Outbreaks of coronavirus infectious disease 2019 (COVID-19) in meat processing plants and media reports of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) detection on foods have raised concerns of a public health risk from contaminated foods. We used herpes simplex virus 1, a non-Biosafety Level 3 (non-BSL3) enveloped virus, as a surrogate to develop and validate methods before assessing the survival of infectious SARS-CoV-2 on foods. Several food types, including chicken, seafood, and produce, were held at 4 °C and assessed for infectious virus survival (herpes simplex virus 1 (HSV-1) and SARS-CoV-2) at 0 h, 1 h, and 24 h post-inoculation (hpi) by plaque assay. At all three time points, recovery of SARS-CoV-2 was similar from chicken, salmon, shrimp, and spinach, ranging from 3.4 to 4.3 log PFU/mL. However, initial (0 h) virus recovery from apples and mushrooms was significantly lower than that from poultry and seafood, and infectious virus decreased over time, with recovery from mushrooms becoming undetectable by 24 hpi. Comparing infectious virus titers with viral genome copies confirmed that PCR-based tests only indicate presence of viral nucleic acid, which does not necessarily correlate with the quantity of infectious virus. The survival and high recovery of SARS-CoV-2 on certain foods highlight the importance of safe food handling practices in mitigating any public health concerns related to potentially contaminated foods. Coronaviridae. SARS-CoV-2 is transmitted mainly via respiratory droplets generated during normal activities , although airborne transmission has been reported depending on the situational context [50), can be recovered up to 72 h post-inoculation (hpi) on stainless steel and up to 24 hpi on cardboard held at room temperature with constant humidity under laboratory conditions [Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of the ongoing coronavirus infectious disease 2019 (COVID-19) pandemic, is an enveloped, single-stranded, negative-sense ribonucleic acid (RNA) virus belonging to the family  context ,4. In on context . Transmi context ,7. Given50), methods that do not assess for infectious virus (nucleic acid amplification tests), or methods using conditions that are not common to real-world environments  [https://www.foodsafetynews.com/2020/11/china-reports-further-food-related-coronavirus-findings/ accessed on 17 March 2021; https://apnews.com/article/beijing-tianjin-coronavirus-pandemic-wuhan-china-a75ec51c11338190c483c78d65c3348d accessed on 17 March 2021) [https://www.food.gov.uk/research/research-projects/qualitative-risk-assessment-on-the-risk-of-food-or-food-contact-materials-as-a-transmission-route-for-sars-cov-2 accessed on 17 March 2021; https://www.fda.gov/food/food-safety-during-emergencies/food-safety-and-coronavirus-disease-2019-covid-19 accesed on 17 March 2021; https://www.who.int/publications/i/item/covid-19-and-food-safety-guidance-for-food-businesses accessed on 17 March 2021) [https://www.efsa.europa.eu/en/news/coronavirus-no-evidence-food-source-or-transmission-route accessed on 17 March 2021) [Beyond contamination of common surfaces such as packaging materials and counters, SARS-CoV-2 has been recovered at refrigeration (4 °C) and freezing temperatures (0 °C and −80 °C) on some meats and food contact surfaces. These results may be limited in their translation to real world situations as virus recovery was measured with methods less specific than plaque assays (TCIDumidity) , the resch 2021) ,11. It ich 2021) . Howeverch 2021) ,15 and, ch 2021) ,16.https://thefern.org/2020/04/mapping-covid-19-in-meat-and-food-processing-plants/ accessed on 11 March 2021) [However, reported outbreaks of COVID-19 among the staff of major pork and beef processing plants in the United States have raised concerns over the possibility of contracting SARS-CoV-2 from foods and food products contaminated during processing ,18,19,20ch 2021) ,23,24,25ch 2021) . While tch 2021) . It is wch 2021) ,28. It iDisinfection validation, risk assessment modeling, virus transfer through HVAC systems, and conducting research on SARS-CoV-2 require access to biosafety level 3 (BSL-3) facilities, which can be challenging. Thus, identification of an appropriate surrogate virus to use in place of SARS-CoV-2 to investigate the survival of infectious virus on foods would be useful. Similar to SARS-CoV-2, herpes simplex virus 1 (HSV-1) is an enveloped virus that is shed in saliva and nasal secretions and is capable of being expelled in droplets from infected humans. While HSV-1 is not typically considered a foodborne pathogen, previous studies have shown that it can survive on common foods that are routinely touched before consumption . HSV-1 aHSV-1 strain 17+ was originally transferred from John Hay  to FDA  and propagated in Vero 76 cells (ATCC CRL-1587), and first-passage stocks were transferred to UCSF ; first-passage stocks were then transferred to the Bertke lab . Stocks were propagated on Vero 76 cells and titrated by standard plaque assay on Vero 76 cells in quadruplicate to determine concentration. SARS-CoV-2  was procured from BEI, and then propagated and titrated on Vero-E6 cells (ATCC CRL-1586). Both viruses were thawed on ice and diluted in Dulbecco’s modified Eagle medium  to prepare inocula.® sampling bags . To mimic contamination of the outer skin of apples, apple skin was used with minimal but uniform flesh. Chicken, salmon, and shrimp were cut into ≈1.7 g pieces. Both the skin/shell and the flesh portions of salmon and shrimp were included in samples, considering this is the standard presentation of these items in the seafood section of most grocery stores.Fresh foods were purchased from a local grocery store, maintaining identical brands for each experiment. We selected three broad categories of food types: meat/poultry (chicken), seafood , and produce . Foods were chopped to approximately 1.5 cm × 1.5 cm and placed into sterile 12 well tissue culture plates or 2.5″ × 5″ × 2.25 mL (1 oz) Whirl-Pak® bags and inoculated with 10 µL of HSV-1 (1 × 106 PFU). One set of samples was immediately processed (0 h) and two additional sets of samples were incubated at 4 °C for 1 h or 24 h before processing. DMEM (1 mL) was added to each Whirl-Pak® bag, followed by a thorough hand massage to break the food into particles and detach any virus that might have bound to the food or entered into the food matrix. Total fluid from each Whirl-Pak® bag was transferred into microcentrifuge tubes and stored at −80 °C until titration. As a negative control, uninoculated food samples were processed identically.Massage Method: Method was modified from . Food sa6 PFU) or 20 µL (1 × 105 PFU) of HSV-1, or 20 µL (1 × 105 PFU) of SARS-CoV-2 in multiple droplets across the surface of the food samples. One set of samples was immediately processed (0 h) and two additional sets were incubated at 4 °C for 1 h or 24 h. DMEM (1 mL) was used to wash the surfaces of the food samples five times by pipette. Total wash fluid was transferred to microcentrifuge tubes and stored at −80 °C until titration. As a negative control, uninoculated food samples were washed and stored using the same method.Rinse Method: Method was modified from . Food sa2 for 48 h, followed by fixation with 10% formaldehyde and staining with plaque dye. Plaques were counted after the plates were dried and the results were expressed as plaque-forming units per mL (PFU/mL) recovered for each sample.To quantify the titer of infectious virus in the wash or massage media, a standard plaque assay was performed on each sample. Samples were serially diluted and inoculated onto confluent Vero E6 or Vero 76 monolayers in 24 well plates in duplicate. The inoculum was incubated for 1 h to adsorb virus and then removed. For HSV-1, Vero 76 cells were used, and inoculum was replaced with DMEM containing 8% fetal bovine serum (FBS), 1% penicillin/streptomycin (PS), and 0.4% pooled human serum to neutralize any virus released into the media. For SARS-CoV-2, Vero E6 cells were used, and inoculum was replaced with a 0.5% agarose overlay to limit the spread of the virus, consisting of DMEM with 8% FBS, 1% P/S, and molecular grade agarose. The inoculum for each experiment was back-titrated by plaque assay, in triplicate, to verify inoculum concentration. The infected plates were incubated at 37 °C with 5% COViral genome extraction: Viral DNA (HSV-1) or RNA (SARS-CoV-2) was extracted from the virus inoculum and wash medium samples using TRI Reagent LS  according to the manufacturer’s directions. Briefly, 100 µL of wash medium from each food sample was mixed with an equal volume of TRI Reagent LS Solution. DNA and RNA were separated by the addition of chloroform followed by centrifugation. The top RNA-containing aqueous phase was removed and precipitated in a solution of isopropanol and glycogen for SARS-CoV-2 or discarded for HSV-1. Precipitated RNA was washed post centrifugation with 70% ethanol, air dried, and eluted in molecular-grade water. For HSV-1, DNA in the lower organic layer was precipitated by the addition of 100% ethanol, washed with 0.1 M sodium citrate in 10% ethanol, washed with 70% ethanol, air dried, and eluted in molecular-grade water. Concentrations and purity of the extracted nucleic acids were determined using a NanoDrop 2000 spectrophotometer  reading absorbances at 260 nm and 280 nm.HSV-1 qPCR: To determine DNA genome copy number, 10 μL qPCR reactions specific for the thymidine kinase 1 (TK1) gene of HSV-1 using iTaq Universal Probe Supermix  were run on a ViiA 7 Real-Time PCR system , as described previously . The stahttps://www.fda.gov/media/134922/download accessed on 11 March 2021) [SARS-CoV-2 reverse transcription polymerase chain reaction (qRT-PCR): To determine RNA genome copy number, 10 μL RT-qPCR reactions specific for the nucleocapsid gene of SARS-CoV-2 (IDT) using the iTaq Universal Probe One-Step Kit  were run on a ViiA 7 Real-Time PCR system , as described previously (ch 2021) . The stap < 0.05.All experiments were performed three times, in duplicate, with freshly purchased foods each time. Plaque assay data were converted to log PFU/mL prior to statistical analysis. Statistical analyses were conducted using analysis of variance via the GLIMMIX procedure of SAS v. 9.4 . Least square means were calculated and significant differences between means were detected at the Appropriate preparation of food samples for microbiological testing is essential, as the detected microorganisms may be surface contaminants or integrated into the food matrix. Pathogens also possess different properties, allowing them to bind to food surfaces with varying forces ,35,36. Wp > 0.05) at 0, 1, and 24 h post-inoculation, ranging from 5.8 to 6.3 log PFU/mL (p > 0.05), but was significantly lower than that of chicken at 1 h (3.8 log PFU/mL) and 24 h (2.6 log PFU/mL) post-inoculation (p > 0.05) at each time point. In contrast, virus recovered from both the apple skin and mushroom was significantly reduced (p < 0.05) at 1 h and 24 h as compared to initial recovery at 0 h (n = 3).Using the rinse method, in which medium was used to rinse the virus-inoculated food samples, the recovery of HSV-1 from chicken was similar  using the massage method compared to the rinse method. When virus recovery from apple skin was compared, we observed a 3–4 log PFU/mL difference between the methods at 0 and 1 h post-inoculation. Characteristics of apple skin, or potentially factors released from the apple while breaking up the food particles during the massage method, appear to have had a substantial impact on infectious virus recovery. To avoid similar issues with other food types, we selected the rinse method for further evaluation of virus survival on foods.Comparing the rinse vs. massage methods, HSV-1 virus recovery from chicken was similar for both methods, ranging from 5.8 to 6.3 log PFU/mL. However, recovery of virus from apple skin was significantly lower (p < 0.05) at each time point  compared to that from chicken (p > 0.05). Recovery from apple skin was significantly lower than chicken at 0 h post-inoculation (1.8 log PFU/mL) but was not significantly reduced over time  may generate 1.23 × 105 copies of viruses from a single cough [5 PFU/mL HSV-1. The virus was recovered using the rinse method immediately after inoculation (0 h) to quantify the maximum virus recoverable from each food type, and after 1 h and 24 h incubation at refrigeration temperature (4 °C). p > 0.05), while virus concentrations were significantly reduced by 24 h post-inoculation (5.1 and 5.3 log PFU/mL at 0 h to 4.9 and 4.8 log PFU/mL at 24 h (p < 0.05), respectively). Similarly, recovery from spinach was significantly reduced by 24 h from 4.7 log PFU/mL at 0 h to 3.1 log PFU/mL at 24 h. Virus concentration recovered from mushroom was significantly reduced from 4.2 log PFU/mL at 0 h to 2.2 log PFU/mL at 1 h (p < 0.05), which was further reduced to 0.4 log PFU/mL at 24 h (p < 0.05). No reduction in virus concentration from shrimp or apple skin was observed up to 24 h incubation. Nonetheless, initial HSV-1 recovery from apple skin (0 h) was approximately 3 log PFU/mL lower than HSV-1 recovery from chicken skin, salmon, and shrimp. In addition, HSV-1 recovery was compared between the foods at the three different incubation times (p < 0.05). We also observed that HSV-1 concentrations recovered from mushroom and apple skin were significantly lower than those from chicken skin, salmon, shrimp, and spinach at 0 h, 1 h, and 24 h (p < 0.05).A recent study reported that a patient with a high SARS-CoV-2 load in respiratory fluid (2.35 × 10le cough . To asseon times . At each5 PFU/mL), methods, and incubation times as we did for HSV-1.To validate the use of HSV-1 as a surrogate for SARS-CoV-2 and to assess survival of SARS-CoV-2 on different types of foods, we conducted an identical experiment with SARS-CoV-2, using the rinse method and approximately the same inoculum dose (1 × 10p > 0.05). However, initial virus recovery (0 h) from apple skin and mushroom was significantly lower than that from the poultry and seafood (p < 0.05). By 1 h post-inoculation, infectious virus recovered from the mushroom was near undetectable (0.8 log PFU/mL), and was significantly lower than all other foods (p < 0.05) at 24 h compared to 0 h. The mushroom showed significantly reduced recovery of SARS-CoV-2 by 1 h post-inoculation; by 24 h post-inoculation, infectious virus was undetectable  are performed to determine whether viral proteins or viral nucleic acids are present on foods, which do not necessarily equate to the presence of infectious virus. Although many of the recent reports regarding identification of SARS-CoV-2 on foods have not provided sufficient information to determine the methods used to assess the presence of the virus, it is unlikely that food processing plants and food import facilities are assaying for the presence of infectious virus, as they lack the necessary biocontainment facilities, expertise, or time. Therefore, we compared differences between infectious virus titer (as determined by standard plaque assay) with viral genome copy number (as determined by nucleic acid isolation and quantitative PCR or RT-PCR) for both HSV-1 and SARS-CoV-2 from the wash samples obtained from the foods assayed in the previous experiments .The RNA copy number for SARS-CoV-2 in the inoculum used for the food inoculation studies was 2X greater than the infectious virus titer  [Salmonella or pathogenic Escherichia coli, but this method has also been used to recover norovirus from leafy vegetables [The International Organization of Standards (ISO) developed and validated a standard set of methods for extraction of viruses from select foods (ISO 15216-1) (il 2021) . This stil 2021) . Howeveril 2021) . Massagegetables ,47. Sincgetables . TherefoDuring our method development and validation processes, we found that the recovery method used (rinse or massage) did not significantly impact the recovery of infectious virus from poultry or seafood up to 24 h post-inoculation, which was not true for fruits and vegetables. This may have been the result of weak attachment of the viruses to the surface of poultry and seafood, allowing full recovery of the virus inoculum. The textures of various foods may also contribute to virus attachment and recovery. Understanding the mechanisms and extent of virus attachment on food surfaces and entry into the food matrices is important for assessing infectious virus persistence and recovery, as well as for implementation of infection control and prevention strategies. The mechanisms by which SARS-CoV-2 attaches to foods have yet to be determined, although these mechanisms have been determined for other foodborne viruses. Norovirus attaches directly to oysters via A-like carbohydrates expressed by the oyster and to produce via electrostatic forces ,36. PoliIn addition, our consistently higher recovery of virus from poultry and seafood compared to fruits and vegetables suggests that these foods provide conditions more favorable to the maintenance of viable viruses for at least 24 h post contamination. For example, protein content in medium, in the form of fetal bovine serum, has been reported to prolong infectivity of SARS-CoV-2 . Thus, hBoth viruses were relatively stable on spinach over the 24 h incubation period . This is not surprising given that viable human (229E up to 2 days) and nonhuman (bovine coronavirus up to 14 days) coronaviruses have been recovered on lettuce stored at 4 °C ,49. Inte4 PFU/mL [We recovered significantly less infectious HSV-1 from apple skin and mushrooms compared to poultry and seafood, using both massage and rinse techniques. The massage technique, which generated an extract of apple juice, reduced the infectious viral titer of HSV-1 to a greater extent than the rinse method, which contained minimal apple juice, indicating that the presence of apple juice may have had an antiviral effect. This reduction in infectious virus may be due to the previously reported antiviral activity of apple juice, as observed against poliovirus type 1, hepatitis A virus, and coxsackievirus B5 ,51. Whil4 PFU/mL . HoweverMushrooms, however, demonstrated robust reductions in infectious viral titers for both HSV-1 and SARS-CoV-2, reducing viable HSV-1 to < 1 log PFU/mL within 24 h and reducing viable SARS-CoV-2 to <1 log PFU/mL within 1 h. During the method development process, a slightly greater initial reduction in infectious HSV-1 was observed when using the massage method vs. the rinse method (3.6 vs. 4.7 log PFU/mL at 0 h), although this was not observed at later time points. This observation may suggest the release of substance(s) from the mushroom that contributed to this initial reduction in infectious virus. Previous reports using cell culture have shown that extracts from various mushroom types have some level of antiviral activity against HIV, influenza A and B, HSV-1 and HSV-2, and hepatitis B and C viruses . This anWhen infectious virus titer, determined via standard plaque assay, was compared to viral genome copy number, determined via qPCR and RT-qPCR, a trend was observed for poultry and seafood, as well as for spinach, with viral genome copy numbers being ≈1.5–2.5× greater than infectious viral titer for HSV-1 and SARS-CoV-2, respectively. While this trend of genome copy number and viable virus titer was observed for these food groups, it is uncertain how translatable this trend would be to other food types, other time points, or other storage conditions, and it should therefore not be interpreted as a predictive relationship. This trend was not observed for either virus when mushrooms or fruit were inoculated. The rapid decline in viable SARS-CoV-2 from 3.1 log PFU/mL at 0 h to undetectable by 24 h, with continued detection of >5 log genome copies/mL viral RNA, highlights the limitations of PCR-based testing if the goal of testing is to determine whether a food item is contaminated with infectious virus. It is possible to detect ≈5 log genome copies/mL in complete absence of infectious SARS-CoV-2. The fact that this pattern was also reproducible with HSV-1-inoculated mushrooms  lends further support to this observation. These observations suggest that no standard predictive relationship exists between food type and retention of infectious virus that can be broadly applied across food groups/items. Other findings also support the idea that that norovirus, hepatitis A virus, and human coronavirus 229E that are not infectious could still present intact RNA on foods, resulting in positive RT-PCR results ,56,57. TWe acknowledge several limitations of our studies. First, we tested survival of SARS-CoV-2 on only six different foods. Our focus was on survival of the virus on a broad range of food types to gain an initial understanding of the types of foods that may pose a public health risk. Future studies will more comprehensively address common foods, particularly those consumed raw or purchased ready to eat. Second, we only tested survival through 24 h. Considering high titers of infectious virus remained on the chicken and seafood at 24 h, understanding precisely how long the virus remains viable on foods is necessary. Although the foods with the highest recovery, chicken and seafood, would likely be cooked and consumed within a few days of storage at 4 °C, a variety of foods are held at refrigeration temperatures for longer periods of time. Therefore, additional studies of SARS-CoV-2 viability on relevant foods held at refrigeration temperatures for longer periods of time are needed. Finally, it is important to note that interpretation of these results requires caution. While these results answer our original research question of whether viable HSV-1 and SARS-CoV-2 can be recovered from some types of foods held in cold-chain conditions, it does not answer questions about the infection potential of virus-contaminated foods to humans. As an enveloped virus, which is less stable in the environment than non-enveloped enteric viruses, cooking temperatures inactivate SARS-CoV-2, so properly cooked foods should be safe for eating . These rOverall, viable HSV-1 and SARS-CoV-2 were recovered at comparable levels from poultry and seafood held in cold-chain conditions for up to 24 h. The relative stability of each virus on these food types may have been in part due to the moisture or protein contents of the foods. Recovery of viable HSV-1 and SARS-CoV-2 diverged when applied to produce, however, with apples displaying a significant antiviral effect for HSV-1 but not for SARS-CoV-2. Mushrooms exerted a profound antiviral effect, reducing HSV-1 infectious virus to less than 10 infectious virus particles over 24 h and ablating SARS-CoV-2 to undetectable levels within 1 h. Thus, intrinsic characteristics or components of some types of produce demonstrate antiviral activity, which varies by food type and virus. Our studies suggest that HSV-1 can serve as a reasonable surrogate for assessing survival of SARS-CoV-2 on some foods, but not others. Furthermore, we confirm that PCR-based assays for presence of virus on foods may be used as a screening tool but confirmatory studies must be performed to assess viable infectious virus, as no predictive relationship exists between genome copy number and infectious virus particles on various types of food. Our findings highlight the importance of safe food handling and storage practices, environmental monitoring, and the consistent use of personal protective equipment in poultry and seafood processing facilities as well as in grocery stores, in order to prevent accidental contamination of foods. Although it is not clear which factors within various foods dictate how long infectious virus remains viable, future studies will be expanded to include additional food types and broader storage conditions to determine what food types under what storage conditions allow persistence of SARS-CoV-2 on foods.


The SARS-CoV-2 virus has plagued the planet and caused unfathomable disruptions in every facet of our lives . The undA plethora of hurdles however are still in question. Firstly, the re-opening of enterprise and a country has to be done in a safe manner. To overcome this hurdle the development of multiple vaccinations to this novel virus has been streamlined .The greatest matter in question however, is the efficacy of these vaccines and whether they will be the answer to allow a safe re-ignition of global enterprise as well as countries as multiple cases of reinfections have been recorded, namely reinfections post recovery from the disease ,5.The first case of reinfection by the SARS-CoV-2 virus was recorded 4.5 months post the initial infection which occurred in March of 2020. The patient being a 33-year-old man from Hong Kong who after undergoing genome sequencing was found to have been infected by two different strains with different genome sequences. It was discovered that a total of 24 nucleotides differences existed between the first and second viral infection. In addition to these differences a truncation of the ORF8 protein was noted in the initial infection which was not present in the latter infection. The above findings suggesting that the acquired immunity gained from the wild type may not be viable and effective against various strains of the virus .A study conducted by Laith J and team, tracked 43044 SARS-CoV-2 positive patients for a median of 16.3 weeks. It was found that the reinfection rate amounted to 0.66/10 000 persons. The natural immunity to these reinfections was calculated to have an efficacy of greater than 90%. In addition to proving that reinfections do occur the study concluded that reinfections were less severe than primary infections and none of the reinfections amounted to death. As the pandemic has spread across the planet more data has become available and thus has allowed for far more accurate projections and has allowed for a far greater understanding of the behaviour of the virus. It is thus evident that the risk of reinfection with COVID-19 is quantified to a much smaller degree by Laith J and team as opposed to Parry Jane [A startling series of events is playing out in Brazil, a study of blood donors indicated that 76% of the population had contracted the virus by the month of October 2020. This figure theoretically crosses the lower limit for the induction of herd immunity which should start yielding results at 67% (of a population being infected). It is thus evident by virtue of the fact that there was an outbreak and rampant increase in cases in early 2021 that the immunity conferred to the Brazilians was not protective in nature and did not prevent reinfections. This is of an international health concern as the P.1 variant is able to re-infect individuals, thus posing a risk to all patients both uninfected and convalescent [Sheila F. Lumley and team conducted testing of anti-spike IgG on 12541 healthcare workers and subsequently quantified the relative levels of IgG to that of reinfections. 11364 of the workers tested negative whereas 1265 were positive, 88 of whom in which seroconversion had occurred. A base level of anti-spike IgG was determined in each of the individuals. It was found that the presence of anti-spike/anti-nucleocapsid IgG antibodies correlated with a pronounced reduced risk of a reinfection with SARS-CoV-2 within a period of 6 months. It was additionally evident that healthcare workers with a higher titre of the antibodies displayed milder symptoms or were asymptomatic. It is thus evident that natural antibodies produced against the virus do help combat both reinfection as well as mediate the symptoms of the virus. This finding is poignant and of the upmost value as it acts as a positive yardstick and indicator for the development and efficacy of vaccines against SARS-CoV-2. It must be noted however, that the true duration and extent to which natural acquired immunity offers protection is not well understood and only time and further study will provide the necessary data to draw a sound scientifically based conclusion [It must be noted that this phenomenon of reinfection does not occur exclusively in the SARS-CoV-2 group, but reinfections are a well-established trait in various subsets of Coronaviridae. The Coronaviruses 229E, HKU1, OC43, as well as NL63 cause seasonal reinfections due to the evanescent nature of the established host immunity. It is this evanescence and short lived nature of the host immunity that may be implicated in the reinfections occurring with the SARS-CoV-2 virus [A study by Mohammad Asim and team on using the metabolomic approach to both combat and better understand the virus has based the likelihood of reinfection on an individual basis, namely if an individual has some degree of metabolomic dysfunction it is evident that the particular individual in question will have a greater likelihood of reinfection. Based on this model the quantification of the likelihood of reinfections can be better understood and thus curtailed via the correction of micro imbalances within an individual’s physiology .As in the first recorded case of reinfection, the viral genomes and subsequently the viral proteins of the two infections were markedly different. Mutations among viruses are common and often are detrimental to humans and make the treatment thereof complex and difficult, as in the case of (HIV) human immunodeficiency virus where rapid mutations occur frequently. The influenza virus periodically undergoes antigenic drift and shift, in antigenic drift minor mutations and alterations occurs which allow the natural immunity to be effective against the new variant of the virus, however when antigenic shift occurs it causes the natural acquired immunity to no longer be viable and effective and thus leads to an epidemic. A similar picture could be occurring with COVID where antigenic shift is occurring more rapidly and thus the already acquired natural immunity is no longer effective against the new variant, thus creating the breeding ground for re-infections. It is however vital for extensive sampling in reinfection cases so as for the comparison of the viral genome between the initial and the secondary infection. This data being used to determine whether the reinfection was caused via failure of the acquired natural immunity against the virus. It will also establish whether the reinfection is due to an entire new mutant, where existing immunity is noneffective and will not suffice in mounting an adequate immune response ,12,13,14Immunization has been the gold standard method to both eradicate and diminish the impact caused by the SARS-CoV-2 pandemic. The question and concern however is vaccine efficacy and whether the vaccines that have been synthesized and that are available on the market will be effective against the new mutations. The various mutations of the virus will dictate reinfection rates as well as immunization efficacy. The B.1.351 variant of SARS-CoV-2 AKA “the South African strain” has caused great concern for the international community as a whole and poses a threat to other nations already undergoing mass scale immunization as it has been found that the B.1.351 variant is more resistant. Vaccines such as Oxford AstraZeneca have been labelled as to have “limited efficacy” against this strain and thus has caused the South Africans to opt for alternate vaccines such as Pfizer vaccine as well as Johnson and Johnson. It must be noted however that the Oxford AstraZeneca is not totally ineffective against the strain, as in a small trial of 2026 people in South Africa it was noted that no patient died nor was hospitalized. The pharmaceutical company is well underway in the process of adapting the vaccine to be effective against the new strain ,16, 17.After the initiation of the immunization with both the Moderna mRNA-1273 vaccine and the Pfizer BNT162b2 vaccine, cases of infection with COVID-19 have been reported. Vaccinated healthcare workers in America underwent periodic testing via PCR. A large cohort of data was collected between December 2020 and the beginning of February 2021. A total of 36 659 individuals received the initial dose and 28 184 completed the immunization. 379 individuals tested positive for COVID within the first fortnight after the initial dose. 37 positive cases were recorded after the second dose; 22 of which were registered within the first week after completing the immunization. The decrease of infections after the second dose of the vaccine is promising, however it is clearly evident that infections in an immunized individual is not only a possibility but is occurring and thus the adherence to protocols and minimal exposure should be exercised around the immunization period until a full antibody response has been mounted. It is also evident that new vaccines will have to be synthesized in order to target the numerous variants [The likelihood of underreporting of actual infections and subsequent reinfections critically disrupts the dataset and hinders the generation of accurate projections. Due to the novel nature of this virus all reliable scientific evidence has to undergo peer review, this specific and verifiable process requires reputable evidence such as PCR test results as well as genome sequencing etc. The innate challenges that this yields is that many individuals who were infected in the initial wave of the virus were never tested via PCR. This naturally creates a dearth within the dataset and therefore it can be suspected that the current figures are only a fractional representation of the true global dataset .It is evident that reinfections of the SARS-CoV-2 virus can be attributed to two major benefactors. The first being a decrease in production or failure of natural acquired immunity thus leading to the reinfection by the same strain of the virus rendering the person ill and or asymptomatic. The second benefactor is that of mutations. Mutations of the virus will predispose to reinfections as the already established naturally acquired immunity is not beneficial or useful to combat a mutated strain. It is thus poignant for cases of reinfection to be investigated thoroughly as to determine whether the reinfection was caused due to an innate lack of immunity or due to a new strain where no prior immunity existed. It must however be noted that the cases of reinfections are a rather rare event and even if a reinfection does occur, the case if usually mild and or asymptomatic. The impact of reinfection on immunization at this point of time is difficult to quantify however, initial reports do show positive signs even in cases where the vaccine is not fully effective against a mutated strain.


Salmonellaenterica serovar Infantis (S. Infantis) cochleates protected by encapsulation technology as a potential vaccine and to determine its safety in pullets. Cochleates were encapsulated by two technologies, spray drying and ionotropic gelation at different concentrations (0–15% v/v), and were characterized by physicochemical properties, protein content and Fourier Transform Infrared Spectroscopy (FTIR). The cochleates were white liquid suspensions with tubular shapes and a protein content of 1.0–2.1 mg/mL. After encapsulation by spray drying, microparticles ranged in size from 10.4–16.9 µm, were spherical in shape, and the protein content was 0.7–1.8 mg/g. After encapsulation by ionotropic gelation, beads ranged in size from 1620–1950 µm and were spherical in shape with a protein content of 1.0–2.5 mg/g. FTIR analysis indicated that both encapsulation processes were efficient. The cochleates encapsulated by ionotropic gelation were then tested for safety in pullets. No ill effect on the health of animals was observed upon physical or postmortem examination. In conclusion, this study was the first step in developing a potential oral S. Infantis vaccine safe for poultry using a novel cochleate encapsulation technology. Future studies are needed to determine the effectiveness of the vaccine.The aim of this study was to develop and characterize  Salmonella spp. causing the foodborne disease salmonellosis in human beings. A significant increase in Salmonella has been observed in many countries over the past years, leading to international restrictions on imports and exports of chicken meat [Salmonella is devastating to public health and has a negative economic impact on the poultry industry. In Europe, Salmonella enterica serovar Infantis (S. Infantis) is the most frequently reported serovar from chicken meat (36.5%) and broilers (56.7%) [S. Infantis is one of the most commonly isolated serotypes from sick humans and poultry meat products [Chicken meat is the most widely consumed meat in the world, constituting 30% of global meat production . Poultryken meat . Salmoneproducts .Salmonella infection, vaccination of poultry has become mandatory [Salmonella control vaccines are parenteral [Salmonella serotypes have been developed that use live or attenuated bacteria instead. The main disadvantage is the possibility of a mutation that leads to increased severity of disease or side effects [Since poultry products were identified as the major source of andatory ,6. Most renteral ,8; howevrenteral ,6,7. Ora effects . For thi effects . S. Infantis contains several negatively charged molecules, such as phospholipids, proteins and lipopolysaccharides (which can be used as a source of negatively charged components), to induce calcium-cochleate formation, as has been studied with other Gram-negative bacteria [An innovative strategy in the development of oral vaccines has been the cochleate system. Cochleates have been used to protect and deliver several bacterial compounds such as membranes, proteins and DNA as vaccines by the oral route ,12. Cochbacteria ,12. A disadvantage of cochleates is that they can very quickly release their contents at small intestine level because of their lipidic nature, which leads them to be degraded by enzymes and bile salts ,14. To iS. Infantis cochleates. We hypothesize that it is possible to use S. Infantis membranes to develop cochleates and then encapsulate them by two different methodologies, generating safe vaccine formulations for poultry. The aim of this study was to develop and characterize S. Infantis cochleates protected by encapsulation technology as a potential vaccine and to determine its safety in pullets.Currently, there is no evidence in the literature about the development of oral vaccines from S. Infantis strains were obtained by microbiological isolation from cloacal swabs taken from chickens and layer hens belonging to commercial farms from the Valparaiso and Libertador General Bernardo O'Higgins regions, which are the major poultry production areas in Chile, using Cary Blair medium swabs. The torula was placed in a sterile test tube with 5 mL of Phosphate Buffered (PBS)  with novobiocin (20 ug/mL)  and incubated at 37 °C for 18 to 24 h. invA gene [Salmonella spp. isolates were sent to the Chilean Institute of Public Health for serotyping using the Kauffman—White classification scheme. After incubation time 100 μL from each tube was spread on modified semisolid Rappaport Vassiliadis agar, (MSRV semisolid agar)  smented with novobiocin 20 μg/mL) and incubated for 24 h at 41.5 ºC. Of the samples that presented suspicious growth, a roaster sample was taken and sowed by exhaustion in ; this sample was incubated at 37 ºC for 24 h. Samples that showed black or translucent colonies were isolated by seeding on XLD and McConkey agar, . Once the colonies were isolated, a Polymerase Chain Reaction (PCR) was performed to confirm the genus by amplification of the  μg/mL anS. Infantis in a bioreactor  with automatically controlled conditions  in order to obtain the highest yield of biomass. Th bacteria were centrifuged to obtain the bacterial sediment and washed with sterile Phosphate Buffered Water. The bacterial suspension was subjected to cold sonication pulses at 400 wats for 30 s using an Ultrasonic Processor UP400S sonicator . A bacterial sediment was prepared from g for 5 min and 15 °C. The supernatant was adjusted to a concentration of 5 mg soluble protein/mL by bicinchoninic acid method (BCA) .For the purification of the bacterial membrane, bacterial sediment was suspended in a buffer containing 30 mmol/L of tris(hydroxymethyl)aminomethane (TRIS) at a rate of 1:10 volume of bacterial sediment per TRIS. This suspension was frozen at −80 °C for 2 h and defrosted by continuous sonication at 400 watts and 24 kHz . The defrosted suspension was added to 1.5% (w/v) sodium deoxycholate (DOC) and was stirred at 150 rpm at 20 °C for 72 h and centrifuged at 250× 2 at pH 10). Once the drops were administered, the dispersion was stirred for 3 h, and then a wash solution was added to remove the DOC (containing 0.121% w/v Tris and 0.087% w/v NaCl at pH 10). The solution was centrifuged at 2900× g for 40 min at 4 °C; the supernatant was discarded and the sediment reserved. The sediment was then re-suspended in the washing solution, and the soluble protein concentration was determined by BCA method.Cochleates were characterized by transmission electron microscopy (TEM). To obtain images by TEM, an aliquot of 10 µL of cochleate was taken and deposited on grids  and stained with 1% v/v aqueous uranyl acetate for 1 minute. Cochleates were observed in a transmission electron microscope  operated at 80 kV. The photographs were processed through Megaview G2 Software .Cochleates were encapsulated by two different technologies: spray drying and ionotropic gelation. For spray drying, an encapsulating solution based on 0.5% w/v sodium alginate  plus 20% w/v maltodextrin  in distilled water was prepared. Cochleates were added to the encapsulating solution at 0% , 5% (MP-5%), 10% (MP-10%) and 15% (MP-15%) v/v by magnetic agitation. The blends were then immediately fed to a B-290 Mini Spray Dryer . The inlet and outlet air temperatures were set at 140 ± 5 °C and 95 ± 5 °C, respectively. The air flow, rate of feeding and atomization pressure were 500 L/h, 8 mL/min and 20 psi, respectively.For the ionotropic gelation method, sodium alginate  was used as the encapsulating material. A sodium alginate solution (2% w/v in distilled water) was prepared and different concentrations at 0% , 5% (B-5%), 10% (B-10%) and 15% (B-15%) v/v of cochleates were added to this solution by magnetic agitation. The blends were then immediately fed to a B-390 Encapsulator  with particle diameter set to 1000 μm. Beads were formed by ionotropic gelation using a 5% w/v cross-linking solution of calcium chloride and then deposited in plastic boxes to be dried until reaching a constant weight at a temperature of 40 °C/6 h. Once dried, these beads were removed from their boxes and stored at room temperature.L*: lightness, a*: redness/greenness and b*: yellowness/blueness) with a Konica Minolta CR-300  colorimeter.Microparticles and beads were photographed using a Sony DSC–HX1 digital camera . Color was measured according to the Hunter Lab color scale . Briefly, 0.5 g of the microparticles were placed in the powder jet dry feeder accessory with a pressure of 0.30 mPa. To prepare the bead samples, 1 g of beads were suspended in 10 mL of Milli-Q water and placed in a sample cell under magnetic stirring. During the measurement, a 650 nm laser diode passed through the particle suspension; the scattered light was detected and collected by a silicon photo diode detector. All the samples were analyzed in triplicate at 25 °C.The microparticles and beads were observed by Scanning Electron Microscopy (SEM). The dried samples were mounted on a cylindrical aluminum stub using double–sided tape to sputter coat them with gold (twice) at 20 kV in an argon atmosphere by a PELCO 91,000 sputter coater unit . Coated samples were then examined by an SEM  equipped with an energy dispersive X-ray spectroscopy (EDS) using an accelerating voltage of 25 kV.2SO4 and 2.0 mL of CuSO4/H2SO4. The digestion procedure begins at a temperature of 200 °C which then reaches 400 °C. A sample of 2.0 mL of water was also digested simultaneously as the blank. The samples and blank digests were then alkalinized with NaOH and steam distilled. Released ammonia was absorbed in solutions of 2% boric acid and nitrogen content was determined by titration with 10 mM HCl after blank correction.The protein content of microparticles and beads was determined using a modification of the micro-Kjeldahl method . Briefly, aliquots of 1 g of sample were digested with 500 mg K−1 yielded each spectrum.FTIR analyses were performed on cochleates, microparticles and beads. An ATR/FTIR interspect 200-X spectrometer  provided FTIR spectra for each sample. Spectroscopic measurements were performed directly using the PIKE Miracle TM accessory in a Ge single reflection crystal plate. A concave tip was used for all FTIR spectra. An average of 20 scans over the spectral range of 600 to 4000 cmAll protocols were approved by the Bioethics Committee at the University of Chile (Certificate of Bioethics 2018-18186-VET-UCH). In order to accomplish the committee requirements, all personnel involved in poultry management demonstrated appropriate skills through professional/technical degrees and previous experience. Fourteen one-day-old female Rhode Island pullets received from a commercial hatchery were equally divided into two groups; they were treated with cochleates encapsulated by ionotropic gelation at 0% (B-0% as control group) and 15% (B-15% as treatment group). The birds were kept under strict biosecurity conditions at the Avian Pathology Laboratory facilities for six weeks in two community cages inside two separated isolated rooms; then, they were vaccinated with cochleates encapsulated by ionotropic gelation. Commercial pullet growth diet, formulated according to nutritional requirements proposed by the National Research Council , and watThe beads measured 1950 ± 28 µm, and each dose was prepared in individual Eorf tubes. The pullets’ beaks were carefully opened, and the beads were given; between five and six beads were administered reach the calculated dose. Then, 0.5 mL of PBS was given with a tuberculin syringe to assure bead consumption. Over 5 days, the treatment group was vaccinated with 65 µg cochleates to evaluate the safety of the formulation. The control group was treated with empty alginate beads. During the experiment, all birds were examined daily by the same person for clinical signs according to the parameters proposed by Morton and Griffiths with some modifications ; the temAt the end of the safety study, the birds were euthanized by cervical luxation and immediately necropsied to look for pathological changes. Any unspecific signs in the gastrointestinal tract that could be caused by the vaccine formulation, such as petechial, hyperemia, hemorrhages, inflammation, ulcers and necrosis, were searched for. The birds were examined from tongue to rectum, and other organs  were inspected. p < 0.05). All statistical analyses were run with Statistix 8 software . Data is presented as average ± standard deviation. The safety trial provided group scores, and thus, the data is analyzed as descriptive data. After verifying the normality of the data with a Shapiro–Wilk test, the characteristics of beads and microparticles were analyzed by ANOVA and a Tukey test  and ionotropic gelation (beads) methods, different concentrations of cochleates and encapsulant material were tested. For the microparticles, we used 0–15% v/v cochleates, 0.5% w/v alginate and 20% w/v maltodextrin. For the beads, we used 0–15% v/v cochleates and 2% w/v alginate. The surface morphological characterization by scanning electron microscopy (SEM) and elemental composition by energy-dispersive X-ray spectroscopy (EDS) of selected microparticles (M-5% and MP-15%) and beads (B-5% and B-15%) are presented in Electronic micrographs showed well-formed beads with spheroidal shapes and heterogeneous surfaces except for B-15%, where elongated beads with sharp edges were observed B. EDS anThe protein content in the microparticles and beads is presented in −1, corresponding to O-H bonds. The second most important band corresponds to N-H bending bonds (1640 cm−1). For the microparticles an important band characteristic of O-H stretching bonds was observed (3319 cm−1). Then, less important bands were observed at 2940 cm−1 and 2359 cm−1, corresponding to C-H stretching bonds. Subsequently, bands characteristic of C-O stretching bonds were observed (1371 cm−1 and at 1010 cm−1). All of these bands are related to the alginate and maltodextrin molecular structures. −1). Then, bands of the asymmetric and symmetric bond of the carboxylate group C=O of alginate (1636 cm−1 and 1428 cm−1) were observed. Finally, a band representing the C-O stretching bonds (1009 cm−1) was observed.The FTIR of the beads has a characteristic broad band of O-H stretching bonds  present in bacterial membranes through interaction with positively charged multivalent ions (Ca+2), minimizing their interaction with water and promoting the formation of tubular structures [+2) interact ionotropically with the negatively charged membranes, acting as a bonding agent that generates the rolling of the membranes [The milky appearance of ructures ,14. The ructures . It has embranes . This prembranes . In this study we encapsulated the cochleates to provide them greater stability for their passage through the gastrointestinal tract . The tecA change in the shape of the beads was observed as the concentration of cochleates increased B, varyinThe shape of the microparticles was observed in detail by scanning electron microscopy and was considered to be typical morphology for maltodextrin/alginate microparticles produced by spray drying. Invaginations resulting in folds and protrusions occur during the drying process ,32.Due to the encapsulation method, the sizes of the beads are significantly bigger than the microparticles. As expected, we observed a clear tendency of increased sizes in both systems, beads and microparticles, with an increase in cochleate concentration. Unexpectedly, MP-15% produced smaller microparticles than lower concentrations (MP-5% and MP-10%). Interestingly, other authors have reported that the final particle size, using spray drying technology, is affected by the concentration of the feed solution “but in a non-linear way” . For insThe success of the encapsulation process is demonstrated by the protein content, FTIR and EDS analysis. Protein content is a very important estimate because it is related to the amount of antigens contained in the formulations. As expected, protein concentration was higher in formulations with higher cochleate loads. Interestingly, the beads were able to retain more protein than the microparticles. This could be explained by the fact that ionotropic gelation allows a high retention of the encapsulated compound. This process takes place when a bonding zone is produced between an alginate G-block that is electrostatically bonded to another G-block through the calcium ions, forming a structure known as the "egg box" model, which protects the encapsulated compounds with high efficiency ,36.+2 [The FTIR analysis shows the different interactions that occurred between the cochleates and the encapsulant materials to form the microparticles and beads. In the FTIR analysis of the beads, the main interaction was O-H bonds. These interactions may be the results of hydration of the hydrophilic groups of the alginate, electrostatic interactions between alginate and some groups of the cochleates (such as amino acids) and/or the formation of bonds with adjacent hydroxyl groups . The FTI+2 ,38. FTIR+2 . The preNo nitrogen atoms, typical of protein and indicative of cochleates, were observed by EDS analysis of the surface of the microparticles (MP-15%) and beads (B-15%), confirming an efficient encapsulation of the cochleates by both technologies.in vivo safety trials were carried out. In this study, we tested an oral vaccine prototype with antigens obtained from bacterial membranes, which is different from most effective oral vaccines that are made with live or attenuated agents [S. Infantis where infection is mainly oral, the development of a vaccine given orally is highly desirable.After the development of novel vaccine prototypes, d agents . The chod agents , reducinS. Infantis vaccine prototype is harmless to birds: no physical changes or pathological alterations were found in any segment of the digestive tract or any organ at the necropsy. The next step in this research will be to measure the immune response in the birds and conduct a challenge study against S. Infantis to determine the effectiveness of the vaccine against this pathogen.The vaccine developed by ionotropic gelation, which has a higher retention of the antigens, was tested orally at the maximum concentration in pullets. The safety trial results showed that the novel S. Infantis and then efficiently encapsulated the cochleates using two different technologies, spray drying and ionotropic gelation, to develop an oral vaccine prototype. Encapsulation by ionotropic gelation was more efficient, obtaining millimeter-sized beads (1620–1950 µm) with a protein content between 1.0–2.5 mg/g (reflecting the high content of antigens). Beads containing 15% v/v cochleates were delivered orally to birds for a safety test. During the safety study, no physical or health alterations were observed in the birds. In the post-mortem examination, no pathological alterations or lesions were observed in the gastrointestinal tract or in the organs analyzed. In this study, we took the first step toward the development of a novel prototype oral vaccine against S. Infantis. It is essential to analyze the immune response of the birds and the efficiency of the vaccine in future studies.In this study, we derived cochleates from the bacterial membranes of


The genotype S (G57) H9N2 virus, which first emerged in 2007 with the substitution of the G1-like PB2 gene for F98-like ones, has become the predominant genotype in the past 10 years. However, whether this substitution plays a role in the fitness of genotype S H9N2 viruses remains unknown. Comparison of the PB2 genes of F98-like and G1-like viruses revealed a close homology in amino acid sequences but great variations at nucleotide levels. We then determined if the packaging region, a unique sequence in each segment utilized for the assembly of the vRNA into virions, played a role in the fitness of the S genotype. The chimeric H9N2 virus with PB2 segments of the G1-like packaging regions significantly increased viral protein levels and polymerase activity. Substituting the packaging regions in the two terminals of F98-like PB2 with the sequence of G1-like further improved its competitive advantage. Substitution of the packaging regions of F98-like PB2 with those of G1-like sequences increased the infectivity of the chimeric virus in the lungs and brains of chicken at 3 days post infection (dpi) and extended the lengths of virus shedding time. Our study suggests that the packaging regions of the G1-like PB2 gene contribute to improve the survival advantage of the genotype S H9N2 virus in China. H9N2 viruses have evolved into multiple genotypes (A-W) since its first emergence in China in 1994. These genotypes are divided into five lineages including A/chicken/Beijing/1/1994 (BJ94-like), A/duck/Hong Kong/Y280/1997 (Y280-like), A/quail/HongKong/G1/1997 (G1-like), A/duck/HongKong/Y439/1997 (Y439-like), and A/chicken/Shanghai/F/1998 (F98-like) . More thin vitro and in vivo. Our study unveils a previously unrecognized role of the packaging regions in genotype S H9N2 virus evolution in China.The highly selective genome packaging of influenza A viruses relies on complex RNA-RNA or RNA-protein interactions . The speThis study was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the Ministry of Science and Technology of the People’s Republic of China. All animal experiments were approved by the Jiangsu Administrative Committee for Laboratory Animals (Permission number: SYXK-SU-2017-0007), and by the Institutional Biosafety Committee of Yangzhou University and complied with the guidelines of Jiangsu laboratory animal welfare and ethics of Jiangsu Administrative Committee of Laboratory Animals.https://www.gisaid.org/registration/terms-of-use/. The Isolate IDs can be found in The sequences of PB2 of all H9N2 AIVs isolated between 1996 and 2019 were downloaded from the EpiFlu Database (GISAID): SH14 or PB2AH320 plasmid for each experiment. All results were the means ± SD from three independent experiments.A set of plasmids were generated on the pHW2000 backbone, carrying each segment of A/Chicken/Anhui/AH320/2016 (genotype S H9N2), F98-like PB2 gene from A/Chicken/Shanghai/14/2001 (H9N2), and chimeric PB2 segments, respectively. 293T cells were transfected with 300 ng firefly luciferase reporter, 30 ng of the control pRL-TK vector, which expresses the Renilla luciferase, and 300 ng each of PB1, PA, NP and PB2 plasmids shown in AH320 backbone and 5′-FAM-CGGTAGCAGGTGGAACAA-MGB-3′ for the PB2 genes mutated from PB2SH14 backbone. The PB2 RNA levels shown were as log10 of copy numbers.The protein expression and RNA synthesis were quantified by Western blot and real-time RT-PCR in a four-plasmid expression system or infection conditions, respectively. (i) Triplicate wells of 293T cells were transfected with four plasmids, including PB1, PA, NP, and the chimeric PB2 segment showed in SH14 gene were coinfected with P-WT-AH320 virus, and the viruses containing the mutants from PB2AH320 gene were coinfected with P-AH320-PB2SH14 virus. As described above, the medium was replaced with opti-MEM containing 50 mmol NH4CL to ensure a single cycle of viral replication. At 48 hpi, the infected cells and supernatants were collected for RNA extraction, and the RNA levels were detected with the quantitative real-time RT-PCR as described above.Triplicate wells of MDCK cells were coinfected 1 MOI for each virus. The viruses containing the mutants from PB2Every experiment was run in triplicate wells and repeated at least twice.6 50% egg infective doses (EID50) of each plaque purified mutant viruses. Three chickens were euthanized by CO2 asphyxiation for each group at 3 and 5 dpi, and lungs and brains were collected for virus titration. At 1, 3, 5, and 7 dpi, oropharyngeal and cloacal swabs from five chickens of each group were taken for the detection of viral shedding.Eight 5 weeks old SPF while leghorn chickens were inoculated intraocularlly with 10P < 0.05.Statistical analyses were conducted by using SAS software, version 9.2  (which spread prior to 2006) and G1-like that spread since 2007 (All available PB2 sequences of H9N2 AIV during 1996–2019 were downloaded from the EpiFlu Database (GISAID): nce 2007 . The meance 2007 .SH14 (F98-like) and PB2AH320 (G1-like) genes  harboring the mutant PB2 segment mentioned above were successfully rescued in the background of AH320 virus. However, the attempt to generate AH320-PB2sA and AH320-PB2sAs reassortants was not successful. By serial plaque purification, P-AH320-PB2SH14, P-AH320-PB2aS, P-AH320-PB2Sa, P-AH320-PB2aSa, P-AH320-PB2mutSH, P-WT-AH320, P-AH320-PB2As, and P-AH320-PB2mutAH viruses were obtained from the rescued viruses.The coding and non-coding regions in the two ends of the PB2 gene are involved in genome incorporation into the virion of WSN virus . To invee) genes . There a PB2SH14 , and a P320 gene . Eight rSH14 and PB2AH320 genes .To determine the relative contribution of PB2 vRNA sequences to the prevalence of genotype S H9N2 viruses, we constructed chimeric PB2 segments by exchanging the packaging regions of PB220 genes . We then PB2SH14 . Howeverynthesis , PB2aS.AH320 were reciprocally substituted by the F98-like sequences  had little impact on RNA synthesis, either.The G1-like packaging regions of PB2equences . Except ectively . The lev320 gene . Failure process . The vRN process . The subA levels . ConsistThe observation that mRNA synthesis level of several mutants was inconsistent with their protein expression may be related to the post-transcriptional processing, regulation and translation of PB2 gene .50 values, respectively . Substitution of 3′-terminal packaging region of the PB2AH320 gene reduced the proportion of infectious virus particles to a level similarly to that of AH320-PB2SH14 virus (As). While amino acid substitutions at 44 and 676 positions in PB2SH14 increased the production of infectious virus  increased virus replication to the level comparable to P-WT-AH320 virus  further increased this ratio in the co-infected cells and conditioned media  reduced PB2As/PB2SH14 ratio in the co-infected cells and conditioned media  at 5 dpi : The animal study was reviewed and approved by the Jiangsu Administrative Committee for Laboratory Animals (Permission number: SYXK-SU-2017-0007), the Institutional Biosafety Committee of Yangzhou University, and Jiangsu laboratory animal welfare and ethics of Jiangsu Administrative Committee of Laboratory Animals.XLL and MG designed this study. XLL, YZ, SQ, RG, ZG, and JM performed the experiments. XLL, MG, XX, XW, JH, SH, XWL, SC, DP, XJ, and XFL drafted and revised the manuscript. All authors read and approved the final manuscript.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.


Haemophilia bears substantial humanistic and economic burden on children and their caregivers. Characterising the differential impact of severe versus moderate paediatric haemophilia is important for clinical and health policy decisions. We analysed health-related quality of life (HRQoL), annual direct medical (excluding factor treatment costs), non-medical and societal costs among children and adolescents with moderate and severe haemophilia A or B without inhibitors from the European CHESS-PAEDs study. Information was reported by physicians and caregivers; patients aged ≥ 8 years self-reported their HRQoL. Descriptive statistics summarised demographic and clinical characteristics, costs, and HRQoL scores (EQ-5D-Y). Regression models estimated differences in HRQoL and costs for moderate versus severe haemophilia adjusting for age, body mass index z-score, country, number of comorbidities, and weight-adjusted annual clotting factor consumption.p < 0.001; N = 794), while societal costs were more than half of the predicted costs for children with severe haemophilia . Mean predicted HRQoL scores were 0.74 and 0.69 for moderate and severe disease, respectively .The analytic sample comprised 794 patients with a mean age of 10.5 years; most had haemophilia A (79%) and 58% had severe haemophilia. Mean predicted direct medical costs in moderate patients were two-thirds of the predicted costs for severe disease (€3065 vs. €2047; Children with haemophilia and their caregivers displayed a significant economic and humanistic burden. While severe patients showed the highest direct medical and societal costs, and worse HRQoL, the burden of moderate haemophilia on its own was substantial and far from negligible.The online version contains supplementary material available at 10.1186/s13023-022-02301-0. Haemophilia is an inherited lifelong bleeding disorder characterised by inadequate clotting factor VIII in the case of haemophilia A or factor IX for haemophilia B, affecting primarily males. Haemophilia A has a substantially higher prevalence than haemophilia B—approximately 17.1 versus 3.8 cases per 100,000 males, respectively . Among pPaediatric haemophilia confers a burden not only on the children and adolescents, but also on their caregivers, including both psychosocial and financial challenges . The carUnderstanding the economic impact of paediatric haemophilia requires a broader view of the care to account for haemophilia-related direct medical costs from healthcare resource use, indirect costs such as for medical equipment and professional caregiving help, and societal costs such as diminished work productivity , 9. We eWe conducted an analysis of the ‘Cost of Haemophilia across Europe: a Socioeconomic Survey in a Paediatric Population’ study (CHESS PAEDs) to quantify the differential direct medical and societal costs (including caregiver burden), and patient humanistic burden (HRQoL), of moderate in relation to severe disease paediatric patients with haemophilia A or B without inhibitors.n = 146) and observations identified as outliers according to Cook’s distance  growth chart ), countr direct mRegression models were developed to estimate cost outcomes for patients with moderate versus severe haemophilia using a generalised linear model (GLM) with Gamma distribution and log-link function. The AME were computed to ascertain the effect of each covariate on the outcome of interest. Four models were tested for each analytic sample, controlling for (1) severity only; (2) severity, age, BMI z-scores, and country; (3) model 2 plus number of comorbidities; and (4) model 3 plus total weight-adjusted factor consumption. Total factor consumption was used as a proxy for treatment strategy and to account for access to clotting factor treatment. Ultimately, the categorical value for type of haemophilia treatment  was discarded due to multicollinearity issues (> 80% of patients with severe haemophilia were receiving a prophylaxis regimen). Clinical outcomes such as bleeding events and problem joints were not included in the regression models since there is a known positive correlation between such outcomes and haemophilia severity level. Model 4 demonstrated the best goodness of fit (lowest Akaike and Bayesian information criteria) and was used for all analyses.Self-Reported Only” sample included only children who provided all EQ-5D-Y responses for themselves (aged 8–17 years). The “All Children” sample comprised all children including those whose caregivers provided all or some of the responses as a proxy for the child.Differences in EQ-5D-Y scores across severity groups were estimated using a Tobit model bounded between −0.594 and 1.0; AME were used to show the effect of the covariate on the bounded outcome variable. The same four models described above were tested and model 4 demonstrated the best goodness of fit. We conducted the HRQoL analyses in two samples based on the completion mode of the EQ-5D-Y instrument. The “p < 0.05) for all analyses. No imputation of missing values was performed, and patients with missing responses were excluded from the analysis. All analyses were performed using STATA® 16 .Statistical significance was determined at the 5% alpha level . Table A3: Cost components used in the CHESS-PAEDs study. Table A4: Physician-reported control variable definitions


Our experimental analysis indicates that the S100P-53 complex formation is successfully disrupted by pentamidine, since S100P shares the same binding site for p53 and pentamidine. In addition, we showed that pentamidine treatment of ZR-75-1 breast cancer cells resulted in reduced proliferation and increased p53 and p21 protein levels, indicating that pentamidine is an effective antagonist that interferes with the S100P-p53 interaction, leading to re-activation of the p53-21 pathway and inhibition of cancer cell proliferation. Collectively, our findings suggest that blocking the association between S100P and p53 by pentamidine will prevent cancer progression and, therefore, provide a new avenue for cancer therapy by targeting the S100P-p53 interaction.S100P, a small calcium-binding protein, associates with the p53 protein with micromolar affinity. It has been hypothesized that the oncogenic function of S100P may involve binding-induced inactivation of p53. We used  Residues were assigned as active and passive based on the NACCESS program output. Active residues had ≥40 percent accessible areas, and the remaining residues were considered passive. The residues of S100P consists of T6, G9, V14, S16, M37, G43, Q46, D50, D52, A53, E73, F74, I75, V76, F77, A79, A80, S83, A84, K91, A92, and G93 while p53-TAD residues consist of T18, F19, S20, D21, W23, K24, L25, L26, L28, and N29. The starting PDB file for S100P was obtained from PDB code: 1J55, and for p5317-29 was derived from the PDB code: 1YCR. The rigid-body energy minimization stage resulted in a 2000 structure, and additional 2000/200/200 models corresponding to the it0/it1/water refinement stages were created. At the end of this process, the best 200 models were clustered as per their interface-ligand root-mean-square deviation (RMSD) values with a cut-off of 5 Å, and all the clusters were given a HADDOCK score. PyMOL software (version 2.3.3) was used to visualize the created complexes through the HADDOCK program -1,3-benzene disulfonate) produces a water-soluble formazan dye upon reduction by mitochondrial dehydrogenase enzymes in viable cells. The released soluble formazan in the medium is correlated to the number of viable cells. The more cells, the more dehydrogenase enzymes available to reduce WST-1 to water-soluble formazan. To perform the WST-1 assay, ZR-75-1 human breast cancer cells were seeded at a concentration of 5 × 103 cells/well on a 96-well plate in a day before experiments. Subsequently, the cultured cells were treated with or without increasing concentrations from 5 to 100 μM of pentamidine for 48 h. Afterwards, 1/10 volume of WST-1 reagent was added to the wells and incubated at 37 °C for another 4 h before harvesting. The plate was agitated by gentle shaking to mix the medium in the wells. The absorbance of the released soluble formazan in the wells were measured at 450 nm by the Synergy 2 microplate reader . The relative cell numbers were determined by the relative absorbance comparing to that from control treatment. The statistical analysis was performed by a 2HPO4, 2 mM KH2PO4). Cell lysate was extracted by sonication in RIPA Buffer . The soluble protein extracts were isolated from the supernatant after centrifugation at 15,000× g for 10 min. Equal amounts of protein extracts were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred onto a polyvinylidene fluoride (PVDF) membrane (Millipore). The membrane was blocked with 5% skim milk at room temperature for 1 h, and then hybridized with a specific primary antibody with gentle agitation at 4 °C overnight. After washing with PBST (PBS containing 0.1% Tween-20), the membrane was incubated with HRP-conjugated secondary antibody at room temperature for 2 h. Subsequently, the tested proteins were visualized by the addition of enhanced chemiluminescence (ECL) re-agent (Millipore).Cells were washed twice with phosphate buffer saline  influences the local environment of the nuclei present at or near the interface region thus showing the changes in cross-peaks located in the proximity of the interface region.The versatile NMR method uses chemical shift perturbation data to identify the interface region between protein-protein interactions . In shor1H-15N chemical shift assignments for the free S100P (BMRB 17866) and p531-73 (BMRB 17760) proteins are available. We mapped the interacting p531-73 region and the S100P residues near the interface region that formed the S100P-p531-73 complex and were derived from the chemical shift perturbations and/or intensity decrease detected on the 1H-15N HSQC spectrum. At a 1:1 ratio of the 15N labeled p531-73 to unlabeled S100P, a total of 26 residues corresponding to p531-73 region  were identified and are shown in The 1-73 to the 15N labeled S100P at a ratio of 1: 1 led to the identification of 21 residues  in S100P that interacted with p531-73 as shown in Conversely, the addition of unlabeled p53d). The cross-peaks under consideration were located in the fast exchange in both 1H and 15N dimensions. The 15N S100P HSQC/NMR spectra titrated with unlabeled p531-73 indicated the cross-peaks that underwent significant alterations. The selected residues consisted of S16, M37, D52, A53, A67, I75, and A79. The mean Kd was found to be 4.86 μM as shown in d) was in the micro-molar range.The data derived from the chemical shift perturbation analysis provided the values of the dissociation constants  is a useful tool for defining the protein–ligand binding interface. The overlapping of free 15N S100P and S100P in a complex with pentamidine spectra . The cross-peaks under consideration were located in the fast exchange in both the 1H and 15N dimensions. The 15N S100P HSQC/NMR spectra titrated with unlabeled pentamidine indicated the cross-peaks undergoing significant alteration when compared with the free S100P protein. The selected residues consisted of D13, Q46, A79, S83, and A84. The mean Kd was found to be 48.74 μM as shown in d) was in the micro-molar range.The data derived from the chemical shift perturbation analysis provided the values of the dissociation constants  were consistent with the major destructive chemical shifts of the residues. The binding area of p5317-29 was placed in the hydrographic cleft of S100P, and the S100P binding area included residues T6, G9, V14, S16, G43, Q46, D50, D52, A53, E73, F74, I75, V76, F77, A79, A80, S83, A84, K91, A92, and G93. The docking results show that the interaction between p5317-29 and S100P is regulated via hydrophobic interactions . The comparison analysis indicated that the breast invasive ductal carcinoma cell line ZR-75-1 expresses the highest level of S100P and, therefore, the probability of pentamidine binding would favor S100P.The ZR-75-1 breast cancer cells were treated with varying amounts of pentamidine  for 48 h, and the relative folds of cell numbers were determined by the WST-1 assay. 1H-15N HSQC/NMR spectroscopy was chosen as it provides the mapping of the protein-protein and the protein-ligand binding interface directly to the surface of the protein structure.Several posttranslational modifications, mainly through acetylation, methylation, and phosphorylation, determine the stability and function of the tumor suppression p53 protein. These alterations also establish the associations between p53 and its binding partners, which in turn affect modifications, stability, and behavior toward p53 . The mos1H-15N HSQC titration, and HADDOCK to evaluate the binding of S100P with the p531-73 and pentamidine. The S100P binding region of the p531-73 overlaps with the binding sites of the pentamidine molecules, which shows that pentamidine could block the interaction of the S100P and p531-73 region. In the second part the concept was proved in ZR-75-1 human breast cancer cells. We have shown that blocking the interaction of S100P and p53 by pentamidine results in the stabilization of p53 at the protein level and induction of its down-stream gene, p21, thereby inhibiting cell proliferation (In the present study, we first concentrated on the structural determinants of specific protein-binding interfaces; thus, for that purpose, we performed NMR feration . CapocciIn conclusion, we demonstrated that pentamidine is an effective agent that can be used to inhibit cancer progression. The findings show that pentamidine could help to treat cancer as an anti-cancer agent or as a targeted therapy along with some other approaches. The pentamidine molecule constitutes the first step of a logical drug design project aimed at identifying the S100P protein inhibitor that would bind to S100P at the p53 binding site. In future work, these lead compounds will be developed into higher-affinity S100P inhibitors that could possibly have useful therapeutic properties.


Direct healthcare costs of a serious ADR were based on the number of reimbursed fluoroquinolone prescriptions from the Social Insurance Institution of Finland’s database. Sensitivity analyses were conducted to address parameter uncertainty. A total of 1 831 537 fluoroquinolone prescriptions were filled between 2008 and 2019 in Finland, with prescription numbers declining 40% in recent years. Serious ADRs associated with fluoroquinolones lead to estimated direct healthcare costs of 501 938 402 €, including 11 405 ADRs and 3,884 deaths between 2008 and 2019. The average mortality risk associated with the use of fluoroquinolones was 0.21%. Severe clostridioides difficile infections were the most frequent, fatal, and costly serious ADRs associated with the use of fluoroquinolones. Although fluoroquinolones continue to be generally well‐tolerated antimicrobials, serious adverse reactions cause long‐term impairment to patients and high healthcare costs. Therefore, the risks and benefits should be weighed carefully in antibiotic prescription policies, as well as with individual patients.The aim of this study was to estimate healthcare costs and mortality associated with serious fluoroquinolone‐related adverse reactions in Finland from 2008 to 2019. Serious adverse reaction types were identified from the Finnish Pharmaceutical Insurance Pool’s pharmaceutical injury claims and the Finnish Medicines Agency’s Adverse Reaction Register. A decision tree model was built to predict costs and mortality associated with serious adverse drug reactions (ADR). Severe  clostridioides difficile infections (SCDI), which alone cause more than 95% of the total direct healthcare costs of the ADRs. Accordingly, from the economical perspective, efforts should be focused on limiting the risk of FQ‐associated CDI.In this present study 1 831 537 FQ prescriptions filled between 2008 and 2019 were estimated to trigger 11 405 ADRs and 3,884 deaths, resulting in a total healthcare cost of 501 938 402 € during the twelve‐year observation period. The most impactful of the FQ‐related ADRs are severe  ADRadverse drug reactionCDIclostridioides difficile infectionDILIdrug induced liver injuryFQfluoroquinoloneICinsurance claimLOSlength of staySCARsevere cutaneous adverse reactionSCDIclostridioides difficile infectionsevere SJSStevens‐Johnson syndromeTENtoxic epidermal necrolysis1clostridioides difficile infections (CDI), cardiovascular toxicity, musculoskeletal, renal, and liver disorders, and reactions involving the central nervous system.Fluoroquinolones (FQs) are a group of antimicrobial drugs, which have activity against gram‐positive and gram‐negative bacteria and are used to treat genitourinary, respiratory, gastrointestinal, skin, and soft tissue infections. FQs have been in wide clinical use for over 30 years. In Finland, the number of FQ prescriptions has steadily remained at 5–6% of all antibiotic prescriptions.clostridioides difficile infections (CDI).Serious adverse reactions are defined as events that either result in death, are life‐threatening, require inpatient hospitalization or prolong current hospitalization, or result in persistent or significant disability or incapacity.While it is important to understand the economic burden of ADRs, research of the costs is challenging since most of them remain unreported.22.1clostridioides difficile infections (SCDI), severe cutaneous adverse reactions (SCAR), acute liver injuries, and aortic ruptures were included as serious ADRs in the model. The overall incidence of FQ‐related serious ADRs is estimated to be between 0.1% and 1%.Serious FQ‐related adverse reaction types were identified from the Finnish Pharmaceutical Insurance Pool’s pharmaceutical injury claims and the Finnish Medicine Agency’s Adverse Reaction Register, which were available from 2002 to 2012, except for aortic ruptures, which were yet to be recognized as FQ‐related ADRs. The probabilities of serious ADRs were estimated based on published literature and are depicted in Table Estimates of direct healthcare costs associated with serious FQ‐related adverse effects in Finland of a 12‐year period from 2008 to 2019 were based on the number of reimbursed FQ prescriptions collected from the Social Insurance Institution of Finland’s database. The numbers of prescriptions were multiplied by the probability of a serious ADR and the estimated cost of a single serious FQ‐related ADR episode. Mortality estimates were calculated by multiplying the estimated risk of mortality due to ADR with the number of reimbursed FQ prescriptions. Since there was no valid data source on the risk of FQ‐related SCDI, our estimates combined an overall risk of FQ‐related CDI based on the study from Despande et al. (0.092)2.2Clostridioides difficile bacteria are members of normal gut microflora that are resistant to numerous antimicrobials and can thus colonize the human gut after antimicrobials have altered the proportions of the microbiota. The toxin expression of the bacteria results in gastrointestinal illness.Clostridioides difficile ribotype 027 clone, CDI has become more prevalent, severe, and more difficult to treat due to resistance to many antimicrobial agents.2.3FQs were first connected to tendon injuries in 19832.4Hypersensitivity to FQs can cause rare severe cutaneous adverse reactions (SCAR), which can be life‐threatening and is often preceded by hypersensitivity to beta‐lactams.2.5FQ‐associated hepatotoxicity ranges from mild transient aminotransferase elevations to hepatitis and jaundice and even to severe and acute liver injuries and hepatic failure. The onset may vary and, similarly to SCAR, possibly occurs via immunological mechanisms.2.6The array of FQ‐associated cardiovascular toxicity has amplified in recent years. QT interval prolongation and torsade de pointes in connection with FQs were described in the 1990s in several publications. Additionally, the systemic use of FQs is now known to increase the risk of aortic aneurysms and dissection,2.7The study was conducted from the perspective of the healthcare provider and addresses direct healthcare costs. Outpatient visits and hospitalizations were valued with the unit costs of non‐psychiatric specialized healthcare in Finland and contained all costing items related to the health service use, including laboratory tests, dispensed drugs, technical aids, and medical imaging costs. These costs did not include healthcare client fees, which equates to approximately 5 percent of healthcare costs.2.8The decision tree is a commonly used tool for decision analysis in healthcare decision‐making and economic evaluations of health technologies.FQs are not distinguished from one another in the model as their ADR profile is assumed to be similarAll patients were hospitalized due to serious ADRs and required additional health servicesAbsolute risks were drawn from literature and assumed similar, although they may vary in different countries due to genetic factors, study designs, and healthcare systems. Additional confounders in the model are possible comorbidities, polypharmacy, duration, and dose of FQs, and use of multiple antibioticsData for serious ADR costs and health service use were available from 2002 to 2012 and it was assumed that treatment guidelines matched the FQ‐prescription period from 2008 to 2019.Modeling is a simplification of reality and, thus, entails certain assumptions:2.9Due to varying estimates of FQ‐related ADR and mortality incidence, the decision tree model contains uncertainty about the exact probabilities used as inputs. To deal with parameter uncertainty in the model, sensitivity analyses were conducted to assess their impact on direct healthcare costs and ADR‐related mortality.3A total of 1 831 537 FQ prescriptions were filled between 2008 and 2019. During the twelve‐year observation period, the number of FQ prescriptions fell by 40% from 2008 (n = 184 369) to 2019 (n = 79 926), peaking at 195 806 in 2011. In Finland, the number of FQ prescriptions has declined consistently with all antimicrobial prescribing  and being the costliest FQ‐related ADRs, they were also the most frequent serious ADRs (n = 10 108). The healthcare costs of a patient, who recovered from FQ‐related SCDI were 46 005€. A patient, who did not recover from the infection, had even higher healthcare costs, i.e., 52 324€. Total costs of SCDIs amounted to 487 381 481€. Other serious FQ‐related ADRs were significantly less frequent. Aortic and tendon ruptures tallied up total costs of 7 161 990€ with 330 events and 5 469 228€ with 534 events, respectively. The total estimated mortality associated with serious FQ‐related ADRs between 2008 and 2019 was 3,884, with SCDI being the leading cause of death . No deaths were associated with tendon ruptures or SCAR with the baseline estimates. FQ‐related liver injuries were associated with 210 and aortic ruptures with 135 deaths during the 12‐year observation period. According to baseline estimates, the average mortality risk associated with FQ‐related ADRs was 0.21%.The results of the sensitivity analyses are shown in Figures The declining number of FQ prescriptions has meant that both total medication costs and ADR costs have significantly fallen since 2008 (Figure 4The economic burden of ADRs is often overlooked in decision making, although healthcare resources are limited, and overall medication expenditure is rising. While ADRs remain a notable cause of morbidity and mortality, the number of ADR‐related deaths seems to have decreased in recent years.ADR research relies heavily on timely spontaneous ADR‐reporting, thorough post‐marketing surveillance, and observational research. However, the true extent and nature of all drug‐induced ADRs will continue to elude healthcare professionals and decision makers if reporting levels remain poor. The importance of post‐marketing surveillance from regulatory agencies such as EMA cannot be stressed enough. Without it, there is no incentive for pharmaceutical companies to continue research after marketing authorization. Aortic ruptures and neuropsychiatric ADRs represent types of FQ‐related ADRs identified only recently, which can be difficult to distinguish from other causes but have distinct harmful clinical and economic implications. A particular challenge of the study was the lack of data concerning ADR costs. Due to this, we were unable to estimate the costs of several known FQ‐related ADRs, such as neuropsychiatric events. Therefore, although the costs and mortality presented in this study are significant, they are likely still an underestimation of the full economic impact of FQ‐related ADRs. The shortage of comprehensive cost data directed us to conduct the study from the perspective of the healthcare provider, and, therefore, direct costs borne by other parties, such as patients and employers, were not included, in addition to the loss of productivity. A societal perspective would include all costs regardless of by whom they are borne and would be ideal.The role of pharmacogenomics is becoming more important in the ADR research. In connection to FQs, for example, studies have shown significant pharmacokinetic variability among tuberculosis patients treated with moxifloxacin, which is thought to be connected to variations in genes encoding drug‐metabolizing enzymes and drug transporters.This study drew its estimates from several published sources and databases, which generated parameter uncertainty. Variability in ADR incidence estimates is caused by differences in study populations, geographical areas, and hospitals, in addition to methodological differences in classifying ADRs. Although death is the worst ADR, its precise incidence remains equally difficult to estimate due to similar challenges. Sensitivity analyses were conducted to expose the model to extreme situations. The best‐case scenario probably underestimates, and the worst‐case scenario likely overestimates cases, mortality, and costs, leaving the actual outcomes somewhere in between. The model inputs involving probabilities of serious ADRs and mortality associated with them were obtained from published literature, which enables that part of the model to be generalized and transferred to other countries. Although the full model with the current model parameters describes ADR costs in a setting that is unique to Finland, the cost inputs in the model can be modified independently and utilized to generate internationally transferable results.Tendon ruptures are no doubt the most distinctive and familiar serious FQ‐related ADRs. However, particular emphasis should be on patient groups with an increased risk of developing CDI. SCDIs are the most frequent, fatal, and costly serious adverse reactions associated with the use of FQs, and the need for cost‐effective therapeutic alternatives will continue. According to our study, SCDIs cause more than 95% of the total direct healthcare costs of the ADRs. Although the number of FQ‐associated SCDI initially fell rapidly in Finland until 2020 along with declining use of FQs,5clostridioides difficile infections, which alone cause more than 95% of the total direct healthcare costs of the ADRs. Accordingly, from the economical perspective, efforts should be focused on limiting the risk of FQ‐associated CDI. Nevertheless, the different risks and benefits of these drugs should always be weighed carefully.Although FQs continue to be generally well‐tolerated antimicrobials, serious ADRs can cause long‐term disabilities to patients and high healthcare costs. In fact, on average, FQ‐related ADRs appear to be approximately ten times more expensive than the FQ medications themselves. The most impactful of the FQ‐related ADRs are This is a retrospective observational study with registered data, and neither an ethics approval nor patient consent was required.The authors of this manuscript have no conflicts of interest to declare.All persons who meet authorship criteria are listed as authors. All authors took part in conceptualizing the idea, designing the study, analyzing the data, and writing the manuscript. All authors have seen and approved the final version of the manuscript.


Amblyomma testudinarium larvae. Further epidemiological surveillance and etiological studies are necessary to assess the status and risk of jingmenvirus infection in Japan.Jingmen tick virus (JMTV) and the related jingmenvirus-termed Alongshan virus are recognized as globally emerging human pathogenic tick-borne viruses. These viruses have been detected in various mammals and invertebrates, although their natural transmission cycles remain unknown. JMTV and a novel jingmenvirus, tentatively named Takachi virus (TAKV), have now been identified during a surveillance of tick-borne viruses in Japan. JMTV was shown to be distributed across extensive areas of Japan and has been detected repeatedly at the same collection sites over several years, suggesting viral circulation in natural transmission cycles in these areas. Interestingly, these jingmenviruses may exist in a host tick species-specific manner. Vertical transmission of the virus in host ticks in nature was also indicated by the presence of JMTV in unfed host-questing  Ticks are hematophagous ectoparasites that infest various animals and can transmit a variety of pathogens, including protozoa, bacteria, and viruses, to their hosts during the blood-feeding process. Tick-borne viruses have become the focus of public health attention with the re-emergence of highly pathogenic viruses such as Crimean-Congo hemorrhagic fever virus (CCHFV) and the emergences of new viruses such as severe fever with thrombocytopenia syndrome virus and Bourbon virus .Advances in next-generation sequencing (NGS) technologies have dramatically facilitated the study of tick-borne viruses. A series of recent studies have revealed the ticks harbor a wide variety of viruses through comprehensive analyses utilizing next-generation sequencers . This haRhipicephalus microplus) collected in China in 2010  and was passed through sterile 0.45-μm filters , and then mixed in equal amounts up to a total of 380 µL . This sample was then used for library preparation for NGS analysis as described previously ,26. In b2. A fresh culture medium was then added to each well, and the incubation was continued for seven days, followed by two subsequent blind passages under the same conditions. Culture supernatants after the incubation period were analyzed using next-generation sequencers (as described previously)  or BHK-21 cells  as described by Kobayashi et al. . In brieviously) ,26 to co4], as described previously [Previously, we used BHK-21 cells to isolate viruses from field-caught ticks from various parts of Japan during 2013 to 2014 . The blieviously . The coneviously . Libraryeviously .To identify virus-positive tick pools, reverse transcription polymerase chain reaction (RT-PCR) was performed on total RNA extracted from each filtered tick pool homogenate, as described previously . RT-PCR The virus-positive pools were then used to determine the complete viral genome sequence. Sequence gaps between contigs obtained using RNA virome analyses were filled with subsequent RT-PCRs using primers specific for the viral sequences. The resultant amplicons were analyzed using direct Sanger sequencing using an ABI 3130 Genetic Analyzer  or Genewiz, Inc. , as previously described ,26. The Jingmenviral open reading frames and their encoded amino acid sequences were determined using Genetyx version 14 software .Attempts to isolate Takachi virus (TAKV) in cell cultures failed, which raised the possibility that the TAKV sequence was derived from an EVE in the host tick genome. DNA extracted from the homogenate of TAKV-positive pool was analyzed through PCR using the following four virus-specific primer sets: IMOI-Js1-F and IMOI-Js1-R for segment 1, IMOI-Js2-F and IMOI-Js2-R for segment 2, IMOI-Js3-F and IMOI-Js3-R for segment 3, and IMOI-Js4-F and IMOI-Js4-R for segment 4 , as descMultiple sequence alignments were performed using the MAFFT online service . The GblAmblyomma testudinarium, Haemaphysalis flava, Hae. formosensis, Hae. hystricis, Hae. longicornis, Hae. megaspinosa, Haemaphysalis sp., Ixodes ovatus, I. persulcatus, and I. turdus, were collected in 2018–2020 in Ehime and Ishikawa Prefectures, Japan . Eight pools derived from Am. testudinarium ticks were identified as JMTV-positive using RT-PCR screening , all of which were biting on the skin, in Tsushima City in 2013; the JMTV strains were named T281 and T285 . Thus, the novel jingmenvirus genome likely exists as RNA but is not derived from the EVE in the host tick genome. Therefore, we tentatively named the new virus as Takachi virus (TAKV) after the Japanese name of the host tick species Am. testudinarium ticks, although virus detection was performed on diverse unfed ticks from nine species across three genera. Remarkably, JMTV was detected not only in nymphs but also in unfed larva, indicating the vertical JMTV transmission in Am. testudinarium ticks in nature. In a previous study, an infectious JMTV was isolated from Am. javanense, and JMTV infection experiment in Am. javanense ticks has confirmed the virus accumulation and replication in the salivary glands, suggesting Am. javanense may be a potential vector of JMTV [Amblyomma genus may be important as vectors and reservoir hosts of JMTV. Three Amblyomma species are known to be distributed in Japan [Am. testudinarium, where we detected JMTV in this study, is considered to be critical species as a JMTV vector because of its high preference for humans [Amblyomma testudinarium is widely distributed from India to Southeast Asia and East Asia, including Japan ; this sAm. testudinarium infestation have been described in a resident bird species, Scolopax mira  was shown to be genetically related to wild boar distributed in China and the Indochinese Peninsula, including Laos [The JMTV strains detected in this study were shown to be phylogenetically related to strains from southern China and Laos. This suggests that virus-infected ticks can be transported over a long distance by migratory birds as reported for CCHFV . In JapaYamauchi ), and in species . This tild boars , ticks ming Laos . This sting Laos . Furthering Laos ,14, it iWhile attempting to isolate the virus, we detected JMTV RNA in the supernatant of BHK-21 cell cultures after two blind passages. Although this indicates possible virus replication in the cells, we cannot rule out the detection of viral RNA carryover from the virus-positive tick homogenates for inocula. According to the first report describing JMTV , JMTV geHae. formosensis ticks. This suggests a specific and stable virus-tick host relationship similar to those between JMTV and Am. testudinarium ticks, also demonstrated in this study. TAKV occupies a phylogenetically intermediate position between human-pathogenic JMTV and ALSV . FurtheAm. testudinarium larvae, suggesting the vertical transmission of the virus in host ticks in nature. Further epidemiological surveillance and etiological studies are necessary to assess the status and risk of jingmenvirus infection in Japan.JMTV and a novel jingmenvirus, TAKV, were detected in host-questing ticks for the first time in Japan. JMTV was also detected in the supernatant of BHK-21 cells inoculated with the tick sample. Interestingly, these jingmenviruses may exist in a host-tick species-specific manner. This study also revealed that JMTV was distributed across an extensive area in Japan and was detected at the same collection site for several years. Moreover, JMTV was found in