Orthohantavirus, family Hantaviridae, order Bunyavirales) are enveloped, negative-sense, single-stranded, tripartite RNA viruses that are emerging zoonotic pathogens harbored by small mammals such as rodents, bats, moles, and shrews. Orthohantavirus infections cause hemorrhagic fever with renal syndrome (HFRS) and hantavirus cardiopulmonary syndrome in humans (HCPS). Active targeted surveillance has elucidated high-resolution phylogeographic relationships between patient- and rodent-derived orthohantavirus genome sequences and identified the infection source by temporally and spatially tracking viral genomes. Active surveillance of patients with HFRS entails 1) recovering whole-genome sequences of Hantaan virus (HTNV) using amplicon (multiplex PCR-based) next-generation sequencing, 2) tracing the putative infection site of a patient by administering an epidemiological questionnaire, and 3) collecting HTNV-positive rodents using targeted rodent trapping. Moreover, viral genome tracking has been recently performed to rapidly and precisely characterize an outbreak from the emerging virus. Here, we reviewed genomic epidemiological and active surveillance data for determining the emergence of zoonotic RNA viruses based on viral genomic sequences obtained from patients and natural reservoirs. This review highlights the recent studies on tracking viral genomes for identifying and characterizing emerging viral outbreaks worldwide. We believe that active surveillance is an effective method for identifying rodent-borne orthohantavirus infection sites, and this report provides insights into disease mitigation and preparedness for managing emerging viral outbreaks.Emerging and re-emerging RNA viruses pose significant public health, economic, and societal burdens. Hantaviruses (genus  Orthohantavirus) are enveloped, negative-sense, single-stranded RNA viruses belonging to the family Hantaviridae and order Bunyavirales  is caused by Sin Nombre virus and Andes virus. Approximately 150,000 cases of HFRS are reported annually, with a case fatality rate (CFR) of <1–12% , epidemiological interviews, and targeted rodent trapping. This includes 1) genome tracking using whole-genome sequencing, 2) follow-up of suspected infection sites using patient interview data, and 3) rodent trapping to capture the natural reservoir host. Additionally, this review presents effective preventive and mitigation strategies for rodent-borne orthohantavirus outbreaks using active surveillance.Apodemus agrarius were collected from the trapping sites. The partial genomic sequence of HTNV showed a phylogeographic relationship between US soldiers diagnosed with HFRS and HTNV-positive A. agrarius. The potential rodent exposure sites in HCPS cases in the United States were identified via patient interviews  are employed . In 2005terviews . Partialterviews . Partialterviews . Small mterviews . In additerviews .via repeated transmission from local rodent reservoirs rather than sustained human-to-human transmission. In 2016, genomic surveillance and phylodynamic analyses of Zika virus (ZIKV) genomes from infected patients and Aedes aegypti mosquitoes showed that the Caribbean Islands were the main source of the local ZIKV outbreak in Florida, whereas multiple ZIKV transmissions occurred throughout the Americas  travels, exercises, and outdoor activities; 2) clinical complication progression; 3) contact time of suspected sources prior to the onset date; and 4) vaccination. As interviewees might not remember all the information, we found that the suspected site and timing of infection should be considered carefully while conducting epidemiological interviews and records. Based on the epidemiological interviews, a rapid responsive team collected rodents in military training areas near the putative sites and analyzed the samples for viral genome sequences using RT-PCR and NGS. The HTNV genome sequences obtained from the rodents collected at the suspected site were phylogeographically analyzed with the genome sequences obtained from the samples of patients with HFRS. Phylogeographic analyses showed that four ROKA patients with HFRS from different military bases participated in off-site military exercises within 30 days prior to the onset of clinical symptoms  within a very short distance (approximately 5 km) to track the precise infection site.In the ROK Army (ROKA), active surveillance was implemented by the Armed Forces Capital Hospital to track HFRS outbreaks in military personnel . The potsymptoms   setting up a trash can with a cover, 2) cutting the grass on the ground, 3) spraying water to prevent dust prior to exercises or activities, and 4) installment of public warning signs to prevent human access to endemic areas  . HoweverIn this study, we reviewed the active surveillance for emerging hantaviruses by focusing on concurrent genome sequencing, epidemiological data, and targeted rodent trapping Box 1. WBox 1A 57-year-old man visited the hospital in Yeoncheon-gun with continuous fever and dizziness. He told the doctor that he was a farmer who worked in a field near the Hantaan river and had harvested crops. There was point bleeding in the palate and conjunctival hemorrhage. He was hospitalized in the emergency room. His body temperature was normal, but his blood pressure was 85/55. The blood and urine test results showed high white blood cell counts  and low specific gravity of urine (1.015). He had hypotension and acute renal failure, and he received fluid therapy during admission in the emergency room  and the Research Program to Solve Social Issues of the National Research Foundation of Korea (NRF) funded by the Ministry of Science and ICT (NRF-2017M3A9E4061992).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.


Residual infectious virus in neutralized samples was assessed by passage on cells and evaluation for viral cytopathic effect. No infectious virus was detected in cells inoculated with EBOV/Mak exposed to NaOCl (0.5% or 1%), PCMX (0.12% to 0.48%), or EDS for ≥ 5 min. These results demonstrate ≥ 6 log10 inactivation of EBOV/Mak dried on prototypic surfaces by EDS or formulations of NaOCl (≥ 0.5%), PCMX (≥ 0.12%), or 70% ethanol at contact times ≥ 5 min.Microbicides play critical roles in infection prevention and control of Ebola virus by decontaminating high-touch environmental surfaces (HITES), interrupting the virus-HITES-hands nexus. We evaluated the efficacy of formulations containing different microbicidal actives for inactivating Ebola virus–Makona strain (EBOV/Mak) on stainless-steel carriers per ASTM E2197-11. Formulations of sodium hypochlorite (NaOCl) (0.05–1%), ethanol (70%), chloroxylenol (PCMX) (0.12–0.48% by weight) in hard water, and a ready-to-use disinfectant spray with 58% ethanol (EDS), were tested at contact times of 0, or 0.5 to 10 min at ambient temperature. EBOV/Mak was inactivated (> 6 log This most recent outbreak, and the outbreaks occurring between 2014 and 2016, emphasize the need2 for effective approaches for reducing the spread of the disease from community to community and from nation to nation. Ebola virus disease is listed in the World Health Organization’s List of Blueprint Priority Diseases, as “… given their potential to cause a public health emergency and the absence of efficacious drugs and/or vaccines, there is an urgent need for accelerated research and development…”3.The Ebola virus has continued to re-emerge in lethal outbreaks, with the most recent occurring in the Democratic Republic of the Congo, Africa in May 20184. For instance, objects in the vicinity of infected patients  have been shown to be contaminated with Ebola virus RNA5. Considering this, an important intervention for limiting viral dissemination may involve the use of an effective virucidal agent for disinfecting surfaces contaminated with Ebola virus, thereby mitigating the risk of transmission of the virus to healthy individuals, including health-care workers.The Ebola virus may be transmitted, in part, by contact with environmental surfaces (fomites) contaminated with secretions and excretions from infected individualsFiloviridae family, and is an enveloped virus. As such, the Ebola virus should be relatively susceptible to a variety of microbicidal inactivation approaches6. In view of the lethality of the virus, the United States Centers for Disease Control and Prevention (CDC) offers the following guidance7: “…selection of a disinfection product with a higher potency than what is normally required for an enveloped virus is being recommended at this time. EPA-registered hospital disinfectants with label claims against non-enveloped viruses  are broadly antiviral and capable of inactivating both enveloped and non-enveloped viruses.” The United States Environmental Protection Agency (EPA) requires that claims for efficacy of a product for an emerging enveloped virus include that the product be approved for inactivating at least one large or one small non-enveloped virus8.The Ebola virus is a member of the The virucidal efficacy of microbicides for Ebola virus is usually determined in studies involving virus suspended in a liquid matrix. In addition, many studies have made use of surrogate viruses such as bacteriophages, enveloped viruses , or non-enveloped viruses such as caliciviruses or picornaviruses. As a result, there are few reports of the efficacy of inactivation of Ebola virus dried onto carriers . The testing of virucidal efficacy for Ebola virus surrogates theoretically should ensure efficacy for inactivation of the Ebola virus. Despite this, testing conducted specifically with Ebola virus itself is needed to confirm the results obtained with surrogate viruses. In this study, virucidal investigations were performed using fully pathogenic Ebola virus at the Canadian Science Centre for Human and Animal Health Biosafety Level 4 (BSL-4) facility.9  and a formulated disinfectant spray containing 58% ethanol (EDS) to data published previously11, at the same testing facility and using the same methodology, for 70% ethanol and for varying concentrations of sodium hypochlorite (NaOCl). We have reported both sets of results within this article in order to facilitate direct comparisons of virucidal efficacy for the various microbicidal actives.In this paper, we have conducted studies on the inactivation of Ebola virus–Makona variant (EBOV/Mak), dried onto steel carriers in the presence of an organic load, per American Society for Testing and Materials (ASTM) method ASTM E2197-1150 (TCID50) assay performed in Vero E6 cells per the ASTM standard9.In addition to the methodology described in the ASTM standard, we also evaluated any residual infectious virus following exposure to microbicides through inoculation of undiluted neutralized test sample into cultures of Vero E6 indicator cells. This was done to evaluate the possibility of virus being present at levels lower than the limit of detection of the tissue culture infectious dose11, and that NaOCl could be neutralized by 1% sodium thiosulfate11.During the evaluation of possible neutralizing agents, it was determined that PCMX could be neutralized with Letheen broth, and the disinfectant alcohol spray could be neutralized with DMEM + 10% FCS + 10 units/mL penicillin/streptomycin see . It was 9. Three lots of PCMX were evaluated at three concentrations each  in hard water, with contact times of 0.5, 1, 5, and 10 min at ambient temperature (~ 21 °C). An initial virus load of ~ 6.9 log10 tissue culture infectious dose50 (TCID50) was dried on the carriers and then exposed to PCMX. At each concentration evaluated, the infectious EBOV/Mak titer recovered from the carriers was reduced by > 5 log10 to the LOD of the assay within five min . An initial virus load of ~ 6.3 log10 tissue culture infectious dose50 (TCID50) was dried on the carriers and then exposed to EDS. The infectious EBOV/Mak titer recovered from the carriers was reduced by > 5 log10 to the LOD of the assay within 5 min  for inactivating EBOV/Mak dried within an organic load on stainless steel carriers was evaluated per ASTM E2197-11The results of the plate safety test for EDS are shown in Table 9. Contact times of 0, 1, 2.5, 5, 7.5, and 10 min were evaluated11 at ambient temperature (~ 27 °C). An initial virus load of ~ 6.8 log10 tissue culture infectious dose50 (TCID50) was dried on the carriers in the presence of the tripartite soil load9 and then exposed to the ethanol solution. Infectious EBOV/Mak titers recovered from the carriers were reduced by > 5 log10 within 2.5 min  in hard water, with contact times of 1, 2.5, 5, 7.5, and 10 min at ambient temperature (~ 27 °C). An initial virus load of ~ 6.8 log10 tissue culture infectious dose50 (TCID50) was dried on the carriers in the presence of the tripartite soil load9 and then exposed to NaOCl. At each concentration evaluated, the infectious EBOV/Mak titer recovered from the carriers was reduced by > 5 log10 to the LOD of the assay within five min  are contaminated during outbreaks, these might serve as sources of infection of healthcare workers and other non-infected persons through the intermediacy of the hand. Targeted decontamination of such HITES therefore represents an opportunity for interrupting the spread of the virus.Filoviruses such as Ebola virus are capable of persisting for weeks on non-porous surfaces such as glass and plastic14 and the lethality (~ 41%) of the associated hemorrhagic disease15 mean that risk mitigation in the form of surface decontamination must be effective beyond the typical expectation of a 3–4 log10 reduction in viral titer. The US Environmental Protection Agency (EPA) stated in its 2012 disinfectant product guidance16 that “The product should demonstrate complete inactivation of the virus at all dilutions. If cytotoxicity is present, the virus control titer should be increased to demonstrate a ≥ 3 log10 reduction in viral titer beyond the cytotoxic level.” Most microbicides and/or the neutralizing agents used in determining inactivation kinetics are cytotoxic to the cells used in TCID50 assays used to determine efficacy. This impacts the sensitivity (limit of detection of the TCID50 assay). In the absence of cytotoxicity, a 4-log10 reduction in viral titer is considered to be effective. These requirements were recently modified in the 2018 revision of the EPA guidance17, with the new requirements being: In the revised guidance, a valid test requires (1) that ≥ 4.8 log10 of infectivity per carrier be recovered; (2) “the product should demonstrate a ≥ 3 log10 reduction on each surface in the presence or absence of cytotoxicity”; and (3) “if cytotoxicity is present, the virus control titer should be increased if necessary to demonstrate a ≥ 3 log10 reduction in viral titer on each surface beyond the cytotoxic level.” Note that in this revised guidance, an efficacious product does not need to demonstrate complete inactivation at all dilutions. That is, some residual infectious virus may be allowed.The low minimum infectious dose of the Ebola virus in humans (estimated to be 1 to 10 infectious units)50 assay, an alternative means must be used to demonstrate complete inactivation . In our experiments, this has been accomplished through the method referred to as the “plate safety assay”. This abrogates the issue of cytotoxicity through inoculation of undiluted neutralized test samples into 6-well cultures of Vero E6 cells, and then by conducting serial passages of the inoculated cells. A negative result in this method provides evidence of complete inactivation of any infectious virus.In the case of the Ebola virus, we believe that the efficacy of a microbicide must be demonstrated by reduction of viral load to undetectable levels. When cytotoxicity to detector cells occurs in the TCID11 on the efficacy of 70% ethanol and varying concentrations of sodium hypochlorite against EBOV/Mak. Each of these data sets were obtained from the same testing facility using the same methodology. As a result, the virucidal efficacy results should be directly comparable. The carrier inactivation data for EBOV/Mak presented here demonstrate that a variety of microbicides should be useful for effective inactivation of Ebola virus on stainless steel surfaces. These microbicides include 70% ethanol at contact times ≥ 5 min, NaOCl at concentrations of 0.5% or greater, at contact times ≥ 5 min, PCMX at concentrations of 0.48% and contact time of ≥ 5 min, and the EDS used as supplied at contact time ≥ 5 min. Under these conditions, no residual EBOV/Mak virus was detectable (≥ 6.3 log10 inactivation) as indicated by the TCID50 assay and the plate safety assay.Here, we present our data on the virucidal efficacy of varying concentrations of PCMX and a single concentration of a formulated ethanol spray against the Makona strain of Ebola virus (EBOV/Mak), together with previously published data11 previously examined the efficacy of 70% ethanol and several concentrations of sodium hypochlorite against three variants of the Ebola virus . The authors found that the Makona variant was somewhat less susceptible to the lower concentrations of sodium hypochlorite, while being similarly susceptible to 70% ethanol and higher concentrations of sodium hypochlorite11. We therefore expect that the efficacy data obtained here for the Makona variant should apply also to other outbreak variants of Ebola virus.Cook et al.18 as an example of a suitable disinfectant solution for hemorrhagic fever viruses. Smither et al.19 use a similar experimental design  to study the disinfection of Ebola virus Yambuku-Ecran (EBOV-Ecran) on aluminum carriers by 0.75% NaOCl in tap water for 10 min contact time. In this case, complete inactivation of the 1.5 × 105 TCID50/mL dried on the carrier was achieved19. No organic matrix was used in the studies for EBOV/Ecran. Additional studies by Smither et al.20 evaluated disinfection of EBOV/Mak dried in a blood matrix on stainless steel or aluminum surfaces. In this study, 0.5 and 1% NaOCl inactivated EBOV/Mak dried in tissue culture medium or deposited in wet blood to the LOD of the assay (10 TCID50/mL) in 15 min. This suggested that inactivation of EBOV in the presence of dried blood was particularly challenging for NaOCl, more challenging that is, than virus dried in tissue culture medium. Only a 5-min contact time with 5% peracetic acid solutions was capable of completely inactivating the EBOV/Mak dried in blood in that study20.Sodium hypochlorite 0.5% solutions are recommended by the CDC11 using 70% ethanol and NaOCl solutions and from the present study using PCMX solutions and EDS demonstrated ≥ 6 log10 inactivation of EBOV/Mak dried on steel by EDS or solutions of NaOCl (≥ 0.5%), PCMX (≥ 0.12%), or 70% ethanol at contact times ≥ 5 min. Higher log inactivation values might actually have been possible, but were not able to be determined in the present study due to limitations in the amount of challenge virus that could be applied. These four disinfectants display sufficient inactivation efficacy for Ebola virus at reasonably short contact times that may be practically achieved in the field. Use of these disinfectants for surface decontamination in the field or in healthcare settings therefore has the potential to reduce infectious Ebola virus load on those surfaces and to reduce spread of virus from infected to non-infected individuals.These results from Cook et al.11 and have not been reproduced here.Methods for the previously reported Ebola variant carrier inactivation studies involving ethanol and sodium hypochlorite were described in Cook et al.2 in Dulbecco’s modified Eagle medium  supplemented with 10% fetal calf serum  and 10 units/mL penicillin/streptomycin (Gibco). Ebola virus Makona variant  was obtained from a clinical isolate. For the studies on PCMX and EDS, the isolate was biotechnologically engineered to express green fluorescent protein (GFP).African green monkey Vero E6 cells  were maintained at 37 °C/5% COg) for 10 min. The supernatants were pooled and overlaid onto 20% w/v sucrose cushions prepared in Tris-NaCl-EDTA buffer. After centrifugation at 133,907×g for 2 h, the resulting viral pellets were resuspended in virus culture medium  overnight at 4 °C. The resuspended virus was pooled and aliquoted into usable amounts and frozen at − 70 °C until needed. Stock virus titers were determined to be > 8.4 log10/mL by TCID50 assay, with titer calculation following the Reed-Muench method21. All EBOV/Mak manipulations were carried out in a BSL-4 laboratory at the Canadian Science Centre for Human and Animal Health, Winnipeg, Manitoba, Canada.A characterized stock of EBOV/Mak virus was prepared by infecting five T-175 flasks of Vero E6 cells expressing green fluorescent protein (eGFP) at ~ 80% confluency at a multiplicity of infection (MOI) of 0.01. The eGFP was evident by day 3 post-infection. At ~ 9 days post-infection, marked cytopathic effects (CPE) were also observed, at which time the flasks were frozen at − 70 °C. The conditioned medium from thawed flasks was clarified by low-speed centrifugation  on the day of assay performance. The resulting solutions were inverted to mix and were used within 4 h of preparation. The EDS formulations containing 58% ethanol were used without further dilution. Three independent lots of EDS  were evaluated.Three concentrations  of three lots of PCMX  were prepared from the ready-to-use commercial product by dilution in 440 ppm hard waterMethods for evaluating neutralizing agents are described in the 9. Inocula for stainless steel carriers were prepared fresh on the day of assay by mixing previously frozen concentrated EBOV/Mak eGFP virus with a tripartite soil load10. The virus in tripartite soil load (10 µL) was added to sterile scored stainless steel test carriers and air dried at ambient temperature for 1 h. After drying, carriers were placed in 6-well plates and 50 µL of microbicide was added to each carrier and incubated for 30 s, 1 min, 5 min, and 10 min contact times. Input virus titers determined for the various virucidal efficacy experiments may have varied as a result of differences in the titers of the virus stocks used in the individual studies. Such differences had no impact on the validity of the studies. Per the standard followed, ASTM E2917-11, at least 4 log10 reduction of virus should be exhibited to demonstrate efficacy, and the input titers used were high enough to allow demonstration of this log reduction after accounting for possible toxicity. A positive control (50 µL of VCM in lieu of microbicide) was used for each assay performed. At the end of each exposure time point, the microbicide was neutralized by adding 950 µL of appropriate neutralizing agent to the test carriers and to the positive controls last. The carriers were rinsed via pipetting 20 times with neutralized solution to elute any dried virus from the carriers into the solution. A 500-µL portion of the positive control and each neutralized test solution was ten-fold serially diluted in VCM, and 50 µL of the resulting dilutions were added to 96-well plates containing Vero E6 cells (n = 5 replicates per dilution). After a 45-min adsorption period, 150 µL of VCM were added to each well. The Vero E6 cell wells were scored 9–14 days post-infection for green fluorescence and cytopathic effect (CPE) and virus titers (TCID50) were calculated according to the Reed-Muench method21. Similar titers for the Virus Positive Control condition were obtained regardless of the readout used (GFP vs. CPE)  neutralized PCMX and EDS solutions displayed toxic effects in the Vero E6 cells and could not be evaluated for viral titer in the TCID50 assay. A plate safety test was employed to evaluate surviving EBOV/Mak virus in these samples. Triplicate 500-µL aliquots of each undiluted neutralized test sample were added immediately following neutralization to 7 mL of VCM in a six-well plate of Vero E6 cells at ~ 80% confluency. The cultures were incubated 9–14 days and scored for presence of green fluorescence and CPE.Inactivation efficacy testing for microbicides was performed in carrier studies Fig.  conducteCPE) see . The logSupplementary file1


Viruses require a host for replication and survival and hence are subjected to host immunological pressures. The complement system, a crucial first response of the host immune system, is effective in targeting viruses and virus-infected cells, and boosting the antiviral innate and acquired immune responses. Thus, the system imposes a strong selection pressure on viruses. Consequently, viruses have evolved multiple countermeasures against host complement. A major mechanism employed by viruses to subvert the complement system is encoding proteins that target complement. Since viruses have limited genome size, most of these proteins are multifunctional in nature. In this review, we provide up to date information on the structure and complement regulatory functions of various viral proteins. Viruses face constant challenges from the host due to host resistance, and the environment because of variations in temperature and humidity. Nonetheless, they adapt to these dynamic changes to become the most successful pathogens . The sucThe concept of host resistance to infection was first documented by the Greek historian Thucydides, who provided an eye-witness account of the plague that struck Athens between 430–427 B.C. 28], and Zika virus (ZIKV), an arthropod-borne virus, is a small (~50 nm) enveloped, single-stranded, positive-sense RNA virus. It is one of the prominent members of the family Flaviviridae. Infections caused by this virus are asymptomatic or cause mild symptoms such as fever, rash, red eyes, headache, and joint/muscle pain. However, in some cases, it has also been shown to be associated with neurological and autoimmune complications .The ZIKV E protein (~53 kDa) is one of the three structural proteins of the virus. It is involved in virus entry and hence is a major target for neutralization antibodies. Its ectodomain contains three modules: a central β-barrel module (DI), a finger-like module (DII), and a C-terminal immunoglobulin-like module (DIII). These modules are linked by flexible hinges and the protein is connected to the viral membrane via a helical anchor. On the viral surface, it forms a raft-like structure where 90 E-dimers compact to create the icosahedral symmetry [ZIKV activates the classical pathway due to the binding of IgM antibodies and C1q to the viral surface. Interestingly, though the virus is susceptible to neutralization by high serum concentration (50% serum), it is relatively stable at low serum concentration (10%) . ExaminaHCV encodes six non-structural proteins: NS2, NS3, NS4A, NS4B, NS5A, and NS5B. Among these, the NS3/4A protease complex and NS5A protein have been shown to modulate complement.Structurally, NS3 protein has a serine protease domain at the N-terminus and a helicase domain at the C-terminus. NS4A, on the other hand, contains an N-terminal membrane-spanning region and a C-terminal cytosolic region. The NS3/4A complex is formed as a result of non-covalent binding of the N-terminal serine protease domain of NS3 to NS4A molecule, where the latter serves as a cofactor. This serine protease complex is required for the proteolytic cleavage of HCV polyprotein precursor. Functional characterization of the protease complex showed that it is necessary for viral replication as well as disruption of the host innate immune system to establish a persistent infection. For example, it is responsible for cleaving host cellular targets involved in the IFN induction pathway.Its role on the complement system was investigated because, during a proteomic analysis, a fragment of C4 was found to be abundant in HCV carriers. The study specifically examined if NS3/4A protease complex is responsible for the C4 cleavage. In vitro proteolysis assay showed that the protease complex cleaved the γ-chain of C4 and thereby inhibited the classical pathway . In addiThe NS5A protein, like that of NS3/4A complex, is also a multifunctional protein. It is necessary for HCV replication, modulation of cellular processes and assembly of infectious virions. It is a zinc-binding phosphoprotein which is rich in proline and is formed by three domains wherein domain I is structured, whereas domains II and III remain unfolded . Domain Because HCV was shown to inhibit expression of various complement proteins, along with the core protein (discussed above), NS5A was also examined for its ability to transcriptionally downregulate the expression of complement proteins. Initially, NS5A was tested for transcriptional repression of C4. The results showed that it downregulates the expression of C4 by attenuating the expression of interferon regulatory factor 1 (IRF-1), which is significant for IFN-γ-induced C4 expression . Later, Hepatitis B virus (HBV) is a small (42 nm) enveloped double-stranded DNA virus. It is known to cause acute as well as chronic hepatitis and hepatocellular carcinoma (HCC). The current estimate suggests that globally over 2 billion people are infected with hepatitis B infection .The HBV X protein (HBx) is one of the four proteins of the virus. This 17 kDa soluble protein consists of two domains: a negative regulatory domain (1–50 amino acid residues) at the N-terminus and a trans-activating domain (51–154 residues) at the C-terminus . HBx intBecause of HBx’s proposed role in HCC development, a study sought to determine if it is involved in overexpression of CD59, which can provide resistance to infected cells against complement-mediated cytolysis and thereby participate in the tumor cell growth. Initial analysis showed that CD59 is upregulated in clinical HCC specimens. Further study with HBx revealed that it promotes the overexpression of CD59, and this is owing to its binding to the CD59 promoter and down-regulation of let-7i . Thus, tAs discussed above, Flaviviruses are small (50 nm) enveloped, single-stranded, positive-sense RNA viruses that are majorly transmitted by arthropods and cause mild febrile illness to severe hemorrhagic manifestations. The prominent members that cause human infections, apart from Zika virus (discussed above), are Dengue virus, Japanese encephalitis virus, Yellow fever virus, West Nile virus, and tick-borne encephalitis virus.NS1 is the most studied and conserved proteins across flaviviruses. It is also highly conserved among the members. It contains three functionally distinct domains namely a hydrophobic β-roll, an α/β Wing domain and a central β-ladder. The entire structure is stabilized by six disulfide bonds ,100,101.An early study conducted on the NS1 protein by Brandt et al.  showed tVaccinia and variola viruses are two well-known orthopoxviruses. Both are large brick-shaped (~350 by 250 nm) double-stranded DNA viruses. Among these two viruses, variola virus, the causative agent of smallpox, killed about 500 million people. Vaccinia virus, on the other hand, does not cause disease in immunocompetent individuals but is known to cause repeated outbreaks in dairy cattle in India and Brazil ,114. NotVaccinia virus (VACV) encodes a homolog of human RCA proteins dubbed vaccinia virus complement control protein (VCP). It is one of the 20 immunoregulatory proteins of VACV. Incidentally, it is also the major protein secreted by the virus-infected cells . The chaOriginal functional characterization of VCP was performed following its purification from the culture supernatant of VACV-infected cells. The protein effectively blocked the classical pathway hemolytic activity . Later, The importance of VCP in VACV virulence was addressed using rabbit, guinea pig and mice intradermal models. Together, these studies established that VCP contributes to virulence by inhibiting complement, which then results in decreased antibody and T cell responses ,119. TheThe complement inhibitor encoded by variola virus was named smallpox inhibitor of complement enzymes (SPICE) . BecauseThe first effort to understand the functional advantage of SPICE over VCP was attempted using an electrostatic modelling approach. The study showed that E108K and E120K substitution in VCP significantly augmented its C3b cofactor activity . A systeCowpox virus (CPV) is a zoonotic virus which is closely related to vaccinia and variola viruses. It exhibits a broad host range  and is kThe genome analysis of CPV showed that like that of vaccinia and variola, it also encodes an RCA homolog . Like VCLike other orthopoxviruses, monkeypox virus is also a brick-shaped (~200 by 250 nm) double-stranded DNA virus. It is important to mention here that currently, it is recognized as the most critical reemerging zoonotic orthopoxvirus infection in humans. Though the virus is restricted to West and Central Africa’s rainforest regions, its outbreak occurred in the United States in 2003 .The monkeypox virus complement regulator monkeypox inhibitor of complement enzyme (MOPICE) is a 24 kDa soluble protein secreted by the virus-infected cells. It is an ortholog of VCP with a single nucleotide deletion causing a frameshift mutation in CCP4. As a result, it contains only three CCP domains . AnalysiMOPICE targets complement proteins C3b as well as C4b . ComparaBecause MOPICE is present only in the virulent form of monkeypox virus, it was hypothesized that it might play a significant role in the monkeypox pathogenesis. Thus, to validate this assumption, in vivo infection studies were undertaken by two groups ,133. InfEctromelia virus is another Orthopoxvirus which causes the disease mousepox. The virus was originally isolated in the 1930s from the laboratory strain of mouse and found to easily transmit between the wild and the laboratory mice . It is vStructurally, ectromelia virus inhibitor of complement enzymes (EMICE) shares 90% homology with the other poxviral RCAs with a major difference in CCP1 . DespiteHerpesvirus saimiri (HVS), a gammaherpesvirus, is a T-lymphotropic tumor virus found in its natural host squirrel monkey. It is a large (170 nm) double-stranded DNA virus that promotes acute leukemia and T cell lymphoma in several species of New World primates such as owl monkeys, common marmosets, and cottontop tamarins ,136. It HVS encodes two homologs of the complement regulatory proteins that inactivate complement pathways at distinct steps: (i) a homolog of RCA encoded by ORF4, termed complement control protein homologue (CCPH)  and (ii)Studies by Fodor et al.  on the iKaposi’s sarcoma-associated herpesvirus (KSHV) or the human herpesvirus 8 (HHV-8) is etiologically linked to Kaposi’s sarcoma. Additionally, it is also linked with pleural effusion lymphoma and multicentric Castleman’s disease . Like otGenome sequencing of KSHV revealed that its ORF 4 encodes a protein that is structurally similar to the human RCA proteins . As a reThe recombinant soluble form (4 CCPs) of the KSHV ORF 4 protein was found to block cell surface deposition of C3b, inhibit complement-mediated lysis of erythrocytes, and inactivate both C3b and C4b with the help of factor-I. Hence, the protein gained importance as a complement regulator and was named as Kaposica (KSHV inhibitor of complement activation) . A paralRhesus monkey rhadinovirus (RRV) is a gammaherpesvirus which is genetically related to the KSHV. Its natural host is rhesus macaque but is also identified in squirrel monkeys, chimpanzees, gorillas, and pigtail macaques, to name a few. Because of its high genomic relatedness to KSHV, RRV endows an in vivo animal model of KSHV infection . InteresThe primary sequence analysis of H26–95 and 17,577 strains revealed that ORF4 of both these isolates encode RCA-like proteins . Using mMurine gammaherpesvirus 68 (γHV68/MHV68) is closely related to Epstein-Barr virus (EBV), HVS, and KSHV ,151. OriComplete sequence analysis of the γHV68 genome exhibited the presence of an open reading frame sharing homology with HVS and KSHV ORF-4 . γHV68 RIn addition to a homolog of RCA proteins (discussed above), HVS also encodes a homolog of human CD59. In humans, CD59 functions as a potent inhibitor of complement-mediated cytolysis. It exerts this effect by tightly binding to C5b-8 and C5b-9 and inhibiting the incorporation of multiple C9 molecules into the membrane attack complex . AdditioOverall, the structure of HVS CD59 is similar to that of human CD59; however, specific differences are apparent. Like that of human CD59, it is also formed by 77 amino acids and contains a C-terminal glycosyl-phosphatidylinositol signal and attachment site. Moreover, the ten cysteines that form five disulfide bonds and stabilize the structure are also conserved in HVS CD59. The protein conversely lacks the N-linked glycosylation site and exhibits 48% amino acid identity to human CD59.HVS CD59 has not been studied well like viral RCA proteins. Nevertheless, it was examined for its ability to block the MAC-mediated lysis of cells. In a transfection experiment, a comparison of its complement regulatory activity with human and squirrel monkey CD59 showed that it is as potent as these counterparts in inhibiting the human and monkey complement-mediated cytolysis, respectively ,24. InteSuccessful infection of hosts by viruses is contingent upon their ability to subvert the vital arms of innate immunity. Consistent with this premise, viruses have been shown to employ both structural as well as non-structural proteins for evasion of complement. As described here, some of these proteins show homology to cellular genes. It is believed that such genes have been acquired from the host by horizontal gene transfer . Viral pIt is conceivable that generation of optimum innate and adaptive immune responses are essential for controlling viral infections and therefore, viruses subvert these to ensure their own survival within the host. However, it is also a fact that occasionally immune responses against virus-infected cells become overtly tissue damaging. Therefore, the pertinent question to be asked is, do some viruses encode complement regulators to reduce the complement-mediated tissue injury? This question has been addressed by two in vivo studies. Miller et al.  noticed It is clear from the data discussed here that viruses encode diverse molecules to evade complement attack, which directly target complement proteins, enzymes, and complexes, and also inhibit the synthesis of complement proteins. Viruses, however, have additional means to counteract immune responses. For example, they are known to encode microRNAs to downregulate innate immune effectors’ expression and modulate the cellular microRNAs to enhance their replication . LikewisUp until recently, the complement system was defined as a humoral system. However, the system has been shown to operate intracellularly affecting T cell effector function . FurtherUnderstanding of the intracellular complement system, dubbed the complosome, is still limited. We now know that inside the cell, complement system crosstalks with MAVS  and infl


Background: Children with cerebral palsy (CP) place a considerable burden on medical costs and add to an increased number of inpatient days in Taiwan. Continuity of care (COC) has not been investigated in this population thus far. Materials and Methods: We designed a retrospective population-based cohort study using Taiwan’s National Health Insurance Research Database. Patients aged 0 to 18 years with CP catastrophic illness certificates were enrolled. We investigated the association of COC index (COCI) with medical costs and inpatient days. We also investigated the possible clinical characteristics affecting the outcome. Results: Over five years, children with CP with low COCI levels had higher medical costs and more inpatient days than did those with high COCI levels. Younger age at CP diagnosis, more inpatient visits one year before obtaining a catastrophic illness certificate, pneumonia, and nasogastric tube use increased medical expenses and length of hospital stay. Conclusions: Improving COC reduces medical costs and the number of inpatient days in children with CP. Certain characteristics also influence these outcomes. Cerebral palsy (CP), the most common childhood-onset and permanent physical disability in numerous countries, is characterized by a neurodevelopmental disorder that affects muscle tone, movement, and motor skills. By international consensus in 2005, CP describes a group of permanent disorders of the development of movement and posture causing activity limitation, that are attributed to non-progressive disturbances that occurred in the developing fetal or infant brain. CP affects approximately 1 in 500 newborns with an approximate prevalence of 17 million people worldwide . We applThe average lifetime economic cost per patient with CP assessed by the US Centers for Disease Control (CDC) was US$921,000 in 2003 . A Dutch“Doctor-shopping”, implicating frequent attendances and switching of physicians, has become common because of easy accessibility to local clinics or hospitals in Taiwan . PatientContinuity of care (COC) comprises two core elements: care of an individual patient and care over time. It comprises three types of care: informational continuity, management continuity, and relational continuity. By applying COC, a trusting and responsible therapeutic association can be established between the patient and physician ,12,13. CThe effect of COC on children with CP has not been investigated. Therefore, we designed a retrospective population-based cohort study to investigate the association of COC, inpatient days, and medical costs among children with CP in Taiwan. Furthermore, we surveyed the possible characteristics relevant to these areas.We retrospectively extracted clinical data from the Taiwan National Health Insurance (NHI) Research Database (NHIRD), a nationwide population-based administrative database of NHI, the beneficiaries of which include approximately 99.9% of the population  of Taiwan . NHIRD iThe current study was approved by the Institutional Review Board of Kaohsiung Veterans General Hospital, Kaohsiung, Taiwan (VGHKS15-EM10-02). The board also waived the need for written informed consent because all personal identifying information used had been de-identified. Outpatient records were traced from the index date. Data were collected regarding outpatient visits one year after the index date, different physicians one year after the index date, age on the index date, gender, inpatient days over five years after the index date, medical costs over five years after the index date, inpatient visits one year before the index date, residential area, urbanization level, hospital level where children with CP obtained the catastrophic illness certificates, comorbidities, and mortality. The residential areas were classified as Northern, Central, and Southern-Eastern Taiwan. Urbanization level was dichotomized into urban and nonurban. Hospital levels, where children with CP obtained their catastrophic illness certificates, were categorized as medical centers and regional or district hospitals. The comorbidities considered in this research were asthma (ICD-9-CM 493), preterm labor (PTL) and small for gestational age , perinatal complications , epilepsy (ICD-9-CM 345), pneumonia , gastroesophageal reflux disease , and nasogastric tube use (ICD-9-CM 47017C and 47018C).We compared total medical costs and inpatient days with a different COC index (COCI) and other characteristics.T is the total number of CP outpatient visits , in is the number of times the patient visited a pediatric specialist, and k is the total number of pediatric specialists visited. In our study, the one-year COCI was applied to compare the medical costs and the inpatient days of children with CP over five years.We applied the COCI developed by Bice and Boxerman to evaluate the COC status . Severalp value of < 0.05 was considered significant. All statistical analyses were performed using SPSS  and SAS .The outcome variables of interest were medical costs and inpatient days over five years from the index date. Medical costs involved fees paid by both caregivers of patients and the NHI Bureau in Taiwan. The distribution of variables was described by the chi-squared test. The continuous variables were compared using a one-way analysis of variance. The relationship between medical costs and inpatient days, as well as other variables, was analyzed using multiple linear regression and residual analysis. Multiple linear regression was not appropriate because of unequal variance assumptions . The effAs illustrated in p = 0.016). The other characteristics associated with increased medical costs in both univariate and multivariate analysis were younger age, more inpatient visits, medical center, pneumonia, and nasogastric tube use. We performed a stratified analysis for medical costs after adjusting characteristics (p = 0.002).The hospital-level random-intercept model HGLM, displayed in eristics A,B. The p < 0.001). The other characteristics associated with increased inpatient days in both univariate and multivariate analysis were younger age, more inpatient visits, living in Southern and Eastern Taiwan, no PTL or SGA, pneumonia, and nasogastric tube use. The hospital-level random-intercept model HGLM, displayed in This study is the first large scale population-based cohort study on COC application among children with CP using NHIRD. We demonstrated that a low COC was associated with an increased number of inpatient days and more medical costs among children with CP. Furthermore, outpatient medical costs were higher than inpatient.No studies had been conducted on COCI among children with CP regarding medical costs and inpatient days. Directly comparing the cost-effectiveness of different treatments and interventions is challenging. A systematic review conducted by Shin, et al. reported that the diversity of the decision contexts and health care systems complicated examining cost-effectiveness. Prevention is a critical strategy for cost-effectiveness . A studyCurrently, COCI related research has demonstrated that high COCI could reduce costs and the number of inpatient days. In Korea, a nationwide cohort study was conducted on 47,433 patients with new diagnoses of hypertension, diabetes, hypercholesterolemia, and complications of these diseases in 2003 and 2004. The results revealed that high COCI was correlated with fewer inpatient days and reduced inpatient and outpatient health care costs . A studyIn the current study, lower COCI was associated with higher medical costs, particularly in ambulatory patients with CP. These findings indicated that changing physicians was correlated with a large increase in medical costs associated with outpatient visits, and, to a lesser extent, increased medical costs resulting from inpatient visits. The five–year emergency department visits after the index date of children with CP are shown in In our multivariate analysis of the characteristics of children with CP, younger age was associated with increased medical expenditure, which is similar to a study performed by Park et al. They reported that annual health care cost per person with CP decreased gradually with growing age before the age of 18 years . Our finThere are some limitations to our study. First, the study was retrospective, and some confounding factors could not be extracted from the NHIRD, including the unrecorded costs of medication and treatments at patients’ own expense. Second, CP severity is often categorized using the Gross Motor Function Classification System. However, we could not extract the degree of CP severity from NHIRD. Tonmukayakul’s systematic review reported a strong relationship between CP severity and expenditure . This liThis population-based cohort study demonstrated that high COCI levels in children with CP decreased medical costs and inpatient days. We suggest that a child with CP should not change pediatricians frequently, in order to decrease medical costs and inpatient days. Moreover, younger age, more inpatient visits, residential area, hospital level, PTL and SGA, pneumonia, and nasogastric tube use exhibited an effect on medical costs and inpatient days. For policy-making and cost-effectiveness regarding children with CP, further long-term and high-quality clinical studies should be conducted.


Fijivirus (family Reoviridae) have caused serious damage to rice, maize and sugarcane in American, Asian, European and Oceanian countries, where seven plant-infecting and two insect-specific viruses have been reported. Because the planthopper vectors are the only means of virus spread in nature, their migration and efficient transmission of these viruses among different crops or gramineous weeds in a persistent propagative manner are obligatory for virus epidemics. Understanding the mechanisms of virus transmission by these insect vectors is thus key for managing the spread of virus. This review describes current understandings of main fijiviruses and their insect vectors, transmission characteristics, effects of viruses on the behavior and physiology of vector insects, molecular transmission mechanisms. The relationships among transmission, virus epidemics and management are also discussed. To better understand fijivirus-plant disease system, research needs to focus on the complex interactions among the virus, insect vector, insect microbes, and plants.Viruses in genus  Plant-infecting fijiviruses have double-shelled, icosahedral particles approximately 70 nm in diameter, with spherical, short surface spikes (A spikes) on each of the 12 vertices of the icosahedron . The outFijiviruses are transmitted by delphacid planthoppers in a persistent-propagative manner . In geneIn nature, plant infecting fijiviruses are only spread by planthoppers vectors except garlic dwarf virus (GDV) through vegetative propagation materials . In mostJuncellus serotinus [Rottb.] C. B. Clarke) and variable flatsedge (Cyperus difformis L.) in family Cyperaceae, oat (Avena sativa L.), rice (Oryza sativa L.), sugarcane (Saccharum officinarum L.), maize (Zea mays L.), barley (Hordeum vulgare L.), rye , wheat (Triticum aestivum L.), and pangola grass (a sterile triploid of Digitaria decumbens Stent) in family Gramineae, and garlic (Allium sativum L.) in family Liliaceae (Eight plant-infecting fijiviruses  and one iliaceae .S. officinarum L.), but other Saccharum species, Sorghum species, and maize can be experimentally inoculated with viruliferous planthoppers  Scopoli, Echinochloa crus-galli (L.) P. Beauv, and Cynodon dactylon (L.) Persoon , including Guyana, Brazil, Peru, Fiji, and Taiwan Province of China (Fiji disease virus (FDV), the first of eight known plant infecting fijiviruses, was reported in 1886 in Fiji . By 1906thoppers . GDV, thnce 1988 . Althougnce 1988 . Maize rnce 1988 . The hos Persoon . Mal de  Persoon . In addi Persoon . Oat ste Persoon , causes  Persoon . Pangola Persoon , is a seof China . Rice blof China . In 2008of China  and causof China . The gloFiji disease virus, Mal de Río Cuarto virus, Maize rough dwarf virus, Pangola stunt virus, Rice black streaked dwarf virus, Southern rice black streaked dwarf virus, Oat sterile dwarf virus and Garlic dwarf virus) in genus Fijivirus and any known vectors are described in Perkinsiella saccharicida Kirkaldy, Perkinsiella vituensis, and Perkinsiella vastatrix . In those cases where hatching did occur, the incubation period was longer by up to 3 days (about 25%) than that of eggs from non-viruliferous females, and the mortality of the resulting larvae was high. Similarly, compared with non-viruliferous S. furcifera, SRBSDV-viruliferous females deposited fewer eggs and the viruliferous nymphs spent longer time developing into adults  Pers. (Bermuda grass) for longer than 4–7 days and does not molt or lay eggs on this plant, but it can survive and breed successfully when the plants are infected with MRDV or the planthopper is already MRDV-viruliferous when placed on the plant  in maize , transmitted by Nilaparvata lugens Stal, has become more frequent in southern China since its co-infection with SRBSDV during 2010s , P2 , P3 (capping enzyme), P4 (outer shell B-spike protein), P8 (minor core protein), and P10 (major outer capsid protein), seven putative non-structural proteins P5-1, P5-2, P6, P7-1, P7-2, P9-1, and P9-2 . These vDrosophila melanogaster and mosquitoes to limit viral infection have been extensively studied  pathway plays a key role in antiviral defense . In vect studied . VsiRNAst vector . Similarle layer . de HaroS. furcifera were identified to interact with P6, P7-1 or P10 of SRBSDV. Five proteins  interacted with SRBSDV P6, and they are mainly involved in gene transcription, protein translation, protein post-translational modification and protein synthesis  exhibited different levels in five organs: neuroglian, MLC2, polyubiquitin and profilin highest in the gut, but ribophorin II and E3 ubiquitin ligase highest in the salivary glands and hemolymph, respectively, which indicated that they might play specific roles in viral progresses in different organs , a major problem for Australian sugarcane industry. The virus epidemics led to serious yield losses during 1970–1990 because FLG-susceptible cultivar NCo310 was planted in a large area and the vector  density . Besidesmportant . On the n region .D. kuscheli does not reproduce on maize, it breeds on MRCV susceptible winter cereals such as oat, wheat and rye, which serve as virus reservoirs  and Honghe Prefecture (Yunnan) in March. In central and eastern China, initial infection usually occurs in late spring and early summer from the viruliferous S. furcifera population that migrates with air flows from southern provinces of China and northern Vietnam or Myanmar  Cooperative research in plant pathology, insect ethology, ecology and other disciplines can help clarify mechanisms underpinning the four-way interactions in the plant–insect vector–virus–environment network. (b) Entomologists, plant virologists, crop breeders, and ecologists can coordinate and cooperate to advance the selection, cultivation and rational distribution of resistant varieties. (c) International exchange and cooperation should be strengthened to solve disease problems from a global perspective, focusing on the main places where the diseases occur, ascertain the migratory pathways of the vectors and the source of foreign insects, determine the source of the virus, and prevent outbreaks of the disease on an international scale. (d) A variety of experimental techniques for molecular biology, biochemistry, and environmental biology can be used to comprehensively study the pathogenic mechanism of viruses and their interactions with vectors and develop virus inhibitors or new technologies to block acquisition and transmission of viruses by the vectors.XW conceived and designed the review. LZ, NW, YR, and XW wrote the manuscript. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Animal intestines are the source of edible sausage casings, which are traded worldwide and may come from areas where notifiable infectious animal diseases are prevalent. To estimate the risks of virus contamination, knowledge about the quantity of virus and decimal reduction values of the standard preservation method by salting is of great importance. A literature search, based on the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines, was performed in search engine CAB Abstracts to determine the viral load of 14 relevant animal viruses in natural casings or intestines. Only a very limited number of scientific publications per virus were found and viral loads in the intestines varied from high for ASFV (five publications), BVDV (3), CSFV (6), PPRV (3), RPV (2) and TGEV (3) to moderate for PEDV (2) and SVDV (3), low for HEV (2) and FMDV (5), very low for VESV (1) and negative for PrV (2) and VSV (1). PRRSV was found in intestines, however, viral titers were not published. Three viruses  with high viral loads were selected to search for their inactivation kinetics. For casings, no inactivation data were found, however, thermal inactivation data of these viruses were available, but differed in quantity, quality and matrices. In conclusion, important data gaps still exist when it comes to the quantitative inactivation of viruses in sausage casings or livestock intestines. The natural casing is the edible container of sausages and is mainly derived from the small or large intestine of either pig, sheep, goat or cattle. For example, to produce a natural casing from the small intestines of pigs and sheep, the intestines are flushed with water and scraped for the removal of the inner and outer layers of the intestinal wall. The remaining submucosa layer will form, after cleaning with water and incubation with saturated salt for preservation, the natural casing. Because natural casings are being produced as well as traded worldwide, contagious animal viruses from all around the world may be present in this product. For this reason, international trade is subject to animal and public health guidelines. In order to estimate the risks of contamination, knowledge about the quantity of virus and the decimal reduction values  obtained by the inactivation process is of great importance. The aim of this systematic review (SR) is to gather information about viral loads present in the intestines of the animal host. Additionally, a literature search concerning the inactivation kinetics of three animal viruses found with high viral loads was performed and if possible D-values were calculated and were meta-analyzed. Overall, the results should provide more insight into the knowledge gaps , and subsequently give an indication of which viruses are of major concern for the natural casing industry.Fourteen animal viruses were selected based on their worldwide relevance and impact on the food and livestock industry. Nine viruses are listed by the OIE , as notifiable diseases, these include African swine fever virus (ASFV), classical swine fever virus (CSFV), Foot and mouth disease virus (FMDV), Peste des petits ruminants virus (PPRV), Porcine reproductive and respiratory syndrome virus (PRRSV), Pseudorabies virus (PrV), Rinderpest virus (RPV), Transmissible gastroenteritis virus (TGEV), Bovine viral diarrhea virus (BVDV) . BesidesThe search concerning the viral loads of 14 viruses in the intestines resulted in a very limited number of publications per virus as indicated in African swine fever virus (ASFV) is a large enveloped double-stranded DNA virus of the genus Asfivirus within the Asfarviridae family . African10 HAD50/g in the ileum at 6 days post-inoculation (dpi). Blood titers on the same day were 8.2 log10 HAD50/mL [10 HAD50/g [10 TCID50-eq/g in the ileum at 7 dpi. Furthermore, high titers were found throughout the entire intestine [ASFV was found present in intestines in relative high titers, up to 6.2 logHAD50/mL . Infectintestine . Becausentestine ,11,12. Antestine .Bovine viral diarrhoea virus (BVDV) is an enveloped positive single-stranded (ss) RNA virus, classified within the family Flaviviridae together with other pestiviruses including classical swine fever virus and border disease virus . BVDV oc10 TCID50/g in the ileum at 6 dpi, while the highest blood titers at 7 dpi were 1.8 log10 TCID50/mL [10 were described at 10 days post-infection (pi), respectively. The virus was also present in the blood samples, but no titers were determined [BVDV was found in intestines in relative high titers up to. 7.3 logCID50/mL . Maximumtermined . The higtermined . BecauseClassical swine fever virus (CSFV) belongs to the same family as BVDV and is an enveloped positive single-stranded RNA virus ,28. Clas10 TCID50/g in the ileum at 25 dpi, while on the same day the blood titers were 4.0 log10 [10 TCID50-eq/mL), after necropsy mean viral loads in the jejunum and colon of 3.7 ± 0.7 log10 TCID50/g were detected [CSFV was found in intestines in relative high titers up to 6.8 log.0 log10 . High vi.0 log10 . Recentldetected . Virus ddetected ,35. Thisdetected . In bladdetected ,36. In bdetected ,33,34,35Foot and mouth disease virus (FMDV) is a non-enveloped positive single-stranded RNA virus, classified within the Aphthovirus genus as a member of the Picornaviridae family . Importa10 pfu/mL, while in cattle sera titers up to 3 log10 TCID50/mL were found [10 RNA copies/g at 2 dpi, respectively [For FMDV, the efficacy of standard salt (NaCl) preservation to alternative P-salt treatment of natural casings derived from experimentally infected pigs, sheep and cattle at storage temperatures of 4 °C and 20 °C was investigated, and initial virus titers in the large and small intestines ranged from negative to maximum 3.0 logre found ,43. Whenectively . Similarectively . In cattectively . Hepatitis E virus (HEV) is a positive, single-stranded RNA virus without an envelope. HEV belongs to the genus Orthohepevirus in the family Hepeviridae. Swine HEV, which is closely related to human HEV, was discovered in the United States in 1997 and since then has been reported worldwide . Two swi3 genome copies/g was found in the intestine of 69 wild boar in central Italy [HEV was found present in the duodenum, jejunum, ileum, colon, caecum at copy numbers lower than 1000 RNA copies/g by PCR while blood titers were not determined . Recentlal Italy . Porcine epidemic diarrhoea virus (PEDV) is an enveloped RNA virus with a single-stranded positive-sense genome. It belongs to the family Coronaviridae which, together with the Arteriviridae family, constitutes the order Nidovirales . The two10 TCID50/mL, whereas blood titers were not determined [PEDV was present in intestine homogenates in a concentration of 2 to 6 logtermined ,59.Peste des petits ruminants virus (PPRV) is an enveloped, negative-sense, single-stranded RNA virus with a single serotype, belonging to the genus Morbillivirus in the family Paramyxoviridae. Peste des petits ruminants (PPR) is an acute contagious disease, affecting mainly sheep and goats and occasionally wild small ruminants. Young animals (goats more than sheep) are the most severely affected, showing diarrhea and clear nasal discharge . PPR occ10 RNA copies/g in the caecum at 8 dpi and in blood, PCR titers ranged between 0 and 2 log10 RNA copies/mL [PPRV was found in intestines in relative high PCR titers up to 7.6 logopies/mL . The mosopies/mL ,66. Porcine reproductive and respiratory syndrome virus (PRRSV) is an enveloped single-stranded positive-sense RNA virus and belongs to the same order Nidovirales as PEDV and TGEV, but is a member of the Arteriviridae family, genus rodartevirus . There aPRRSV was detected in the intestines by immunohistochemistry (IHC) and in situ hybridization (ISH), however, no publications describing viral loads were found ,75,76,77Pseudorabies virus (PrV), also known as Aujeszky’s disease virus is an enveloped double-stranded DNA virus belonging to the family of the Herpesviridae, subfamily Alphaherpesvirinae, genus Varicellovirus. PrV can infect cattle, sheep, cats, dogs and rats causing fatal disease. Pigs, however, are able to survive and are therefore considered to be the only natural host. New born and nursing pigs are most severely affected showing mortality rates of 100% and 50%, respectively. Respiratory and neurological signs (mad itch) in weaned pigs and abortions in pregnant sows are hallmarks of this disease ,79,80. EPrV was not detected in the intestines of infected pigs ,83.Rinderpest virus (RPV) is like PPRV, an enveloped negative-sense single-stranded RNA virus and member of the genus Morbillivirus, closely related to the measles and canine distemper viruses. Rinderpest (RP) was an infectious viral disease of cattle, domestic buffalo, and many other species of even-toed ungulates, including buffaloes, large antelope and deer, giraffes, wildebeests, and warthogs with high morbidity and mortality rates caused by oral and gastrointestinal ulcerations. In May 2011, the OIE declared that the disease was eradicated, making RP only the second disease in history to be fully wiped out, following smallpox ,85. Alth10 in the ileum at 10 dpi), while maximum blood titers were 2.6 log10 TCID50/mL [RPV was found in intestines in relatively high titers  is a non-enveloped positive single-stranded RNA virus and classified as an enterovirus within the family of Picornaviridae. Swine vesicular disease (SVD) is a contagious viral disease of pigs. It causes vesicular lesions in the mouth, on snout and/or feet and mucosa, indistinguishable from those observed of FMD ,90. Outb10 TCID50/mL in the ileum at 3 dpi, and in blood with a maximum titer of 4.6 log10 TCID50/mL [SVDV was found in the intestine at a maximum titer of 5.5 logCID50/mL . These rCID50/mL ,96.Transmissible gastroenteritis virus (TGEV) is, like PEDV, an enveloped virus with a positive single-stranded RNA genome that belongs to the genus Alphacoronavirus of the Coronaviridae family within the Nidovirales. The host is the pig and it leads to a mortality of nearly 100% in suckling pigs due to the replication of TGEV in the villous epithelial cells of the small intestine and lung . Occurre10 pfu/g at 4 dpi), however viral loads in blood samples were not determined [10 RNA copies/g) in jejunum and ileum on the same day of post-infection [TGEV was found in intestines in relative high titers (max. 7.5 logtermined . This wanfection . Replicanfection . Vesicular exanthema of swine virus (VESV) is a non-enveloped, positive-sense, single-stranded RNA virus belonging to the genus Vesivirus in the family Caliciviridae . VESV is10 pfu/g in the colon at 4 dpi), blood titers were not determined [VESV was found in intestines in relative low titers (max. 1.8 logtermined . Vesicular stomatitis virus (VSV) is an enveloped, negative-sense RNA virus and belongs to the genus Vesiculovirus of the family Rhabdoviridae . VSV is VSV was found negative in intestines and no viremia was detected .Summarizing, published quantitative data on virus titers in intestines were found for all 14 viruses. The methodology in these publications was highly diverse on all aspects of research , therefore comparison was only possible by a very general interpretation of results . From the 14 viruses used to search for the viral loads (Q1) in intestines and/or natural casings the following three viruses were selected for Q2: CSFV, BVDV and PPRV. The latter two viruses were selected because of their high viral loads in intestines in combination with low viremia titers indicating that the amount of virus present in the intestines is probably due to the replication of the virus in the intestinal wall. CSFV was selected as a “golden standard” because inactivation of this virus was previously determined in the 3D collagen matrix inactivation model . The search concerning the inactivation kinetics of BVDV, CSFV and PPRV in the intestines, meat or food resulted in 8, 18 and 3 publications, respectively .No literature was found describing the inactivation of BVDV in intestines or casings, therefore it was impossible to determine D-values for processed natural casings or intestines. To get an impression about the inactivation kinetics of BVDV in other matrices, data were extracted from literature and, if possible, D-values were determined providing a starting point for future studies concerning natural casings. D-values of BVDV could be determined from the data provided in seven out of eight publications . Results10 BVDV showed a log10 reduction of 0.0–0.4 when incubated at 2–8 °C (pH 7) for a period of 20 days [10 TCID50 and also this virus suspension, which was not added to any ingredient turned out negative at the end of the incubation period at 4–10 °C [Because BVDV in serum showed no decrease in titer for at least 5 days at 22 °C, no D-values from this publication could be calculated . Muscle  20 days . From th 4–10 °C . RegressNo publications were found concerning inactivation in PPRV in intestines/casings, however, D-values could be calculated from data in three publications, which all investigated the thermostability of PPRV live attenuated vaccines . An alteFrom 16 out of 18 CSFV inactivation publications, D-values could be retrieved from the actual publication publications (3) or converted from published half-life data (3) or by regression analysis or from the data described 10) as indicated in  as indic10 TCID50/mL in defibrinated swine blood. CSFV was inactivated when heated to 69 °C, 68 °C and 66 °C for 30, 45 and 60 min, respectively. Regression analysis indicates D-values ranged from twelve (66 °C) to six (69°C) minutes [10 pfu/g, followed by heating at 69 °C for 15 min, none of the two pigs showed signs of CSFV, resulting in a D-value of 7.3 min [10 TCID50/mL remained stable for 7 months, while for 25 °C and 37 °C D-values could be calculated by regression resulting in 1.35 and 0.97 days, respectively [Recently, D-values of CSFV infected intestines, processed with either saturated NaCl or phosphate-supplemented salt for 60 days at 4 °C, 12 °C, 20 °C and 25 °C were published, and ranged for NaCl from 18.3 (4°C) to 3.4 days (20°C) . Previou minutes . After i 7.3 min . Thermal 7.3 min . CSFV su 7.3 min . Inactiv 7.3 min . At seve 7.3 min . Because 7.3 min . Surviva 7.3 min . Thermos 7.3 min . Further 7.3 min . CSFV sp 7.3 min . A simil 7.3 min . D-value 7.3 min . The staectively . Survivaectively .Summarizing the inactivation results, no publications about inactivation kinetics in intestines were available for BVDV and PPRV, while for CSFV only a limited amount of publications was available. Therefore, the majority of D-values were obtained or determined to achieve an impression about the inactivation kinetics in other matrices. All three viruses showed thermal dependent inactivation. 10 D-values in minutes which enabled plotting against temperature  were not available, while the majority of the natural casings are made of the submucosa layer of the small intestine. The ileum comprises the part of the intestines that was most frequently tested for the presence of a virus. The amount of Peyer’s patches is in the ileum higher than in the other parts of the intestines. Because of their involvement in Bovine Spongiform Encephalopathy (BSE) the ileum section of the small intestine of cattle will not be processed to natural casings, while ileum sections of pigs and small ruminants are not removed [For all 14 viruses, between zero and six relevant publications were found, which include quantitative data on virus titers in intestines. Quantitative data on virus distribution over anatomically distinct parts of the intestines are limited and available (at least to some extent) for ASFV, BVDV, CSFV, PPRV, RPV, SVDV and TGEV. With the exception of VSV, PrV and PRRSV, viral loads of all viruses were detected in tested parts of the intestines. Maximum titers were highest for ASFV, BVDV, CSFV, PPRV (qPCR only), RPV and TGEV. Maximum titers for these viruses reached > 6 log removed ,139. BecThe current SARS CoV2 pandemic has led to the evaluation of whether this virus is also relevant for the casing industry. Experimental studies with SARS CoV2 in farm animals have demonstrated that cattle and pigs were low susceptible and resistant to SARS CoV2, respectively ,141,142.Due to the limited number of publications on intestinal viral loads, it is quite difficult to exactly define which viruses may be of threat for the casing industry, because the majority of these publications were not performed with this specific goal in mind. Besides viruses identified with the highest titers, other viruses might still be of concern. For example, PPRSV was detected in intestines in many publications but none of these publications investigated the viral loads. Additionally, the high diversity in methodology between publications, like animal species, ages, virus strains, inoculation dose, time of euthanasia, experimental set-up and virus titration methods could have a major impact on the amount of virus detected in the intestines. As a result, the amount of virus present in the intestines could be either under- or overestimated, which in both cases could have major consequences for the casing industry by possible trade limitations.Three viruses found in the intestines with high viral loads, CSFV, BVDV and PPRV, were selected for Q2 (inactivation) of the literature search. This search resulted in no findings of inactivation data  regarding these viruses in sausage casings, and therefore relevant data to determine quantitative inactivation parameters in sausage casings is non-existent. For CSFV, a reasonable number of inactivation publications were suitable for statistical analysis, while for BVDV and PPRV much less publications were available. Nonetheless, the obtained data can serve as informative guidance for future inactivation studies specific to the casing industry. Besides differences in virus strains, detection methods, spiking versus infected tissues, animal studies, matrices and storage/incubation methods, extracting data for D-value calculation sometimes required additional assumptions concerning graphical data and/or initial titers. The height of the calculated D-values will be influenced by all these different factors and may lead to possible under- or overestimations. For example, D-values of PPRV will probably be overestimated, because studies were limited to vaccine inactivation experiments in combination with stabilizing ingredients. Another example is the determination of the D-value of BVDV from a publication where the initial titer (day 0) is lower than titers at days 2, 7, 14 and 21 during the inactivation . Using aIn some studies, different individual responses to virus infection were observed, e.g., feeding CSFV infected tissue (casings or ham) to susceptible pigs, showed some inconsistencies for both types of tissues, which might be caused by different initial viral loads of individual infected pigs . AnotherSome publications about viral load and inactivation in languages other than English or German could have been missed for this SR. No relevant German publications were found in both searches. Because English is the common language for publishing, the chance of a possible bias in this SR will be quite low.In conclusion, this SR shows that major gaps exist when it comes to viral loads and inactivation of viruses in sausage casings or even the intestines from which the sausage casings are produced. A systematic research approach will be needed to fill these knowledge gaps and develop efficacious and efficient protocols for the inactivation of relevant viruses in relation to possible trade barriers.Q1. What are the virus loads of 14 selected viruses  in intesQ2. What are the inactivation kinetics  of three viruses with high intestinal viral loads selected from Q1?This SR basically followed the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines, including quality assessment and transparency . The sea50), plaque-forming units (pfu), 50% Haem-absorbing doses (HAD50) or TCID50-equivalents by qPCR, genomic copies by PCR or Ag-ELISA. For Q2, the criteria were: compliance with the Q2 first screening criteria and inactivation data should be quantified by virus titration and expressed in TCID50 or pfu. Or publications should describe regression analysis resulting in D-values or half-lives. The main reason for excluding was the failure of describing intestinal viral loads (Q1) or inactivation kinetics (Q2). The in- and excluded publications and flowcharts of Q1, Q2, listed per virus, and the PRISMA checklist are indicated in S23–S36, S37–S39 and S40, respectively. Data from the relevant full-text publications were recorded and summarized in an Excel spreadsheet. For Q1, the following headings were used: reference, virus, virus strain, animal species, inoculation dose, sample type, number of samples, assay, experimental or field study, days post-inoculation, intestine, duodenum, jejunum, Peyer‘s patches, ileum, colon, caecum, bladder, blood and remarks. Publications describing viral loads by quantitative PCR (qPCR) or ELISA instead of virus titration were included in the Excel sheet with a search result . The full-text publications were retrieved through online availability or via the Library Servicedesk of Wageningen UR. These publications underwent a second screening assessing the quality of the published data (step 2), including only publications where the search question (Q1 or Q2) could be answered. For Q1, these criteria were: compliance to the Q1 first screening criteria and viral loads should be expressed as 50% tissue culture infective dose (TCIDh result .10 decrease of virus titer) as indicated in 10(2). Titers expressed in plaque-forming units (pfu) were transformed into TCID50 by dividing the pfu titer with 0.7 [For Q2, the following headings were added to an excel sheet: type of research, inactivation time, inactivation temperature, pH, submucosal membrane, muscular layer, natural casing, viral load, inactivation method, half-life, D-value  for BVDV, CSFV and PPRV. Besides this analysis, Graphpad Prism version 8 was used to perform descriptive statistics and box and whisker plots combined with individual data from non-transformed D-values of BVDV, CSFV and PPRV at relevant temperatures ranging from 4 °C to 25 °C. For a correlation in temperature, the retrieved or calculated D-values in the different matrices were log-transformed and categorized (in minutes) in an Excel spreadsheet. These log