Oncolytic viruses are a new class of therapeutics which are largely in the experimental stage, with just one virus approved by the FDA thus far. While the concept of oncolytic virotherapy is not new, advancements in the fields of molecular biology and virology have renewed the interest in using viruses as oncolytic agents. Backed by robust preclinical data, many oncolytic viruses have entered clinical trials. Oncolytic viruses that have completed some levels of clinical trials or are currently undergoing clinical trials are mostly genetically engineered viruses, with the exception of some RNA viruses. Reolysin, an unmodified RNA virus is clinically the most advanced oncolytic RNA virus that has completed different phases of clinical trials. Other oncolytic viruses that have been studied in clinical trials are mostly DNA viruses that belong to one of the three families: herpesviridae, poxviridae or adenoviridae. In this review work we discuss recent clinical studies with oncolytic viruses, especially herpesvirus, poxvirus, adenovirus and reovirus. In summary, the oncolytic viruses tested so far are well tolerated, even in immune-suppressed patients. For most oncolytic viruses, mild and acceptable toxicities are seen at the currently defined highest feasible doses. However, anti-tumor efficacies of oncolytic viruses have been modest, especially when used as monotherapy. Therefore, the potency of oncolytic viruses needs to be enhanced for more oncolytic viruses to hit the clinic. Aiming to achieve higher therapeutic benefits, oncolytic viruses are currently being studied in combination with other therapies. Here we discuss the currently available clinical data on oncolytic viruses, either as monotherapy or in combination with other treatments. There has been an increasing interest in using viruses as potential therapeutics for different types of diseases. Two main ways in which viruses are being explored as therapeutics are (1) vectors for gene therapy and (2) oncolytic viruses. While viruses as vector for gene therapy are being studied for a wide range of diseases including cancer, “oncolytic viruses” are specific for cancer treatment. The major difference between viral vector for gene therapy and oncolytic virus is that viral vectors used in gene therapy are non-replicating viruses, whereas oncolytic viruses are replication-competent viruses. Oncolytic viruses (OV) are wild-type or engineered viruses that can selectively replicate in and kill cancer cells while leaving normal cells unharmed. OVs are a novel class of multi-mechanistic therapeutics for the treatment of cancer. Some of the mechanisms through which OVs exert their anti-cancer effect include direct lysis of cancer cells and activation of anti-tumor immunity .The concept of using viruses to kill cancer is not new, there is anecdotal evidence of anti-tumor benefits from virus infections dating back to a century ago . HoweICP34.5 [Herpes simplex virus-1 (HSV-1) belongs to the family herpesviridae. It is an enveloped, double-stranded DNA virus with genome size of approximately 150 kbp that encodes ~89 genes . Many HSICP34.5). Furthermore, immunostimulatory properties of T-VEC have been improved by the deletion of the gene ICP47, an inhibitor of major histocompatibility complex I (MHC-I) mediated antigen presentation, and by the insertion of granulocyte-macrophage colony-stimulating factor (GM-CSF) expression cassettes at both loci of ICP34.5. The deletion of ICP47 enhances immune-mediated destruction of the virus-infected cells, whereas virus-encoded GM-CSF increases recruitment and activities of dendritic cells, ultimately boosting anti-tumor immunity [Talimogene laherparepvec (T-VEC) marketed as Imlygic, is a JS-1 strain of HSV-1 that was originally isolated from a cold sore ,7. The v6 plaque forming units (PFUs) of virus followed by multiple injections of higher doses (107 or 108 PFUs). The treatment was well tolerated with the most common side effects being local inflammation and erythema febrile responses. Overall responses to the treatment were mild; however, it was encouraging to find that the virus was able to replicate in the tumors, produce GM-CSF, cause tumor necrosis and inflammation in both the injected and, in some cases, un-injected tumors. After this Phase I study, an initial Phase II clinical trial was performed that included 50 patients with stage IIIC unresectable or metastatic melanoma [8 PFU at the interval of 2 weeks for up to 24 treatments. Tumor regression was observed not only in the injected lesions, but also in un-injected distant tumors including visceral metastases. The overall response rate (ORR) was 26% with 8 out of 13 responding patients experiencing a complete response. The treatment was well tolerated with the most common side-effects being inflammation at injection sites, flu-like symptoms and nausea. This encouraging Phase II trial was followed by a Phase III trial, which involved 436 patients with stage IIIB to IV melanoma. In this study, efficacy of intratumoral T-VEC treatment was compared with a subcutaneous injection of GM-CSF [In preclinical studies, T-VEC was found to induce strong cytotoxic activities against a wide range of tumor cell lines in vitro and caused tumor regression in murine models ,9. Follomelanoma . T-VEC wf GM-CSF . The ORRClinicaltrials.gov; accessed on 1 March 2021) to determine safety and efficacy of T-VEC either as monotherapy or in combination with other therapeutics, including immune checkpoint inhibitors, in different types of cancers. Some of these studies, which are currently in trials, are mentioned in Puzanov et al. reported the findings of a Phase Ib trial of T-VEC in combination with ipilimumab (a cytotoxic T-lymphocyte-associated antigen 4 (CTLA4) checkpoint inhibitor) in patients with advanced melanoma . The comICP34.5 genes. Additionally, G207 has the ICP6 gene inactivated through the insertion of the Escherichia coli lacZ gene. The ICP6 gene encodes a viral homolog of the cellular protein ribonucleotide reductase (RR), an enzyme required for the production of deoxy-ribonucleotides (dNTPs) [ICP6 restricts virus replication to dividing cells with high levels of RR such as cancer cells. While this virus was originally designed for treatment of brain tumors, preclinical studies have found that the virus is effective against a wide range of cancer types including prostate, breast, head and neck, colon, ovaries, neuroblastoma, osteosarcoma and melanoma [6 to 3 × 109 PFUs of the virus intratumorally. Some patients experienced adverse events, but toxicities were not directly related to the virus. Radiographic and neuropathologic analysis showed some evidence of anti-tumor activity. In another Phase I study, nine patients with recurrent malignant glioma were treated intratumorally with 109 PFUs of G207 followed by treatment with 5 Gy radiation [G207 is another oncolytic HSV-1 that has completed several Phase I trials and is expected to begin a Phase II trial soon. Similar to T-VEC, G207 has deletions in both copies of  (dNTPs) . Deletiomelanoma ,20,21,22melanoma . A totaladiation . This coICP47, which allows for enhanced MHC-I presentation on infected cells [n = 13 patients) was significantly higher (92.3%) than the control group (15%) [G47Δ is a variant of G207 that has also been tested for safety and efficacy in glioblastoma and prostate cancer in Japan. G47Δ has an additional deletion in the gene ed cells . Interimup (15%) . Severe up (15%) .ICP34.5 genes [ICP4, ICP27, UL8), and in ICP34.5, that potently inhibit virus replication in normal cells, but not in cancer cells [UL37 gene that prevent axonal retrograde transport as well as latency, ensuring that virus will not damage neurons. Additionally, ONCR-177 encodes five immune-modulatory transgenes  driven by a dual bi-directional promoter [ONCR-177 is an oncolytic HSV-1 virus developed by Oncorus Inc. Unlike T-VEC and G207, ONCR-177 is not deleted in .5 genes . Target er cells . FurtherRP1 is an oncolytic HSV-1 virus containing deletions in the neurovirulence genes ICP34.5 and ICP47. It has two transgenes inserted in its genome: GM-CSF and gibbon ape leukemia virus glycoprotein (GALV-GP R-), a fusogenic membrane glycoprotein. This virus is currently in Phase I trial as monotherapy for recurrent or advanced cutaneous squamous cell carcinoma (NCT04349436) or in combination with anti-PD-1 antibody (NCT04050436). RP1 has been further modified (known as RP2) to express anti-CTL4, which is also undergoing a Phase I trial (NCT03767348).Vaccinia virus (VACV) is an enveloped virus belonging to the family poxviridae. VACV has a linear double-stranded DNA genome that is approximately 200 kpb in size and encodes ~200 genes . VACV isJ2R, encoding for thymidine kinase, replaced by the expression cassettes for human GM-CSF (hGM-CSF) and lacZ [J2R with the aim of increasing its cancer-specificity, and the hGM-CSF expression cassette was inserted with the aim of increasing anti-tumor immunity through the recruitment and activation of antigen-presenting cells (APCs) and cytotoxic T lymphocytes (CTLs) [9 PFUs of JX-594 intratumorally or intravenously. Compared to the control group, the intratumoral or intravenous injection of JX-594 potently inhibited the growth of primary tumors. Furthermore, while all control-treated rabbits developed lung metastases, none of the rabbits treated with JX-594 developed lung metastases. Interestingly, in the rat model in which human GM-CSF is not compatible, 108 PFUs of JX-594 injected three times (intravenous) at the interval of 2 weeks, resulted in a complete tumor regression in majority of the treated mice. This study by Kim et al. was encouraging, especially because it involved immune-competent orthotopic tumor models, as opposed to other studies, most of which used human xenograft models and intratumoral injections for the evaluation of oncolytic viruses.Pexa-Vec (formerly JX-594) is a Wyeth strain of vaccinia virus that has and lacZ . Pexa-Ves (CTLs) . The cons (CTLs) . In thiss (CTLs) . While bJ2R gene was replaced with expression cassettes for hGM-CSF and β-galactosidase. In this study, a total of seven patients with unresectable melanoma were treated twice weekly with intratumoral injections of escalating doses of the virus for 6 weeks. A maximum dose of 8 × 107 PFU virus was used in the study and no maximum tolerable dose (MTD) was reached. Tumor regression in a fraction of patients was observed in this study, and systemic toxicity was limited to flu-like symptoms that resolved within 24 h. Followed by this pilot study, an open-label Phase I dose-escalation trial was conducted with substantially higher doses with the aim of defining safety and the MTD of JX-594 [8 PFU to 3 × 109 PFU every 3 weeks. Grade 1–3 flu-like symptoms were observed in all patients and 4 out of 14 patients experienced transient grade 1–3 dose-related thrombocytopenia. Furthermore, patients treated with the highest dose (3 × 109 PFU) displayed hyperbilirubinemia, which was considered dose-limiting. From this study, 1 × 109 PFU was determined to be the MTD for JX-594. In addition to finding the MTD, several insights were gained from this Phase I trial. First, the study showed that it is possible for the virus to disseminate from injected to non-injected tumors through blood; virus replication and expression of GM-CSF were also confirmed. Second, tumor responses were observed in both injected and non-injected tumors. In terms of efficacy, out of 10 radiographically evaluable patients, 3 had partial response, 6 had stable disease and 1 had progressive disease.The prototype of JX-594 was used in a pilot Phase I trial in 1998 . The virf JX-594 . This st9 PFU. In this study, virus treatment was found to be safe and anti-tumor efficacy was observed in all patients despite the presence of high levels of neutralizing antibodies. JX-594 was found to replicate in tumors and release of virus in circulation was observed. Additionally, this study showed that the virus treatment elevated the levels of anti-vascular cytokines, resulting in tumor vascular shutdown. Interestingly, JX-594 was also found to suppress the replication of hepatitis B virus in HCC patients. Another pilot study was reported by Heo et al. in 2011 on the combination of JX-594 with the kinase inhibitor, sorafenib, for the treatment of HCC [Liu et al. reported another pilot study of JX-594 in advanced refractory hepatitis B virus (HBV)-associated hepatocellular carcinoma (HCC) . The virt of HCC . In muri9 PFU, which was generally well tolerated. This study included a total of 23 patients. The virus was detectable in tumor biopsies of patients who received virus at the dose ≥109 PFU. This study provided the proof-of-concept regarding the feasibility of the systemic administration of JX-594 for the treatment of tumors.In 2011, Breitbach et al. reported the findings from a Phase I dose-escalation trial of a single intravenous infusion of JX-594 . The max8 PFU (low-dose) and 109 PFU (high-dose) were used for intratumoral infusions on day 1, 15 and 29 in patients with advanced hepatocellular carcinoma. The objective of this study was to compare the safety and efficacy of low- and high-dose treatments and induction of immunity against both cancer cells and the oncolytic virus. The objective response rate (15%) and intrahepatic disease control (50%) were found to be equivalent in the injected and non-injected distant tumors for both the doses. GM-CSF expression and the induction of anti-tumor immunity was observed at both the doses. While the tumor response rate and immune endpoints were similar for the low- and high-dose treatments, survival of patients treated with the high-dose treatment was significantly higher  than that for patients treated with the low-dose treatment (6.7 months).Results from a Phase II dose-finding trial for JX-594 in liver cancer treatment were reported by Heo et al. in 2013 . A total9 PFU) was followed by up to five intratumoral injections. The treatment was generally well tolerated and evidence of immune induction against the virus as well as tumor antigens were observed. However, in this study Pexa-Vec failed to improve the survival of patients; the overall survival of patients treated with BSC alone (4.2 months) was not significantly different from the overall survival of patients treated with BSC plus Pexa-Vec. Currently, Pexa-Vec is being tested in combination with sorafenib in a worldwide Phase III PHOCUS trial in HCC patients.Recently, results from a multicenter Phase IIb trial of Pexa-Vec in sorafenib-resistant hepatocellular carcinoma (TRAVERSE) were published by Moehler et al. . Patient9 PFU. While all these studies have established an excellent safety profile for this virus, the anti-tumor efficacy of the virus remains uncertain.Thus far, more than a dozen clinical trials have been completed with JX-594 (Pexa-Vec) in multiple types of malignancies, in which more that 400 patients have been injected with the virus. The MTD has been determined to be 10J2R gene. Additionally, GL-ONC1 is deleted in the F14.5L and A56R genes. The virus encodes three transgenes: β-galactosidase, β-glucuronidase and Renilla luciferase–Aequorea green fluorescent protein [GL-ONC1 (formerly known as GLV-1h68) is an oncolytic VACV based on the Lister strain developed by Genelux Corporation . Like JX protein . In prec protein ,40,41,42J2R and C11R (vaccinia growth factor) genes, and it encodes bacterial cytosine deaminase as well as the somatostatin receptor [J2R and C11R allows for vvDD to selectively replicate in cells with an active E2F and epidermal growth factor receptor (EGFR/Ras) pathway, common features of many cancer cells [9 PFU of virus, the maximum feasible dose, was injected intratumorally without dose-limiting toxicities. The virus was recovered from both injected and non-injected tumors. Although no clinical benefits were achieved in this study, it was encouraging to see that the WR strain of VACV was well tolerated even at the maximum feasible dose of 3 × 109 PFU.The vvDD  is a Western Reserve (WR) strain of VACV that is deleted in receptor . The WR receptor . Deletioer cells ,44,45. Ter cells ,44,45. Fer cells . In this9 PFU virus was injected in patients. The virus was well tolerated with the most common adverse events being grade 1/2 flu-like symptoms. Tumor-specific replication of the virus was observed. Clinical benefits were minimal, but the authors concluded that excellent safety profile of the virus warrants further trials in combination with other therapeutics such as checkpoint inhibitors. The second clinical study using vvDD was published by Downs-Canner et al. in 2016 . In thisClinicaltrials.gov for the term “oncolytic adenovirus” displayed a total of 45 studies in different phases of trials, out of which 11 have been completed and 14 are currently recruiting patients. An oncolytic adenovirus called H101 , is the first oncolytic virus to be approved for the treatment of cancer. H101 is an E1B-deleted adenovirus type 5 that was approved in China in November 2005 for the treatment of head and neck cancer [Adenoviruses are non-enveloped viruses with linear double-stranded DNA genome that ranges from 26 to 45 kbp in size . A searck cancer . Despiteclinicaltrials.gov (accessed on 1 March 2021), DNX-2401 has completed four Phase I trials in patients with brain tumors/glioblastoma and one Phase I trial in patients with ovarian cancer and primary peritoneal cancer. In brain cancer patients, the virus has been studied as a single agent or in combination with other therapeutics such as interferon-y, temozolomide or pembrolizumab. While results from most of the studies have not been published, in 2019 Philbrick and Adamson published the findings of a Phase I study of DX-2401 in patients with recurrent high-grade gliomas [DNX-2401  is an oncolytic adenovirus in which the early gene E1A is inactivated through the deletion of 24 bp in the E1A gene . E1A ina gliomas . In this13 virus particles were administered through intravenous infusion. The most common adverse events of grade 3 were hypoxia, lymphopenia and neutropenia, which were manageable [Kuhn et al. used recombination and a directed evolution approach to create a chimeric group B oncolytic adenovirus called Enadenotucirev . This vinageable . There aLOAd703 is another oncolytic adenovirus that has shown promising results in preclinical studies and is actively being tested in clinical studies for the treatment of a variety of cancers . LOAd70310 to 3 × 1011 virus particles in combination with low-dose cyclophosphamide. No dose-limiting toxicity was observed, and hence, no MTD was identified in this study. In terms of anti-tumor efficacy, 4 out of 10 evaluable patients had stable disease at 3 months. Patients treated with the virus had a short-term elevation in pro-inflammatory cytokines and had an increase in tumor infiltration by lymphocytes, as determined by the immunohistochemistry of tumor biopsies before and after the treatment. In some patients, systemic induction of tumor-specific CD8+ T cells was observed. Furthermore, an upregulation in the levels of PD-L1 in the tumor was observed after virus treatment, suggesting that the efficacy of the virus could be enhanced by combining it with PD-L1 inhibitors. Hence, there are active clinical studies testing the safety and efficacy of ONCOS-102 in combination with PD-L1 inhibitors such as durvalumab (NCT02963831) and pembrolizumab (NCT03003676).Another oncolytic adenovirus being tested in clinical studies is ONCOS-102 . ONCOS-102 is a chimeric adenovirus constructed from adenovirus type 3 and 5, and is armed with the cytokine GM-CSF . Thus faRNA viruses belonging to different families including coronaviridae, picornaviridae, reoviridae, retroviridae, rahabdoviridae and togaviridae have been studied for their oncolytic activities . One comRas and the impairment in type-I interferon (IFN) signaling [Clinicaltrials.gov (accessed in 1 November 2020), a total of 20 clinical trials have been completed with this virus . The virus has been studied as a monotherapy or in combination with other therapeutics for the treatment of a multitude of cancers including metastatic breast cancer, advanced-stage head and neck cancer, metastatic ovarian cancer, malignant gliomas, prostate cancer and metastatic melanoma. Out of the total 20 completed trials, Reolysin was used as monotherapy in 5 studies, and in the remaining 15 studies it was combined with chemotherapeutics such as gemcitabine, irinotecan, docetaxel, carboplatin, paclitaxel and cyclophosphamide  for five consecutive days every 4 weeks. Grade 1 and 2 toxicities included fever, fatigue and headaches, which were independent of the virus doses. Virus-neutralizing antibodies were developed in all patients, with the peak titer detected 4 weeks after the first injection. Evidence of virus localization and replication in tumor was observed in three patients. The study report did not mention anything about anti-tumor efficacy, perhaps because no anti-tumor efficacy was observed.Vidal et al. reported the first clinical study on intravenous delivery of Reolysin . In this8 to 3 × 109 TCID50. Dose-limiting toxicity was not reached, and the common treatment-related toxicities included fatigue and fever. A virus-neutralizing antibody was developed in all patients during the course of treatment. Furthermore, virus shedding was observed in 6 out of 18 patients, and the patients showing virus shedding appeared to be more responsive to the treatment. In this study, the overall clinical benefit rate was 45%: one patient experienced partial response and seven attained stable disease.Gollamudi et al. reported results from another clinical study involving intravenous administration of Reolysin . In this10 TCID50 on days 1–5 of each 4 week cycle. Similar to previous Phase I trials, the treatment was well tolerated. Productive reovirus replication was observed in the tumor biopsies of two patients. Unfortunately, no objective response was observed in this study. Likewise, results from another Phase II trial were reported by Mite et al. (NCT00503295). In this study, 43 patients with bone and soft tissue sarcoma metastatic to the lung were recruited. Patients were treated intravenously with Reolysin every 28 days. Toxicities were mild to moderate, including fever, chills, fatigue, diarrhea, cough, dyspnea, neutropenia and thrombocytopenia. Overall, 14 patients experienced stable disease for more than 2 months, and 5 patients had stable disease for more than 6 months.The first Phase II trial for intravenous monotherapy of Reolysin was initiated in 2008 . In this8 and 1 × 1010 TCID50. In the second stage, patients received a higher dose of radiation (36 Gy) followed by 2, 4 or 6 doses of 1 × 1010 TCID50 virus. The combination treatments were found to be safe with mild (grade 1 to 2) toxicities. Two of seven evaluable patients in the low-dose radiation group showed partial response, and five patients had stable disease. Likewise, in the high-dose radiation treatment, five of seven evaluable patients experienced partial response and two had stable disease. No virus shedding was detected in blood, urine, stool and sputum of the patients. Given the excellent safety profile and better efficacy, compared to previous studies using reovirus as monotherapy, the authors concluded that this combination should be evaluated in newly diagnosed patients receiving radiation with curative intent.While clinical studies with Reolysin as monotherapy have demonstrated that the virus is safe, the lackluster efficacy of the virus as monotherapy has prompted investigators to combine it with other therapeutics. Over a dozen clinical studies encompassing different phases of trials have been completed so far with Reolysin in combination with radiation, chemo- or biological therapies. Harrigton et al. reported the findings of a Phase I trial combining reovirus in patients receiving palliative radiotherapy . In tota10 TCID50 reovirus. Overall, 3 out of 16 patients experienced dose-limiting toxicity, with 2 patients having liver enzymes increased to grade 3, and 1 patient with levels of troponin elevated to grade 3. The increase in liver enzymes was found to be transient and reversible. In this combination treatment, neutralizing-antibody responses to reovirus were delayed and attenuated. Following this study, a Phase II study was initiated to evaluate the safety and efficacy of reovirus in combination with gemcitabine in patients with metastatic pancreatic cancer (NTC00998322). As of today, this study has been completed but findings of the study have not been published. A variety of other chemotherapeutics including carboplatin, irinotecan, cyclophosphamide, paclitaxel and docetaxel have been studied in combination with Reolysin in Phase I and/or Phase II trials against a multitude of cancers. Finally, a Phase III trial of Reolysin in combination with carboplatin and paclitaxel has been completed in patients with head and neck cancer. Findings of the study have not been reported yet.Lolkema et al. reported results of the first clinical study combining reovirus with a chemotherapy . In thisReolysin has also been studied in combination with other biological therapeutics. The first biological therapeutic to be combined with Reolysin was bevacizumab, a monoclonal antibody against vascular endothelial growth factor. In a Phase I trial, colorectal cancer patients with mutant KRAS were treated with Reolysin and bevacizumab together with the chemotherapeutics irinotecan, leucovorin and 5-fluorouracil (NCT01274624). The study has been completed, but the results have not been published yet. Additionally, several clinical studies combining reovirus with checkpoint inhibitors (CKIs) are currently underway .The field of oncolytic virotherapy is rapidly growing, with thousands of preclinical studies reported, and over 100 clinical trials performed thus far. The approval of T-VEC by the FDA in 2015 amplified the interest of cancer researchers and clinicians in this novel therapeutic approach. Apart from T-VEC, there are many oncolytic viruses that have completed different phases of clinical studies. From all the clinical trials performed so far, oncolytic viruses appear to be safe at maximum feasible doses, and in most cases, the maximum tolerable doses have not been reached. While OVs have shown excellent safety profiles, their anti-tumor efficacies are modest at best. Nevertheless, for most viruses, the MTD has not been reached, and it is likely that their anti-tumor efficacies could be increased with future advancements in virus manufacturing technology. Alternatively, novel viruses with higher anti-tumor potency could be generated, which may allow researchers to achieve maximum anti-tumor benefits at doses that are feasible with the current manufacturing process. For example, our group created a chimeric poxvirus through recombination among nine poxviruses, encompassing different strains of VACV and different species of poxvirus . The chiMost of clinical trials, including those for T-VEC, have used intratumoral route for virus administration. The reason for using intratumoral administration is to avoid virus neutralization by antibodies and virus sequestration by macrophages, which could severely impede OVs, especially during repeated injections. The downside of the intratumoral route for virus injection is that the virus could not be administered in tumors that are inaccessible for direct injections. Furthermore, once a cancer has been metastasized, it is not possible to find all the metastatic nodules and inject them. While some studies have shown that injection of virus in one tumor can lead to regression of distant un-injected tumors, this phenomenon of abscopal effect has been observed only in small subset of patients. Recently, some clinical studies have used the systemic route for the delivery of oncolytic viruses, showing the feasibility of systemic injections of OVs in patients. The anti-tumor efficacy achieved through systemic injections of viruses has been modest, just as is the case for intratumoral injections of OVs.Cancer is a complex disease that often requires a complex treatment comprising a combination of several therapeutic approaches. Results from clinical trials suggest that oncolytic viruses as monotherapy are less likely to achieve optimum therapeutic benefits. However, oncolytic viruses appear to be good candidates for combination with other therapeutic approaches, especially immunotherapy. Oncolytic viruses have been shown to subvert immune-suppressive microenvironments in tumors, making them conducive for the effectiveness of immunotherapeutics (reviewed in ). A numbIn summary, the field of oncolytic virotherapy has made dramatic strides in the last two decades, culminating in the approval of the first OV in the Western world. With many oncolytic viruses having completed Phase II trials, it is likely that more oncolytic viruses, perhaps in combination with immunotherapeutics, will soon receive approval from regulatory agencies for the treatment of different malignancies.


I thank all authors, reviewers and the editorial staff who contributed to this special issue. The articles published in this special issue highlight how molecular biology has revolutionized virology. Molecular biology is nowadays largely used for virus detection in routine diagnosis, for virus typing and phylogenetic analysis. It is also useful for the discovery of new viruses that may be pathogens or members of our healthy virome. An example of how central the role of molecular biology has become to virology, is a recently discovered virus that became a public health concern, the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). This virus was first identified by next generation sequencing . One art


There are limited data in the literature on the indirect costs associated with skin and soft tissue infections (SSTIs) in the pediatric population. This study aimed to conduct a systematic review of the indirect costs associated with SSTIs in children.The search was conducted in PubMed, SCOPUS, and Web of Science up to January 2020. Thirteen search strategies were designed combining MeSH terms and free terms. SSTIs were defined as bacterial or viral infections, dermatomycoses, and parasitic infestations. Only primary studies were included. All analyzed costs were converted to 2020 Euros.Varicella-zoster virus: lost workdays by outpatient caregivers ranged from 0.27 to 7.8, and up to 6.14 if caring for inpatients; total productivity losses ranged from €1.16 to €257.46 per patient. Three studies reported indirect costs associated with acute bacterial SSTIs (community-associated methicillin-resistant Staphylococcus aureus) in children: total productivity losses ranged from €1,814.39 to €8,224.06 per patient, based on impetigo, cellulitis, and folliculitis. One study of parasitic infestations (Pediculus humanus capitis) reported total indirect costs per patient of €68.57  plus €21.41 due to time lost by parents in purchasing treatment.Thirteen of the identified publications presented indirect costs of SSTIs in children and were conducted in Argentina, Australia, Brazil, Hungary, New Zealand, Poland, Spain, Taiwan, and the USA. Nine studies described indirect costs associated with infection of The economic burden of SSTIs is highly relevant but underestimated due to the lack of studies reporting indirect costs. Further cost studies will allow a better understanding of the magnitude of the financial burden of the disease.The online version contains supplementary material available at 10.1186/s12913-021-07189-3. The global impact of skin diseases, both social and economic, is significant, with 4.86 billion incident cases of skin and subcutaneous diseases estimated worldwide in 2019 [Though taxonomies may vary, skin and soft-tissue infections (SSTIs) as a category include a broad set of pathological conditions that involve the skin and its underlying structure and range from uncomplicated superficial infections to more severe infections that extend to the subcutaneous tissue, fascia, or muscle , 3. Skinacute bacterial skin and skin structure infections (ABSSSIs). These infections can pose a challenge for the medical community, particularly with the increase in antimicrobial resistance, and are associated with high direct and indirect costs to both the healthcare system and society. Antimicrobial resistance is a growing concern and the increase in the incidence of infections caused by bacteria that have developed resistance to previously effective antimicrobials, such as methicillin-resistant Staphylococcus aureus (MRSA), has led to higher rates of complications, hospitalizations and, occasionally even death [Many SSTIs are caused by bacteria and are referred to as en death , 6. The en death , 8.Varicella-zoster virus (VZV), which is the causal agent of chickenpox and shingles [Other SSTIs are caused by viruses, most notably the shingles . Despiteshingles .Pediculus humanus capitis) is an obligate human ectoparasite that causes head lice infestation (pediculosis capitis) and is estimated to affect more than 100 million people worldwide every year [Parasitic infestations are common among children and adolescents. The head louse  can be deeply affected by these infections. Among the indirect costs associated with childhood diseases are time taken off from work by parents or other caregivers when a child must stay home from school due to illness, time spent traveling to and from hospitals and other healthcare facilities, and the time spent at the healthcare facility while the child receives treatment. Quantifying the burden for a child affected by the SSTI, however, is difficult and has not been estimated. It would need to account, for example, for lost activity, the inability to perform certain tasks, and the cost of missed educational opportunities due to school absenteeism.There are many people worldwide who are vulnerable to the economic consequences of illness, the costs of which can be either direct or indirect. The indirect cost of disease is an important factor and should be taken into account when measuring the additional impact of diseases beyond the traditional direct medical costs. Though these indirect costs are difficult to quantify due to the lack of quality data, they could represent a significant fraction of the total cost associated with many diseases. Indirect costs are the loss of earnings resulting from an adverse health outcome. Decreased productivity can arise from three situations: absence from paid work (absenteeism), including sick leave, early retirement, premature death and reduced employment (or unemployment); reduced productivity during paid work (presenteeism); and changes in unpaid work . In the There are limited data in the literature on the indirect costs associated with SSTIs in the pediatric population. Therefore, we aimed to conduct a systematic literature review to collect and summarize current data on the indirect costs associated with SSTIs in children worldwide.https://osf.io/wrvap/). The methods and findings of this systematic review were reported following the Preferred Reporting Items for Systematic Reviews and Meta-Analysis (PRISMA) guidelines [We carried out a systematic review to retrieve studies reporting the indirect costs of SSTIs in the pediatric population. This study followed the guidelines for conducting systematic reviews of economic evaluations studies published by the Centre for Reviews and Dissemination – CRD  and the idelines . The PRIA PICO approach, the details of which are available in our study protocol, was used to formulate the following research question: What is the estimation of indirect costs in managing SSTIs in the pediatric population aged 1 to 12 years? . The datWe included primary studies reporting health economic analyses of the impact of SSTIs  in the pediatric population from 1 to 12 years of age. Studies with a broader age range were included so long as there was overlap with our specified age range (1 to 12). We included studies that measured indirect costs, defined as lost earnings and productivity of both patients and their informal caregivers, and excluded studies that reported only direct costs as well as those focused on the diagnosis, treatment, epidemiology, or non-economic aspects of SSTIs. The complete list of reasons for the exclusion of articles is included in Fig. The references identified from the initial search were screened using the criteria described above to exclude irrelevant studies based on title and abstract. The full texts of the remaining articles were independently reviewed by two researchers, following the selection criteria described earlier, and disagreements regarding inclusion or exclusion of studies were resolved by consensus.For each study we carried out a quality appraisal using an adapted version of a previously developed critical appraisal checklist for cost-of-illness (COI) studies . Each poThe search yielded 503 unique references, of which 466 were excluded through title-and-abstract screening; 37 articles were reviewed in full text. Twenty-nine articles were excluded based on full-text evaluation, and 5 articles identified from a review of references were added, bringing the final total to 13 studies. The complete eligibility process is described in a PRISMA flow chart in Fig. The studies were carried out in 9 different countries and published between 1992 and 2018. Five were conducted in the United States , 28, 31 Of the studies in this review, the majority were on chickenpox, with a total of 9 studies reporting indirect costs associated with VZV infection , 29–33. Three studies reported the estimated total annual productivity loss costs due to SSTIs at the national level, being €34,623,835.89 in Poland , €17,382Two cost-effectiveness analyses of vaccination programs against VZV estimated indirect cost saving due to vaccination. Hsu et al. reported an estimated reduction of 98.9% in indirect costs with the implementation of vaccination programs (from €32.00 million to €0.34 million) , while ip < 0.04) [p < 0.001), with a €294.58 reduction in median hospital indirect costs (p < 0.001) per patient and episode [Three studies from the United States reported indirect costs associated with ABSSSI in children , 8, 28.  episode .A cost-effectiveness analysis from the United States was reviewed . From thIn this systematic review we collected and summarized all the current data on the indirect costs associated with SSTIs in children between 1 and 12 years of age, encompassing 3 highly contagious skin diseases: ABSSSIs, chickenpox and head lice . With a similar incidence of VZV and cost of lost workdays, these differences were mainly due to the differences in the number of VZV cases reported in 2015 in the 3 countries  reduced indirect costs due to a decreased number of school days and workdays lost compared with treatment with erythromycin  .Finally, the indirect costs reported by Wright et al. in the treatment of subcutaneous abscess in children were higher with traditional packing than with MI drainage, because the latter reduced hospital length of stay . Howeverpediculosis capitis is the most frequent ectoparasitic disease worldwide, and its treatment is costly, indirect costs associated with head lice were found in only 1 cost-effectiveness study, which compared 3 pediculosis capitis treatments [Although eatments . While heatments . Nevertheatments , represeOne important limitation of this study is the scarcity of cost-of-illness studies in SSTIs in the pediatric population, and of those that exist, many are based on data from clinical practice dating from 2005 or earlier. This could underestimate the true extent of the disease burden. In addition, the indirect costs included in this systematic review derived from a heterogeneous group of studies with different methodologies. We included studies related to different diseases, which may not therefore be directly comparable. In this regard, the studies included were conducted in different countries in which indirect costs are estimated according to the country-specific features of the labor market, which makes it difficult to directly compare costs across studies. Furthermore, the methodology used in some studies was not clearly described, or the results were presented as aggregated costs, and thus could not be analyzed in detail. Hence, new studies that analyze the cost-of-illness of SSTIs in pediatric patients should be designed and should present results not only as aggregated costs. This would enable the implementation of tailored strategies and policies. Additionally, these studies should include data from as many different SSTIs as possible so they can be evaluated as a whole.Aside from the scarce data on indirect costs of SSTIs in the pediatric population, the QoL of these patients is also underreported. These skin disorders may cause several disturbances, stigmatization, and psychosocial consequences that affect the lives of both the patients and their families. Therefore, it is of utmost importance to assess to a greater extent the impact of these diseases on the overall QoL in pediatric patients by designing studies evaluating QoL outcome measurements , 45. ThiIn conclusion, there is no doubt that productivity losses attributable to SSTIs in children are extremely important. The economic burden of SSTIs, though highly relevant, is underestimated due to the lack of studies, particularly in bacterial SSTIs. Although many cost-of-illness studies acknowledge the importance of indirect costs and benefits, they are seldom included in economic assessments. New, improved cost studies using a precise, standardized indirect cost estimation methodology will give us a better understanding of the magnitude of the economic disease burden and allow us to determine the efficacy of health policies and develop programs to improve public health.Additional file 1 : Supplementary Table 1. PRISMA Checklist.Additional file 2 : Supplementary Table 2. Search strategies.Additional file 3 : Supplementary Table 3. Result of the quality appraisal.Additional file 4 : Supplementary Table 4. Risk of bias in observational studies included in the systematic review.Additional file 5 : Supplementary Table 5. Risk of bias assessment of included model-based economic evaluations (based on the ECOBIAS checklist).


Tombusviridae. Bio-indexing and RNA-seq experiments indicate that CYVaV can induce yellow vein symptoms independently of known citrus viruses or viroids.Citrus yellow-vein disease (CYVD) was first reported in California in 1957. We now report that CYVD is associated with a virus-like agent, provisionally named citrus yellow-vein associated virus (CYVaV). The CYVaV RNA genome has 2,692 nucleotides and codes for two discernable open reading frames (ORFs). ORF1 encodes a protein of 190 amino acid (aa) whereas ORF2 is presumably generated by a −1 ribosomal frameshifting event just upstream of the ORF1 termination signal. The frameshift product (717 aa) encodes the RNA-dependent RNA polymerase (RdRp). Phylogenetic analyses suggest that CYVaV is closely related to unclassified virus-like RNAs in the family  For instance, non-coding satellite RNAs are incapable of all three functions  tree was graft-inoculated with YV-920, and upon expressing yellow vein symptoms it was held at the CCPP as the CYVD source YV-920C. Since the first report of CYDV in Eustis limequat [Citrus aurantifolia (Christm.) Swing. x Fortunella japonica (Thunb.) Swing.] in California by via dodder or by arthropods. It is readily graft-transmissible to multiple citrus species with the exception of Poncirus trifoliata (L.) Raf. Sensitive indicators such as “Etrog” citron (C. medica L.) and Mexican lime [C. aurantifolia (Christm.) Swingle] develop bright yellow discoloration of petioles and veins. In some cases, the symptoms extend into the young flushes and stems. The yellowing is persistent and visible in older leaves and branches  accession was introduced by Dr. L. G. Weathers most likely from Tulare County, California, into the disease bank of the Citrus Clonal Protection Program (CCPP) at the University of California, Riverside (UCR) under the name YV-920. The original 1969 YV-920 isolate was used in a series of bio-indexing experiments on various citrus indicators, resulting in consistent expression of yellow vein symptoms while testing negative for other graft-transmissible diseases of citrus, such as exocortis, psorosis, tristeza, and concave gum (CCPP bio-indexing records). In a bio-indexing experiment in September 1985, an “Etrog” citron  showed pronounced psorosis and suppressed yellow vein symptoms. Weathers noted that CYVD “… is caused by a virus or viruses…” that have “…some type of interaction with the unrelated viruses of psorosis and vein enation.” Most importantly, however, Weathers indicated that CYVD “… is different from any previously described diseases of citrus”  enhanced yellow vein symptoms and caused stunting and occasional plant death , which is capable of inducing yellow vein symptoms independently of other known citrus viruses or viroids.Citrus plants  used in this study were graft-inoculated with YV-920C as original source of inoculum and maintained in the CCPP quarantine greenhouse at 22–29°C and 16 h light regime.Stems with yellow vein symptomatic leaves were harvested 2.5 years after inoculation, and double-stranded RNA (dsRNA) was extracted from 20 g of bark scrapings using two-cycles of CF11 column chromatography as previously described . dsRNA wTaq DNA polymerase was used to add adenylate residues to the 3' ends of the double-stranded cDNAs, which were then ligated into pGEM-T easy vector . Recombinant colonies were randomly selected, and recombinant plasmids were Sanger-sequenced at the UC Riverside Genomics Core Facility. The sequences of 48 cDNA clones were assembled using the ContigExpress tool (Vector NTI Advance 10-InforMax) and compared with the NCBI nucleotide and protein databases. Two assembled contigs with non-contiguous sequences  with a low E-value to viruses were obtained by BLAST analysis. The sequence gap between the two contigs was filled by real-time polymerase chain reaction (RT-PCR) with specific primers based on sequences flanking the gap  was used to construct a cDNA library using random primers according to the manufacturer’s instructions.  the gap . The seq the gap . Potenti the gap . The CYV49-69, 5'-CCA GAC AGG TGT TTC GAG CAT-3'; R1566-584, 5'-CAA TCA CTG CAA ATC GCG A-3', 536 nt amplicon) and 3' end  of the JX101610 sequence. Reactions were performed with the OneStep RT-PCR Kit  with the same final concentration of buffers and primers (6 μM) per manufacturer’s instructions. The RT step was carried out at 50°C for 30 min followed by a 15 min denaturation at 95°C for activation of the hot-start Taq polymerase. The PCR conditions were as follows, 35 cycles at 94°C for 30 s, 56°C for 30 s, 72°C for 1 min, and a final extension step at 72°C for 10 min. PCR products were analyzed by electrophoresis on 1.5% agarose gels buffered in TAE  and visualized by UV light after ethidium bromide staining and sequenced at the UCR Genomics Core Facility.Two sets of CYVaV specific RT-PCR primers were designed at the 5' . A Maximum likelihood phylogenetic tree for the nucleotide sequence of CYVaV-related viruses and virus-like RNAs was constructed using the Tamura-Nei substitution model for complete nucleotide sequence with rates among sites gamma distributed with invariant site (G + I). Bootstrap values were calculated using 1,000 pseudo replications. Clades with less than 70% bootstrap support were collapsed.Citrus yellow-vein associated virus sequence identities were analyzed by comparing with sequences in GenBank using BLASTn and BLASTx. Nucleotide and amino acid sequences retrieved from GenBank were aligned using CLUSTAL W . IllustrC. aurantium L., vein enation indicator), and Mexican lime (yellow vein and vein enation indicator) were used per experiment. The 2009 (experiment #3163) graft-transmission experiment used the YV-920C as the source of inoculum. In the 2016 experiment (experiment #3358), a 2009 yellow vein symptomatic “Etrog” citron plant (experiment #3163‐ tree 02) was used as the source of inoculum. In the 2020 experiment, a 2016 Mexican lime plant (experiment #3358‐ tree 70) expressing only yellow vein symptoms was used as the source of inoculum. Non-inoculated indicators and vein enation controls (CCPP Disease Bank isolates VE 701 and 709) were included in all experiments. The indicators were maintained under cool greenhouse conditions (24–27°C) and observed periodically for symptoms expression , sour orange (pression .Clonorchis sinensis (GCF_000317415) using Bowtie2 version 2.3.4.1  and the CCPP Disease Bank at the Rubidoux Quarantine Facility , respectively, were also tested. Libraries were constructed using the TruSeq Stranded Total RNA Sample Library Prep with plant rRNA depletion  using TRIazol®  -extracted RNA. The libraries were sequenced using Illumina HiSeq 4000 or NextSeq 500 platforms (2 × 150 bp). Quality control and low-quality reads were filtered out using Fastp , and gen 2.3.4.1 . Reads t 2.3.4.1 , and the 2.3.4.1 .via a −1 ribosomal frameshift leading to a fusion protein product of 717 aa (81 kDa). Sequence analysis of the overlap region between ORF1 and ORF2 revealed the presence of a slippery haptanucleotide, GGGUUUU560-566 typically responsible for the −1 frameshifting event , sugarcane umbra-like virus , and Ethiopia maize-associated virus .BLASTn analysis of ORF 1 did not identify any accessions currently deposited in the GenBank with similar nucleotide sequences. Based on BLASTx analysis, this protein is an apparent ortholog of proteins encoded by unclassified virus-like RNAs such as opuntia umbra-like virus  only to the OULV ortholog. Based on BLASTp analysis, CYVaV RdRp shares the highest aa sequence identities to the OULV orthologs 79%  and to the orthologs of EMaV  and SULV . Lower aa sequence identities (>50%) were shared with orthologs of unclassified virus-like RNAs, such as papaya virus Q , babaco virus Q , and papaya meleira virus 2 . Low aa sequence identities (<40%) were shared with orthologs of umbraviruses, carmoviruses, tombusvirus-like associated RNAs (tlaRNAs), and other members of the family Tombusviridae  and recovering the expected size and sequence amplicons (data not shown).Analysis of the sequence at the 3' end of the CYVaV genome revealed two hairpins in similar locations as in umbravirus and carmovirus, known as H5 and Pr . The putSimilar to umbraviruses, CYVaV has a short (8 nt) 5' UTR containing a perfect carmovirus consensus sequence , the YV-920C “Etrog” citron plant used in the dsRNA analysis served as the source of inoculum. The two subsequent experiments (2016 and 2020 experiment #3358) were graft-inoculated from a selected plant indicator expressing only yellow symptoms . In all three experiments, the cumulative total number of yellow vein indicators expressing typical yellow vein symptoms was 6 of 6 for “Etrog” citron and 22 of 22 for Mexican Lime. On the other hand, none of the vein enation indicators, sour orange (0/16) and Mexican lime (0/16) expressed vein enation symptoms. In all experiments, positive controls began expressing symptoms within 7 weeks post graft-inoculation whereas the non-inoculated controls remained asymptomatic for the duration of the experiments.VirFind based bioinformatic analysis for the identification and discovery of RNA-seq reads related to all potential viruses or viroids, on “Etrog” citron (2009 experiment #3163‐ tree 02) and Mexican lime (2016 experiment #3358‐ tree 70) expressing yellow vein symptoms, identified several contigs of 2,686–2,692 nt length containing the near full-length genome of CYVaV. VirFind analysis did not identify sequences of any known graft-transmissible viral pathogens of citrus . MultiplTo determine the nature of the CYDV pathogen, we performed multiple screenings of citrus trees with characteristic symptoms. This demonstrated that a novel RNA molecule, termed CYVaV, is associated with CYVD symptom development independently of any known graft-transmissible viral pathogens of citrus.Tombusviridae. For example, the CYVaV RNA genome of 2,692 nt is smaller than the umbra-, carmo-, and tombus‐ viruses  as well as the tlaRNAs . In addition, while CYVaV contains two ORFs, like some tombusvirids, encoding the required replication components, it is missing the overlapping movement protein ORFs found in all umbraviruses .Despite its distinct genome size and organization CYVaV shares important RNA structural features on its 3' and 5' ends with members of the family sviridae , 5. CYVasviridae . The penrus TCV;  and tombrus TCV; . In CYVa1-3A/U3-8. This sequence usually begins with three guanylates as in the case of CYVaV ; Gof CYVaV . The CYVRecent studies on various plant hosts, reported small viruses and virus-associated RNAs of approximately 2.8–4.5 kb in size containing two or more ORFs . CYVaV hTombusviridae codes for a replication required protein that is involved in membrane reorganization and interaction with host factors  while any helper virus was not moved forward to the plants of the YV-920C isolate used in our experiments. This could also explain the positive and negative biological indexing results for vein enation recorded previously for the yellow-vein isolates at the CCPP (data not shown). Further studies are required to prove such hypotheses and more importantly to characterize sequence and structural features that may support, the conjectural CYVaV encapsidation and insect transmission but also to exclude the possibility that one or more unknown virus(es) are supporting the CYVaV biology and the phenotypes observed in this study  field trees, that appeared to be free of either psorosis or exocortis and it was not contaminated with other known viruses , 1961. His study .Tombusviridae. On that basis, additional research efforts are currently focused on fulfilling Koch’s postulates using full-length infectious clones, studying the replication and systemic movement of CYVaV in plants, setting up field trials to evaluate CYVaV effects on tree growth and yield in single and mix infections with other citrus pathogens, developing CYVaV as a virus-induced gene silencing (VIGS) expression system, a potential RNA interference (RNAi) based approach for citrus pest and disease management  can be found in the article/S-JK, IT, AS, and GV conceived and designed the experiments. S-JK discovered and S-JK, SB, KG, TH, FO, and MR characterized the CYVaV RNA. AS performed structural sequence analysis. SB, KG, and GV conducted bio-indexing experiments and TD performed RNA-seq experiments. S-JK and GV wrote the first draft of the manuscript and all authors contributed to manuscript revision, read, and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Brassica napus and Brassica rapa for artificial synthesis interspecific hybridization of F1. The F1 self-fruiting rate was particularly low. By comparing the fertilization rate and seed setting rate of nine crosses and selfing combinations of interspecific hybrid progeny F1 and control B. napus, the results proved that the genetic stability of egg cells was greater than that of sperm cells, so the F1 could get seed by artificial pollination with other normal pollen. Based on these results, interspecific maternal inbred offspring (induced F1) from egg cells was obtained by emasculation and pollination with the pollen of DH inducer Y3380. It was found through morphological analysis, flow cytometry identification, and meiotic observation of induced F1, the plants had most normal fertile tetraploid and the meiosis was normal. The FISH results showed that the induced F1 were B. napus , 20 A and 19 C chromosomes. The results of SNP chip detection and genetic cluster analysis found that the genetic variation between interspecies could be preserved or broadened in the induced F1. The use of DH inducer created special breeding resources for interspecific hybridization and distant hybridization of rapeseed while shortening time, improving efficiency, and providing a new insight into innovate breeding resources.Interspecific hybridization of rapeseed is an important way to innovate breeding resources. This research used  Brassica species or between closely related genera is one of the most important means to innovate the resources of B. napus. It can help to improve the traits of B. napus, such as the hybrid offspring was rich in genetic diversity and higher yield [B. napus [B. napus [B.napus, etc. [1 is low and there are other methods that cannot only shorten the breeding period, but perhaps can maintain the genetic distance of hybrid offspring.Distant hybridization between er yield ,2, broadB. napus , create B. napus , signifiB. napus , breed aB. napus , and incus, etc. . Howeverus, etc.  and chrous, etc. , but conus, etc. , and theus, etc. ,12,13,14us, etc.  and cultus, etc. ,17. MicrBrassica crops (Brassicaoleracea and Brassica napus) [B. napus, referred to as DH induction line (DH inducer). Through the observation of the induction phenomenon, the phenotype of the offspring, and the detection of SNP chip, it was speculated that the induction process was due to the zygotes produced by the inducible sperm cells and the maternal parent eggs, the paternal chromosome being eliminated during the mitosis of the zygote, and then the maternal chromosome doubling and forming DH offspring [1 continuous backcrossing with the parent to double the chromosomes of interspecific F1 egg cells form induced offspring with genetic diversity? Therefore, this study used the hybridization of B. napus  and B. rapa  to obtain F1. By comparing the genetic stability of the pollen mother cell and the egg cell of F1, it was found that the genetic stability of the egg cell of the interspecific hybridization was greater than the sperm cell. The results laid a theoretical foundation for the induction of interspecific hybrid F1 by DH inducer to obtain normal offspring. The progeny of the interspecific hybrid F1 induced using the DH inducer was called the induced F1. The chromosome composition was analyzed by chromosome fluorescence in situ hybridization, and the homozygous site rate of induced F1 and the genetic distance from the parent were analyzed by SNP to determine whether induced F1 was stable new rapeseed, and whether its genetic variation had broadened. The research aims to solve the shortcomings of the long cycle of creating new germplasm in rapeseed, and to provide new insights and methods for rapeseed resource innovation.In recent years, Fu ,19 syntha napus) , a largeffspring ,22. Can B. napus, AACC, 2n = 4x = 38) were used as female parents, and Y6 and HZ32  as male parents, respectively, and interspecific hybridizations were carried out  of interspecific hybridization could normally produce tetraploid progeny induced by induction lines, also indicating that the heredity of female gametophytes was more stable than that of male gametophytes.The pollen mother cells (PMCs) of induced Faphase I i. The chaphase I i; the ceaphase I k. Duringaphase I l. The me1 could not self-bred was that the germination rate of triploid pollen was low and could not germinate normally in the stigma, therefore the pollen tube could not enter the ovule to complete fertilization. However, using the DH inducer Y3380 pollen to pollinate T19-4 and T19-5, the fertilization rate and the seed setting rate were much higher than those with self-pollen. This also confirmed why T19-4 (T19-5) × Y3380 could obtain a large number of induced progenies.As the egg cells were not easy to separate and the number of egg cells was sufficient, they were difficult to use for cytological research. It was necessary to determine how to compare the genetic stability of sperm and egg cells with interspecific hybrids of rapeseed. In this study, we observed the pollen adhesion of nine cross combinations of T19-4 and T19-5 after pollination for 24 h. By comparing the fluorescence intensity of the stigma with the amount of fluorescence, the results showed that the fluorescence intensity of the pollen of the inducer lines Y3380 and ZS11 at the stigma of T19-4, T19-5, and ZS11 was stronger a, and po1 emasculation and pollination, induced F1 offspring were obtained. From the chromosome number and flow cytometric identification, it was proven that part of induced F1 was tetraploid, but it was unclear whether the constitution of chromosomes A and C was consistent with that of normal B. napus. In order to obtain the chromosomal constitution of interspecific hybridization F1 and tetraploid-induced F1, fluorescence in situ hybridization was performed on T19-4, T19-5, W58, and W59, and ZS11 was used as a control. The result showed that ZS11 had 38 chromosomes, 18 C genome chromosomes , and 20 A genome chromosomes  (1 (T19-4) of interspecific hybridization, nine chromosomes of C genome , and 20 chromosomes of A genome  (1 chromosome of interspecific hybridization was AAC (2n = 3x = 29). The result of induced F1 was the same as that of ZS11, with a total of 38 chromosomes: 18 C genome chromosomes , and 20 A genome chromosomes   induced by the DH inducer Y3380 was a tetraploid B. napus . This meant that the matched structures of chromosomes 20 A and 18 C were relatively stable during induction. In addition, we selected some hexaploid plants from crossing by interspecific hybridization F1 and octoploid induction line Y3380 to observe by fluorescence in situ hybridization: Hexaploid W58 and W59 had about 54 chromosomes with 27 C genome chromosomes , and 27 A genome chromosomes   a. There  signal) b. The co signal) c. Theref signal) d.1 generation, the surviving 13 strains of W58 line and five strains of W59 line were analyzed by 50 k SNP chip. In total, 42,090 effective sites were detected, classified, and counted as AA, BB, AB, respectively. AB represented the heterozygous site; AA and BB were the homozygous sites; and the percentage of homozygous sites was counted. The homozygous rate per plant of W58 line was 74.24%–81.63% (1 generation of interspecific hybridization induced by the DH inducer Y3380 could quickly select more homozygous sites and provided a higher homozygous rate (greater than 70%) for a stable single plant. In general, the homozygous rate of hybrid offspring was about 60%. For the parents, by comparing F1, F2, induced F1, BC1F1, differences in SNP loci between the two materials, an n × n matrix could be obtained. The smaller the difference, the smaller the value of the matrix elements, and the closer the distance between the two samples. Thus, all samples were effectively clustered together, and a cluster diagram of the genetic distance of the samples was drawn , it was found that the hybrid offspring of B. napus  and B. rapa  T19-4 and T19-5 have abnormal meiosis. This resulted in low pollen fertility and low pollen vigor [B. napus and B. rapa for self-pollination to obtain a large number of stable genetic offspring.The hybrid sterility of interspecific hybridization F obvious , and the obvious ,24,25. Ten vigor . In addi1 through DH induction lines, the key question was whether the interspecies hybridization F1 megaspore mother cells could produce normal megaspore gametes. In order to determine whether the megaspore mother cell had abnormal meiosis and whether it could be fertilized normally, the normal tetraploid pollen was crossed with the interspecific cross F1 (triploid), the ovule was stained with aniline blue to determine whether the ovule was fertilized, and the seed setting rate was analyzed    b. Tetrap8, AACC) c. This rcidental .1 was induced by the inducible line Y3380, it was more likely to form normal tetraploid B. napus . Again, this proved that the meiosis of the egg cells of interspecific hybrid F1 were relatively normal, and it was easy to form normal gametes.Therefore, through the observation of the ploidy change and FISH of the progeny induced by interspecific hybridization, we found that after the interspecific hybrid F2 (W97), backcross offspring BC1F1 (W99) and the parent  of interspecies hybridization. The chromosomes of the inducible line were lost or incomplete lost after the interspecific hybridization FB. napus were used in this study: P3-2 bred by Fu of Chengdu Academy of Agricultural and Forestry Sciences  [Brassica napus L., AACC, 2n = 4x = 38). Two common Brassica campestris: Y6 , HZ32  were also used. The octaploid Y3380  artificially synthesized by Fu of Chengdu Academy of Agricultural and Forestry Sciences in 2017 was a DH inducer in B. napus [1 developed in May 2019. In October 2019, F1 was planted in the network room of Chengdu Academy of Agriculture and Forestry Sciences, with equal row spacing and even seeding. In March 2020, F1 was bagged and self-bred to obtain F2; F1 were pollinated with Brassica DH inducer Y3380 to induce F1; F1 was backcrossed with female parent P3-2 or ZS11 to obtain BC1F1, T19-4, and ZS11 hybridized obtained CK1, ZS11 and T19-4 hybridized obtained CK2, and ZS11 and T19-5 hybridized obtained CK3, ZS11 self-bred obtained CK4. The schematic diagram of each hybridization combination is shown in Two samples of common 4x = 38) , the conB. napus . The abo1 and induced F1, planted at the seedling stage, selecting representative plants after pod harvest, taking photos with SLR camera (Canon E0s 200D), and making statistics on the seed setting rate of F1, BC1F1, induced F1, and CK [1, and induced F1 was determined [1 and induced F1 pollen and calculated the germination rate after 4 h. As pollen germination occurred, the length of the pollen tube was larger than or equal to 2 times the diameter of the pollen grain. Germination rate percentage was calculated by following formula: germination rate % = germinated pollen number/total pollen number × 100.The morphological observation of this experiment mainly included the morphological characteristics of parents, F, and CK ,22. The termined . The poltermined  method t1, and induced F1 to determine the ploidy according to the reference, from 8:00 to 11:00 am. We took the fresh young leaves of the tested plants, washed them with distilled water, and dried them with filter paper; avoided the main leaf veins, took the leaves with a hole punch with a diameter of 8 mm and placed them in a pre-cooled petri dish, and added 0.5 mL of pre-cooled cell lysis buffer . We used a sharp razor to quickly chop the leaves to very fine particles and filtered with a 300-mesh filter into a 2 mL EP tube. After that, 1.5 mL PI staining solution (5% propidium iodide and 5% RNase) was added in the dark for 30 min and then measured on the machine to collect 20,000 cell particles. Before testing the samples, we used ZS11 as a reference for calibration , and used this as a reference for ploidy determination. We used a flow cytometer  to measure the processed material. The PI-stained cell nucleus suspension entered the flow chamber and emitted fluorescence through laser irradiation. Then, BD AccuriC6 Plus was used to determine the fluorescence value  at the position of cell division G1. Sample ploidy = reference material ploidy ×  at the position of sample G1 peak/fluorescence value  at the position of G1 peak of reference material).Flow cytometry has been widely used in the determination of DNA content ,36. In tMitosis was observed with the root tip. The rapeseed seeds were sterilized in 75% alcohol for 30 s at 9 a.m., 0.1% mercury liters for 5 min, and sterilized water was rinsed more than three times. The seeds were placed in a sterilized germination box and cultured in the dark at 25 °C for 24 h in an incubator. At 9 a.m. the next day, the germination box was placed in an incubator at 15 °C for 12 h in the dark, and at 9 p.m., the germination box was placed in an incubator at 25 °C for 12 h in the dark according to the method of Li  and LangBrassica stigma. We counted the fertilization rate 48 h after pollination. Fertilization rate percentage was calculated by the following formula: Fertilization rate % = the number of ovules with fluorescent dots in the silique/total number of ovules in the silique × 100.We took the pistils of different cross combinations at 24 h and 48 h after pollination. We took 3 siliques from each material for three repetitions, fixed them with Carnoy’s solution (ethanol: acetic acid = 3:1) for more than 24 h, then transferred to 70% ethanol for 30 min, rinsed with distilled water for 30 min, then transferred to 1 M NaOH solution for softening treatment for 12 h, then We took the material out and rinsed it three times with distilled water, each time for ten minutes. We used 2% modified aniline blue dye solution , 1 M (pH 9.5) glycerin, and distilled water mixed in a volume ratio of 5:8:7) for 3–5 h  under coBrassica C genome was provided by Huazhong Agricultural University and had been introduced into the E. coli plasmid vector. The bacterial solution of E. coli was thawed and activated in LB medium (25 μg/mL chloramphenicol), and cultured at 37 °C. We shook the bacteria overnight and scribed to LB medium plate (25 μg/mL chloramphenicol), cultured overnight at 37 °C. A single colony of Escherichia coli was selected in liquid LB medium (25 μg/mL chloramphenicol) and cultured overnight at 37 °C. The DNA probe of the overnight culture broth was extracted using the Gold HiEndofree Plasmid Maxi Kit instruction method to extract the probe DNA of the specific rapeseed C genome sequence BNIH123L05 in the BAC library. The probe was labeled using the Nick Translation method [2) twice, and then 10 μL hybridization solution [The DNA special probe BNIH123L05  in the Bn method  and the solution  (labeledBrassica 50K SNP (single nucleotide polymorphism) chip  to extract parents, F1, induced F1, BC1F1, Brassica DH inducer Y3380. The genomic DNA was sent to Wuhan Shuang lv yuan Research Institute for final data analysis. According to the theta value of the sample, the genotypes were classified into three types: AA, BB, and AB. AA and BB represent two homozygous genotypes, and AB represents a heterozygous genotype. Based on the B. napus genome Darmor4, the frame of each chromosome was constructed. The ordinate was the length of the chromosome (Mb), and the abscissa was the chromosome name (the corresponding random chromosome on the right). We marked the sites with heterozygous genotypes (AB genotype) in blue on the map . By comparing the loci difference between the two samples, an n × n matrix was obtained, and the Test Maximum L of MEGA 11 software was used to calculate the Nei genetic distance and construct the genetic cluster map.SNP chip detection was completed by Wuhan Shuang lv yuan Research Institute, using B. napus and B. napus. It was found that the disorder of pollen mother cell meiosis easily produces abnormal gametes, which leads to the low seed setting rate of selfing offspring. After backcrossing, self-crossing, and induction, the average fertilization rate and average seed setting rate of the offspring of hybridization between B. napus and B. napus were calculated. The genetic stability of egg cells was greater than that of sperm cells, which laid the foundation for DH induction. In view of this result, the DH inducer was used to induce the chromosomes of the egg cell (AC) to double which formed the self-progeny of the egg cell. Induced offspring could form tetraploid B. napus . At the same time, the genetic distance between some individual plants and the parent was greater than that of hybrid F1, backcross offspring, and the genetic distance between selfing offspring and parent, which enriched the genetic variation of offspring. This research provided a new insight and perspective for interspecific hybridization of rapeseed.In this study, pollen fertility statistics, pollen vitality statistics, and pollen mother cell meiosis observations were performed on two pairs of hybridization between


Under the serious DVT scenario (0 mmHg vs. 150 mmHg), m10, m40, and m50 achieved accuracies of 73%, 76%, and 73%, respectively. The experimental results show that this proposed examination system may be practical as an auxiliary tool to screen DVT in homecare settings.Deep vein thrombosis (DVT) of lower limbs can easily arise from prolonged sitting or standing. Elders and pregnant women are most likely to have this disease. When the embolus of DVT comes to pass the lung, it will become a life-threatening disease. Thus, for DVT disease, early detection and the early treatment are needed. The goal of this study was to develop an examination system to be used at non-medical places to detect the DVT of lower limbs with light reflection rheography (LRR). Consisting of a wearable device and a mobile application (APP), the system is operated in a wireless manner to control the actions of sensors and display and store the LRR signals on the APP. Then, the recorded LRR signals are processed to find the parameters of DVT examination. Twenty subjects were recruited to perform experiments. The veins of lower limbs were occluded by pressuring the cuff up to 100 mmHg and 150 mmHg to simulate the slight and serious DVT scenarios, respectively. Six characteristic parameters were defined to classify whether there was positive or negative DVT using the receiver operating characteristic curves, including the slopes of emptying and refilling curves in the LRR signal, and the changes of venous pump volume. Under the slight DVT scenario (0 mmHg vs. 100 mmHg), the first three parameters, m Deep vein thrombosis (DVT) occurs when a blood clot forms in one or more of the deep veins in the human body, which will change the blood flow of veins and result in poor blood circulation. Detection of DVT is usually a clinical challenge for doctors of all medical specialties, and deterioration of such disease may make the treatment process more complicated. Moreover, DVT can happen even in the absence of serious disorders. As investigated, thrombosis can occur in any part of the venous system, but it appears most frequently at the deep veins of the legs . Post-thTraveler’s thrombosis is caused by sitting still in a transport vehicle for a long time, and blood clots are formed and deposited inside the deep veins of lower limbs, which constitutes DVT . Scurr eClinical examinations of DVT are currently performed using ultrasound or venography, and they all need to be carried out by professional technicians or physicians in a hospital ,6. MoreoAlthough the impedance plethysmography has good sensitivity in detecting venous thrombosis in the hip, thigh, and popliteal area, it is not very sensitive to calf vein thrombosis ,11. The In recent years, people have been fond of using wearable devices to monitor their bodies’ condition in daily life. In various wearable devices, an exercise watch or band can be utilized to measure the heart rate and the number of steps ,18, an eLRR, a noninvasive and non-ionizing radiation measurement technique, can be applied to observe whether a lower limb has DVT by means of reflective PPG sensors. In the examination procedure, the physician has to look for an appropriate position for placing the PPG sensor to obtain a reliable PPG signal, and guides the patient to perform the dorsiflexion at the ankle to push the blood in the vein of the calf back to the heart. However, it is hard for users, at home, to check the vein condition by such medical grade apparatus. The aim of this study is to develop an examination system, available for use in non-medical places, to detect the DVT of a lower limb. The system included a wearable device with commercial reflective PPG sensors and an accelerometer to measure LRR signals, and a mobile application (APP) to control the works of sensors, guide the rhythm of foot action, and display and store the LRR signals in real time. An array sensor embedded with three reflective PPG sensors was designed to detect the change of venous blood flow during the calf pumping. An accelerometer was placed at the foot to simultaneously monitor the dorsiflexion action at the ankle. In order to verify this prototype, twenty healthy subjects were included to participate in this study. The veins of a lower limb were partially or totally occluded by inflating the cuff up to 100 mmHg or 150 mmHg to simulate the stenosis phenomenon in slight or serious DVT scenarios, respectively. The identifiable rules for determining the quality of the LRR signals from the different PPG sensors were studied. Thus, the LRR signal with the best quality and sensitivity was chosen. Six characteristic parameters regarding the LRR signal were defined as the slopes of emptying and refilling curves in the LRR signal and the change of venous pump volume. Those parameters were used to classify whether the DVT occurrence was positive or negative by a receiver operating characteristic (ROC) curve. The results showed that the prototype of the examination system has the potential for screening for DVT in non-medical environments.x-axis signal of the accelerometer that are uploaded to a personal computer for further smoothing of the LRR signals and for acquiring the characteristic parameters from the LRR signals with Matlab R2015a version.x-axis signal of the accelerometer  is sampled by the MCU with a 12-bit analog-to-digital converter. The MCU of the main board is a 16-bit microcontroller , which reconciles the workflow of the MAX30102 and ADXL325, performs the signal processing to obtain the DVT-related parameters, and transmits the data to the APP by a Bluetooth module . The power circuit includes a Li battery (150 mAh), a charging integrated circuit (IC) (TIBQ24072), and a regulator IC . The size of the main board is 54 mm × 70 mm. x-axis signal is shown in The LRR signal measured by PPG sensors is a direct current (DC) component of the PPG signal, representing the change of venous blood flow. However, The PPG signal not only contains the venous flow, but also contains the pulses of arterial blood flow. Thus, the proposed system applied a fourth-order Butterworth low-pass filter with a cut-off frequency of 0.5 Hz to filter out high-frequency noise and used a three-point interpolation technique to smooth the LRR signal. The zero-crossing points of the differential filtered signal were used to find the peaks and valleys of the LRR signal. Then, the median point between the neighbor peak and valley was found in the LRR signal. The continuous three median points were used to build the LRR interpolation signal by a second-order polynomial function for approximating the smooth LRR signal. x-axis signal has larger alternating amplitudes in the emptying phase than those in the resting and refilling phases. Thus, the first maximum negative and last positive changed points of the x-axis signal is used to define the start and end points, Xstart and Xend (red and black star symbols), in the emptying phase. Then, the start maximum point of the smooth LRR signal, LRRstart (red circle symbol), is found between before and after 50 points of Xstart, and the end maximum point of the smooth LRR signal, LRRend (black circle symbol), is found between before and after 50 points of Xend, as shown in start corresponds to the resting baseline (short green dash line), and the average within the five seconds before the ending measurement represents the refilling baseline (long blue dash line). In the study, six characteristic parameters from the LRR signal were defined to identify the DVT, as follows. VP1 is defined as the change of venous pump volume between the LRRend and resting baseline, VP2 is the change of venous pump volume between the LRRend and refilling baseline, m10 is the slope of the empty curve from the LRRend to LRRstart, m40 is the slope of the refilling curve from the LRRend to the point at 40 s, m50 is the slope of the refilling curve from the LRRend to the point at 50 s, and m60 is the slope of the refilling curve from the LRRend to the point at 60 s.The average of the LRR signal within five seconds before LRRx-axis signal is larger than 90°, such an LRR signal is considered of low quality, as show in 1 or VP2 is negative, the LRR signal is determined to be of low quality, as show in Two fundamental rules for determining the quality of LRR signals on the different PPG sensors were formulated. The first rule states that, according to LRR, the PPG sensor should detect the downstream of venous blood flow in the emptying phase. If the PPG sensor detects the upstream of venous blood flow, the sensor may have been placed at a wrong position. Thus, when the phase angle between the filtered LRR signal (red line) and In the study, an APP program is designed to control the functions of the wearable device a, to insThis study recruited twenty healthy subjects  without cardiovascular disease or injured limbs. Their age was between 20 and 24 years , weight between 38 and 80 Kg (60.9 ± 10 Kg), and height between 150 and 180 cm (164.4 ± 9.2 cm). The vein of one lower limb was occluded by pressuring the cuff up to 100 mmHg and 150 mmHg to simulate the slight and serious DVT scenarios. This experiment was approved by the Research Ethics Committee of China Medical University & Hospital (No. CMUH107-REC3-061), Taichung, Taiwan.Subjects were requested to comfortably sit on a chair with the foot resting flat on the floor, and the angle between calf and thigh was about 110°, as show in In the study, the terms, true positive (TP), false positive (FP), true negative (TN) and false negative (FN), refer to the result of our experiments and the correctness of the classification. TP means that a subject was correctly diagnosed as DVT, FP means “incorrectly diagnosed as DVT”, TN means “correctly diagnosed as non DVT”, and FN means “incorrectly diagnosed as non DVT”. Here, the performance of the proposed method was evaluated using accuracy, (TP+TN)/(TP+FP+FN+TN); precision, TP/(TP+FP); sensitivity, TP/(TP+FN); and specificity, TN/(FN+TN).t-test to assess the performances of six parameters for the sensitivity of DVT detection. A p-value of 0.05 or lower is considered statistically significant (represented in red) between the negative (0 mmHg) and positive (100 and 150 mmHg) groups. We find that all parameters have significant differences between the negative and positive groups. However, under the same scenario comparison (0 mmHg vs. 150 mmHg), p-values of the six parameters in the IR-light measurement are lower than those in the red-light measurement. Moreover, the absolute mean values of six parameters all decrease as the colluded pressure increases. To select the best sensitivity value of each parameter from three LRR signals, we analyzed the maximum, median, and minimum values of parameters under the different venous occluded scenarios in the red-light and IR-light measurements, as shown in p-value, which was the optimal value to detect DVT. We find that m10, VP1, and VP2 should select the maximum values; m50 and m60 should select the minimum values; and m40 should select the median values. An ROC curve was used to evaluate the performances of six parameters in the IR-light measurement in the various DVT scenarios. 10, m40, and m50 in the slight DVT scenario (0 mmHg vs. 100 mmHg), having accuracies of 72%, 69%, and 69%, respectively. The first three parameters in the serious DVT scenario (0 mmHg vs. 150 mmHg) are m40, m50, and m10, with accuracies of 76%, 73%, and 73%, respectively. The first three parameters with a larger ROC area in the slight DVT scenario are m40, m10, and VP2, with areas of 0.82, 0.79, and 0.78, respectively. The first three parameters with larger ROC area in the serious DVT scenario are m40, VP2, and m50, with areas of 0.86, 0.81, and 0.81, respectively. Therefore, m40 and m10 have the best performances to detect the DVT conditions in the serious DVT scenario, meaning that the cut points are −1.79 mV/s and 3.05 mV/s. 40 in the slight DVT scenarios, and DVT is a prevalent disease with an occurrence of between 2.5% to 5% in the total population, and about 30−40% of DVT patients will finally suffer from pulmonary embolism . The dia40 and m10 in the serious DVT scenario are 76% and 73%, and 0.86 and 0.78, respectively. Moreover, for various venous occluded scenarios, the performance of m40 is significantly lower using the occluded pressure of 100 mmHg than the occluded pressure of 150 mmHg, with sensitivity and specificity of 100% and 10% vs. sensitivity and specificity of 100% and 20.2%, respectively. The present results seem no better than previous ones [The LRR technique has been used in several studies for the noninvasive examination of patients with suspected lower limb DVT ,29. The ous ones ,15,16. T40 and m10. In the second rule, the values of both VP1 and VP2 parameters should be greater than zero. If the value of either the VP1 or the VP2 parameter is negative, the baseline drift phenomenon must exist in the LRR curve operation.The study has successfully developed a DVT examination system that may be available in non-medical places and help to examine whether users have DVT of lower limbs. To do so, an obvious challenge is how to obtain an optimal position for the LRR probe in order to apply the system. In the first rule proposed in the study, both the accelerometer and the LRR signals in the emptying phase are used to determine if the PPG sensor detects the upstream or downstream venous blood flow. Once the LRR curve is obtained from the downstream of the venous blood flow, it can be further utilized to find two characteristic parameters, mThis study aimed to develop the prototype of an examination system used at non-medical places for detecting DVT in lower limbs using LRR. The prototype of this DVT examination system shows that the commercial reflective PPG sensors and accelerometer could be used for deep vein thrombosis (DVT) detection in lower limbs at a non-medical place. Moreover, based on a wireless technique, this measurement system can be operated to control the actions of sensors and display and store the LRR signals on the APP of a smartphone. To comply with the Subject Consent Form in the approved Institutional Review Board (IRB) project, the authors were only allowed to recruit healthy subject to do the related clinical trials. The DVT with stenosis phenomenon in unhealthy participants was reasonably simulated by inflating the cuff up to 100 mmHg or 150 mmHg. Although two characteristic parameters, m40 and m10, in the PPG signals measured with the prototype are available for distinguishing the negative from the positive group, we did not prove that these two parameters would become standard parameters for the DVT examination. The reason for this is that the recruited subjects were all healthy adults. In the next study, the authors will again apply the IRB for clinical trials including healthy and unhealthy subjects based on the current findings in applying the prototype.In the system, an array of optical sensors and a motion sensor were designed. Users are asked to only put the two kinds of sensors at the proper places, and then operate the APP. The APP will automatically connect the wearable device to a smartphone and test the LRR signals, and the users should follow the instruction of the APP to move their foot. Finally, the examination reports will be promptly displayed by means of the APP. Moreover, the approved IRB only allows us to perform the clinical trial on healthy subjects. Thus, the included subjects all were the healthy young subjects . However, based on the current results in this study for DVT detection, the authors will apply the IRB for the clinical trial on patients with DVT in the next study.The wearable device must be user-friendly and easy to operate, and it must be usable at non-medical institutes. Based on the LRR, the examination system has been developed for screening tests in subjects with lower limb DVT and holds several advantages over previous LRR instruments. The proposed system includes a wearable device and APP program. The wearable device not only has an array probe with three reflective PPG sensors, but also has an accelerometer to detect the foot action conditions. The wearable device can compensate blood displacement for different cutaneous optical densities because of skin color, skin thickness, and soft tissue, and it permits quantitative evaluation of vein position by the quality of the LRR signal. The APP program can control the functions of the wearable device, instruct the rhythm of the foot action, and display and store the LRR signals. Twenty healthy subjects were recruited to perform experiments to verify the performance of this prototype. One important finding is that the two characteristic parameters, m40 and m10, both can be applied to detect the DVT of loer limbs without missing any positive cases. This suggests that the prototype of the examination system has the potential for screening for DVT in non-medical environments.


Blood samples were individually collected from 1432 pigs at slaughter age. Viral genomic material, including RNA of a sample process control virus was isolated from food samples using a QIAamp® Viral RNA Mini Kit. Virus-specific IAC-controlled real-time PCR methods were used for detection of target viruses. HEV RNA was found in 6  out of 246 samples of tested foodstuffs. The virus was detected in pig’s blood  and liver  with no significant differences observed in the frequency of its occurrence between the two by-products ; however PAdV was detected more frequently in pig’s blood than in liver . The HEV strains belonged to the 3f and 3e subtype groups and the pAdV strains were assigned to serotype 5. PAdV was detected in pigs regardless of the farm size from which they originated. The number of animals raised on the farm (the farm size) had no influence on the occurrence of HEV or pAdV infections in pigs . Although HEV was detected in pig’s offal only sporadically, consumers cannot treat its occurrence with disregard as it demonstrates that HEV-contaminated pig tissues can enter the food chain.Pig’s blood and liver are valuable edible slaughter by-products which are also the major ingredients of offal-derived foodstuffs. The aim of the study was an evaluation of the occurrence of hepatitis E virus (HEV) and porcine adenovirus (pAdV) as an index virus of faecal contamination in pig’s blood and liver for human consumption. In total, 246 samples of retail liver ( Foodborne transmission of hepatitis E virus (HEV) to humans was described for the first time in 2003 and infection was linked to consumption of raw deer meat  in edible pig’s blood and liver used for production of offal-derived foodstuffs. In addition, virus prevalence in these food ingredients was analysed considering the size of the farm which slaughtered animals originated from, farm husbandry and feeding systems.7.1 TCID50/ml was used as a sample process control virus (SPCV) added to liver and blood samples before analysis. It belongs to the virus collection of the Department of Food and Environmental Virology at the National Veterinary Research Institute in Poland. Preparation of the virus suspension in cell culture was previously described by Rzeżutka et al.  with a titre of 10a et al. .n = 100) and pooled pig’s blood (n = 146) were analysed for the presence of HEV and pAdV. The liver was purchased through regular retailers and originated from different production batches which were sold during the years 2016–2017, while the blood was collected during multiple visits to two slaughterhouses during 2018 and 2019. The blood came from animals aged from 3 months (porkers) to 24 months (backfatter sows). Finishers aged 5–6 months constituted 99.7% of the sampled pigs. The animals came from farms of different sizes breeding fewer than 25 pigs in the case of the small farms, between 25 and 50 pigs if the breeder was medium-sized and more than 50 for the large operations. Farms were located in Mazovia, Kujawy-Pomerania, Lubelskie, Łódzkie and Podlaskie, which are central and eastern voivodeships of Poland. Characteristics of the farms and husbandry details in relation to the age and weight of slaughtered pigs and the scheme of sampling are presented in Table In total, 246 samples of liver n = 00 and po(lobus hepatis dexter) or caudate lobe (lobus hepatitis caudatus) was sampled. Samples were frozen at − 20 °C until use.Samples of pig’s liver (~ 100 g) were purchased from two local butcher’s shops in Puławy, Poland selling an assortment of meats. They were supplied with pig’s liver by slaughterhouses operating in Lubelskie province. Throughout the sampling period, each shop was visited at least twice a month. The peripheral part of the right liver lobe g for 5 min. at 2–8 °C. Isolation of viral RNA from 100 μl of liver homogenate or frozen whole blood was performed using the QIAamp® Viral RNA Mini Kit  according to the manufacturer’s instructions. Nucleic acids were suspended in RNA- and DNA-free water in a final volume of 100 µl. RNA was directly used for testing or stored at − 80 °C. To confirm correct sample processing and assess the efficiency of virus recovery from each analysed liver and blood sample, an SPCV was used. The virus recovery was assessed based on a comparison of the Cq (quantification cycle) values obtained for the SPCV after sample processing with those generated for isolated viral RNA derived from the virus inoculum used for sample spiking  was directly placed on the sample. After virus adsorption a 1 ml of PBS was added followed by a mechanical tissue disruption . The obtained homogenate was centrifuged at 14,000 × 1 to 105 G.C./µl of HEV RNA of known concentration. It was obtained during transcription of the plasmid DNA (pDrive Cloning Vector) containing a 70 bp fragment of the ORF3 genome of HEV gt3 pig strain. The HEV genome copy number present in samples was determined by HEV RT-qPCR in duplicate reactions . The identification of pAdV serotypes was performed based on their sequence alignment with the reference sequences of human and animal adenoviruses representing different serotypes available in GenBank. Analyses were performed using BLAST (https://blast.ncbi.nlm.nih.gov/Blast.cgi).For identification of the HEV subtype or pAdV serotype of detected virus strains in edible blood and liver the ORF2 fragment  was used to assess the differences in frequency in occurrence of the viruses in tissues (liver and blood samples) of slaughtered animals originating from small, medium and large farms. Calculations were made with Statgraphics Centurion version XV .The presence of statistical differences in the frequency of HEV and pAdV detection in liver and blood samples was assessed by Student’s t = 1.33; p = 0.182 > 0.05). The viral load in blood ranged from 11 G.C./ml to 9.0 × 104 G.C./ml, whereas in liver it was as low as 16 G.C./g. PAdV was detected more frequently in pig blood than in liver  with an overall 13% (95% CI: 8.0–19.5%) prevalence among the tested samples .HEV RNA was found in 6  out of 246 samples of tested foodstuffs. All blood samples showed a 100% (very good) extraction efficiency. In the case of livers the mean extraction efficiency was good (24.07  ±  30.15%) with values ranging from 1.1 to 100%. None of the samples showed poor virus recovery. Virus was detected in five samples of pig’s blood  and in only one liver sample  (Table A specific PCR product corresponding to the HEV ORF 2 region was obtained for five virus strains. Four strains belonged to the 3f virus subtype (previously designated 3 l (p) subtype, EU360977, bootstrap value >84%), and one to the 3e subtype   out of 146 samples of pig’s blood and in one liver, which constituted a mere 1% of the tested liver samples. There were no statistically significant differences observed between the virus’ occurrence in liver samples and blood samples. HEV has also been detected in livers of locally raised pigs in Africa , dinuguan (Philippines), mykyrokka (Finland), tiết canh (Vietnam) or svartsoppa in Sweden. Besides whole blood, its fractions, i.e. sediment of red blood cells and plasma are also used in the meat industry  contaminating food items  was used had no significant impact on the frequency of infections in slaughtered animals. HEV detection being only sporadic in slaughtered pigs could mainly be associated with animal age: active infection mostly occurs in young animals, whereas the majority of pigs at the time of slaughter are virus-free  related to the detection methodology used and (ii) an underestimation of the results of virus occurrence in pig’s blood tested as pooled samples. The suitability of the silica-membrane-based RNA extraction method for extraction of HEV RNA from pork slaughter by-products such as pigs blood  or specific farm husbandry and feeding practices. Although the virus was only sporadically detected, its occurrence still remains consequential for consumers as it demonstrates that HEV-contaminated pig tissues can enter the food chain. Of note is that they are the major food ingredients for production of the variety of edible offal meat products which may not undergo sufficient heat-treatment (if any) to inactivate the virus (Colson et al.,