigated because of its strict link with human activities. Thanks to selection programs and crossbreeding processes, a great variety of horse breeds developed all over the world, especially for farming, transport, and military purposes.The development of mechanization focused on animal productivity has drastically changed the management of different species leading the current animal husbandry to take care of high-productivity breeds paying special attention to selection programs. Local native breeds have been subjected to a selective crossbreeding in order to preserve their peculiarities . Among thttp://www.anacaitpr.it) reports that at the beginning of 20th century, the mares resulting from this selection program were crossed with Norfolk-Breton stallions, officially creating the first generation of Italian Heavy Draught Horse recorded in a Studbook in 1927. Thereafter, the French Breton was used to improve the heavy draft abilities of this breed. These crossings produced bulky yet also agile horses, with the adults weighing between 700 and 900 kg and the preferred withers height between 156 and 162 cm for males and 153–160 cm for females, which made this breed also perfect for farming. Thanks to the remarkable development and to the milking attitude of the mares, foals can reach a weight of 400 kg at 7–8 months. Even though the Italian Heavy Draught Horse is particularly adapted for agricultural work with minimum environmental impact, the improvement of mechanization reduced the use of these horses, and they became particularly suitable for meat production especially thanks to their fast growth speed and muscle mass development (http://www.anacaitpr.it).In the last decades in Italy, as well as in many other developed countries, the traditional uses of horses have been abandoned, and many breeds have registered a decrease in population size . One of elopment . At the www.fao.org/dad-is). In this scenario, where phenotypic traits and genealogical data are often insufficient to ascertain horse history and origins, morphometrical approach and traditional breeding strategies are supported by molecular analyses that provide a needful and reliable tool for an efficient management of genetic resources  aiming to understand its maternal origins and the relationships with other continental European heavy horse breeds. The knowledge of genetic variability of the maternal founding lines represents an indispensable requirement both for the productive performance and the enhancement of autochthonous genetic resources . HistoriSamples consisting of 1,2 ml of peripheral blood were collected from 81 specimens belonging to Italian Heavy Draught (IHD) horse breed in Central Italy. When the genealogical information was available, we traced back to a maximum of three generations. The Italian name of the breed is CAITPR . All experimental procedures were reviewed and approved by the Animal Research Ethics Committee of the University of Perugia (Comitato Universitario di Bioetica Prot. n. 2017-01) in accordance with the European Union Directive 86/609.® Blood & Tissue Kit, Qiagen) or automated (MagCore®Automated Nucleic Acid Extractor) extraction protocols. Only for maternally unrelated individuals the mitochondrial control region was amplified and sequenced by referring to the protocols reported in Total DNA was extracted by following manual  were assembled and aligned to the Equine Reference Sequence , Belgian Draught , Breton , Croatian Coldblood , Franches-Montagnes , Haflinger , Hungarian Draught , Italian Heavy Draught , Lithuanian Heavy Draught , Noriker , Percheron , Polish Heavy Draught , Rhineland Heavy Draught , Vladimir Heavy Draught , and Vyatskaya   .https://www.fluxus-engineering.com/). The first network was constructed by also considering data from other published Heavy Draught breeds by uniforming the reading range  for a total of 620 sequences. The second network included all the control-region sequences of IHD horses from this study (N = 52) and those available from GenBank (N = 27)  .Different mtDNA sequence variation parameters were estimated by using DnaSP 5.1 software .http://www.cran.r-project.org/).Pairwise Fst calculations were carried out using the Arlequin v3.5 software package , and intIn order to graphically display (and summarize) the mitochondrial relationships among the analysed samples, Principal Component Analyses (PCA) were performed using Excel software implemented by XLSTAT, as previously described .The 81 IHD samples were collected from different farms in Umbria (55), Latium (20), and Abruzzo (7) regions. When available, we accurately evaluated their pedigree and excluded the maternally related individuals, thus the final dataset was represented by a total of 52 specimens  .The analysis of 610 base pairs from the control-region sequences showed a high haplotype diversity (Hd = 0.969) with a total of 30 haplotypes and 53 polymorphic sites (S) detected . Overallπ) of 0.030  to allow the comparison between all samples, we obtained a total of 120 haplotypes, with 49 polymorphic sites (excluding gaps and ambiguous sites) and a Nei’s nucleotide diversity . As for the haplotype diversity, without considering the breeds represented by only three animals per breed , the highest value for the haplotype diversity (Hd>0.96) was found in Italian Heavy Draught and Croatian Coldblood which is the most numerous among the autochthonous draught horses originating from Croatia.It is thought that CRC was created by crossing Croatian native warmblood mares with Noriker stallions under a significant influence of the English Thoroughbred and the Belgian Coldblood. Nowadays it is bred in central and north-western Croatia and is characterized by a great adaptability to maintenance conditions . A studyhttp://www.fao.org/dad-is/transboundary-breed). In particular, BED has played a considerable role in the development of other heavy breeds, and the foundation stock that originated from Belgium was originally known as the Brabant (www.agraria.org). In the early 20th century, the mares continued to look like the original Belgian type, and then they changed from the “working” to the “heavy” type . NOR is considered to be indigenous to the central Alpine region of Europe, and its agricultural use started during the industrialisation period in the 20th century. By the time of the Middle Ages, a small, heavy, and compact horse had already been developed within the NOR breed, and it is currently considered one of the indigenous horses “of limited distribution” (http://www.aia.it). HAF developed from the original native Tyrolean ponies in Austria and Northern Italy (in the South Tyrol region) during the late 20th century. Since it was largely used to improve several local horses, it achieved the aim of a pure breed, and its standards are controlled by the World Haflinger Federation. PER appears to derive from ancient native horses originating from the Perche district, in France; nowadays it is mainly used for draft work, meat, and sport (http://www.fao.org/dad-is). In the early 20th century, PER was the most popular draft horse with the largest breed registry up to the New Continent.Furthermore, our analysis showed the lowest values for haplotype diversity in Belgian Draught, Franches-Montagnes, Noriker, and Percheron, giving evidence of a reduced variability in their partial mtDNA control regions. This finding could be due to the strict selection that involved these breeds, as they were used for crossbreeding and improving different local horses. Together with HAF, they are currently distributed in different continents  were unique (found in only one sample), one (3.85%) was shared between two IHD samples, and 21 (80.77%) were shared with other breeds . If compThe analysis of mutational motifs identified so far  has permThe network analysis of all mitochondrial control regions belonging to the IHD from this study and those from The second network showed a high percentage of our haplotypes shared above all with both BAR and HUD followed by RHD . BAR is Rhenish German Coldblood (RHD) originated from the Rhineland area of western Germany and developed in the 19th century by interbreeding local working horses with other draught horses from neighbouring countries. Then, due to the political division of Germany, gene flows between breeds from East Germany and the RHD were impossible for four decades; as a result of this reproductive isolation, a significant genetic differentiation between RHD and the East German horses was observed .https://blast.ncbi.nlm.nih.gov/Blast.cgi), we verified that there were no identical sequences to four of them among all equine mitochondrial published data. The only exception was HT8, classified in I haplogroup, identical to the mtDNA control region from a Welsh pony (HQ439496).As mentioned above, the haplotype with the highest frequency among our samples  was shared with different European breeds. In the second network, we noticed five haplotypes found only in our samples which were not shared with the other breeds here considered  . Those nhttp://www.fao.org/dad-is).Through the analysis of pairwise genetic distances between populations, we obtained a plot where IHD showed a very high variability within the breed, but a low Nei’s distance with the other populations here analysed, except for the CRC, which together with PER, showed the highest genetic distance from the other breeds . FurtherIn order to summarize the information embedded in the haplogroup frequencies, we performed a principal component analysis (PCA). After reducing the variables (haplogroups) to PCs, we reported the coordinates of the observations for the European Heavy Draught Horse breeds in a two-dimensional graph based on haplogroup frequencies from control-region data . We obseIn order to better explore the peculiarities of IHD samples and to verify the clustering degree, we repeated the PCA analysis by considering single samples so that each individual sequence was represented by a point . The oveOur findings pointed out that the ancestral local mares harbored a very high genetic diversity, verified the autochthonous origin of the breed  and confirmed that the documented contribution of other European coldblooded breeds was restricted to stallions.Appropriate evaluation and characterization of animal populations both at phenotypic and genotypic levels are crucial steps in the development of conservation strategies. Most of the European Heavy Draught horses seem to derive from crossbreeding local mares and heavy draught stallions imported from other countries, and the genetic diversity of native horse breeds could be preserved only by considering also molecular data. For the first time, this study provides a phylogenetic assessment of the mitochondrial distribution within the Italian Heavy Draught Horse breed. The high mtDNA haplotype variability found in our IHD samples reflected the multiple maternal origins of the horses which underwent horse domestication . Our resThese findings reflect the overall mtDNA legacy of the ancestral mares that were probably used at the initial stages of breeding selections a long time ago. Those mitochondrial lineages were preserved during the sex-biased breeding practices that often involved the intensive use of few selected external stallions .In conclusion, our study represents the first mitochondrial characterization of Italian Heavy Draught Horse breed sampled in Central Italy and can be considered a preliminary analysis for further investigations aiming to preserve the genetic peculiarities of local stocks.10.7717/peerj.8996/supp-1Figure S1NC_001640.1).The asterisk indicates the haplotype identical to ERS  were grClick here for additional data file.10.7717/peerj.8996/supp-3Figure S3The comparison between the Italian Heavy Draught (IHD) to a wider range of horses then considered in Click here for additional data file.10.7717/peerj.8996/supp-4Figure S4n = 52 from this study; n = 27 from All Italian Heavy Draught samples ( Click here for additional data file.10.7717/peerj.8996/supp-5Table S1Click here for additional data file.10.7717/peerj.8996/supp-6Table S2Click here for additional data file.10.7717/peerj.8996/supp-7Table S3Click here for additional data file.10.7717/peerj.8996/supp-8Table S4Click here for additional data file.10.7717/peerj.8996/supp-9Table S5Source data for the PCA of European horse breeds including haplogroup frequencies and breed’s geographic originClick here for additional data file.10.7717/peerj.8996/supp-10File S1Click here for additional data file.10.7717/peerj.8996/supp-11Supplemental Information 1Click here for additional data file.