We use an individual based model and national level epidemic simulations to estimate the medical costs of keeping the US economy open during COVID-19 pandemic under different counterfactual scenarios. We model an unmitigated scenario and 12 mitigation scenarios which differ in compliance behavior to social distancing strategies and in the duration of the stay-home order. Under each scenario we estimate the number of people who are likely to get infected and require medical attention, hospitalization, and ventilators. Given the per capita medical cost for each of these health states, we compute the total medical costs for each scenario and show the tradeoffs between deaths, costs, infections, compliance and the duration of stay-home order. We also consider the hospital bed capacity of each Hospital Referral Region (HRR) in the US to estimate the deficit in beds each HRR will likely encounter given the demand for hospital beds. We consider a case where HRRs share hospital beds among the neighboring HRRs during a surge in demand beyond the available beds and the impact it has in controlling additional deaths. As states push to end social distancing and reopen businesses, it is important to understand the cost of opening in terms of lives lost and medical costs incurred. A premature opening will likely cause more deaths and infections as the healthcare system will likely get overwhelmed, and may wipe out all the gains made in the initial shutdown. We use an agent-based model and simulation framework to estimate the immediate medical cost of COVID-19 under different mitigation scenarios. The scenarios consider social distancing with different durations and varying compliance levels. The simulation framework uses a detailed representation of the US population and their social interactions to study the spread of COVID-19. An SEIR (susceptible-exposed-infected-recovered) model captures the time varying health states of the individuals. The infected individuals arrive at one of the three health states i.e. medically attended, hospitalized, or ventilated before getting to the final health state i.e. recovered or dead, as shown in Fig. Medical costs are applied based on the three health states i.e. medically-attended, hospitalized and ventilated. In addition, if an infected individual dies, then the “value of statistical life” is used to estimate the cost of death. We also estimate the shortage of hospital beds that is likely to occur in each Hospital Referral Region (HRR) given the demand for hospital beds and the number of available beds in each HRR in the US. Data on the number of beds in each HRR is obtained from the American Hospital Association (AHA) and a fraction of them are assumed to be available for COVID-19 patients. We consider cases where neighboring HRRs share or do not share hospital beds during a surge in demand.This information is then used to calculate additional deaths and medical costs for each of the mitigation scenarios. Policy makers can apply this kind of analysis to decide where the temporary hospitals may need to be built to offset the deficit in demand for beds. Our goal is to use this knowledge to provide guidance to public health officials and policy makers on the trade-offs between the length of lockdown, compliance to social distancing, infections, deaths and the medical costs. Our scenario-based analysis estimates the burden of the disease in monetary terms, and helps rank-order mitigation strategies.1 consider potential health care costs and resource usage under different attack rates which vary from 20 to 80%. However, it does not consider any interventions or mitigation strategies. Our research focuses on counterfactual mitigation scenarios and their respective costs. We use recent cost estimates for COVID-19 available from the Kaiser Family Foundation (KFF)2 which uses cost of pneumonia cases as a proxy. Our detailed network based model, that captures heterogeneous social interactions and contact times among the individuals in the population, is one of the unique features of the analysis. Additionally, no other research has provided an estimation of medical costs for such detailed mitigation scenarios for the entire US.In related work, authors inOur results show that (1) Without mitigation, the total medical costs would be a significant fraction (5%) of the US GDP; (2) a lockdown of just 2 months, if done early in the epidemic, and with sufficient compliance, could have reduced the medical costs by more than 90%; (3) if 90% compliance could be achieved, then even a 45 day lockdown period would have been enough to contain the epidemic and the medical costs; (4) if HRRs do not share hospital beds with other HRRs, a significant deficit of beds will cause medical costs to skyrocket, through increase in deaths. However, if HRRs shared beds with their neighboring HRRs, the bed-deficits and additional deaths could be reduced to almost zero; and (5) a sensitivity analysis of the parameters shows the costs are most sensitive to the duration of the stay-home order.9 using an individual level synthetic social contact network10—which represents each individual in the population along with their demographic attributes , and their social interactions. The main steps in the first-principles based construction of synthetic populations and social contact networks are: (1) construct a synthetic population by using US Census and other commercial databases; (2) assign daily activities to individuals within each household using activity and time-use surveys ; (3) assign a geo-location to each activity of each person based on data from Dun and BradStreet, land-use, Open Street Maps etc.; and (4) construct a dynamic social co-location based social contact network that is induced when people simultaneously visit locations. These networks have been validated and used for numerous public health analyses before such as16. For details on the construction of social networks, see17.We build on our modeling and simulation framework for epidemic spreadbest guess 2020-04-14 version of “COVID-19 Pandemic Planning Scenarios” document prepared by the Centers for Disease Control and Prevention (CDC) SARS-CoV-2 Modeling Team18. The sequence of health-state transitions and possible paths are shown in Fig. 19.The SEIR disease model and parameters used here for COVID-19 have been defined in the 2. The costs for each health state are shown in Table To estimate the medical cost of treating COVID-19 patients, we use the average cost of treatment for pneumonia, paid among “large employer health insurance” plans, as a proxy22. We use an estimate of $2 million dollar as the value of life to convert deaths into costs23.The total medical costs can be measured through multiple criteria in terms of number of deaths and treatment costs, or a single criterion in dollar terms by converting deaths into dollars using “value of a statistical life”. There are various ways in which the value of a statistical life has been measured. US federal government uses $10 million dollar per life lost regardless of a person’s age while others have used estimates in the range of $160k to $2.4 million based on metrics like average lifetime earnings of a college graduate, 9/11 victim compensation, wrongful death claims, insurance policy value etc.Voluntary home isolation (VHI): Symptomatic people choose to stay at home (non-home type contacts are disabled) for 14 days.School closure (SC): Schools and colleges are closed (school type contacts are disabled).Stay home (SH): People follow public health “stay-home” directive (non-home type contacts are disabled).25. Once closed, schools are assumed to remain closed until end of August after which they reopen. Other social distancing interventions stop at 30, 45 or 60 days from the start date of the intervention, depending on the SH duration. Note that this implies all mitigation efforts end by the end of summer 2020.School closure and stay-home interventions start on different days in different states as stated inWe consider a number of mitigation scenarios that comprise of various social distancing strategies, compliance levels and durations of stay-home order. The following social distancing strategies are used: Durations of stay-home order vary from 0, 30, 45 to 60 days. Compliance to stay-home and voluntary home isolation vary from 60%, 70%, 80% and 90%. Table We use hospital bed capacity data available for each HRR in the US from AHA, to calculate the deficits that are likely to be encountered by each region. We assume three scenarios regarding the use of hospital beds: (1) All COVID-19 patients who need a bed, will have one available i.e. there is no shortage of hospital beds, (2) bed capacity in each HRR is limited and beds cannot be shared among hospitals in other HRRs to accomodate the surge in demand for beds and (3) bed capacity in each HRR is limited but beds can be shared among hospitals in the neighboring HRR regions to accomodate the surge in demand.26.Other factors considered in these scenarios are the average length of hospital stay of patients who are hospitalized, and the percentage of beds dedicated to COVID-19 patients in each HRR. Length of stay is 7 days or 14 days, and the percentage of beds available to COVID-19 patients is 70%, 80%, 90% or 120%. More than 100% bed capacity (120%) has been considered since many hospitals are able to temporarily increase their capacity beyond normal levelsEach day the demand for the number of beds in each HRR is determined by the simulations. The simulation results provide the counts of individuals who are in “hospitalized” state and depending upon the assumed duration of the hospital stay (7 days or 14 days), the demand for beds per day is calculated. Note that this includes individuals who are in “ventilated” state as well since everyone in “ventilated” state has to be in the “hospitalized” state first according to our disease model, see Fig. We assume that patients who need a hospital bed and cannot get it, will die. This is a strong assumption and hence four different values of dedicated bed capacities have been considered to show its sensitivity. Note that only the death counts and overall costs will change in scenarios (2) and (3) when limitations to bed capacity are considered. This assumption of “all bed deficits result in deaths” provides an upper bound on the medical costs resulting from the shortage of beds. If only a fraction of them die then the additional deaths and the costs can be appropriately scaled down.This section reports the simulation results and the medical costs that are likely to incur under various mitigation scenarios. For each of these scenarios we also consider four different possibilities for the availability of dedicated hospital beds for COVID-19 patients. Note that the hospital beds are considered as a post-processing step, after the simulations have been run and infected individuals have arrived at “hospitalized” state.Figure Figure 27. If the demand for beds exceeds the supply, patients who do not find a bed, will die. Each additional death results in extra costs at the rate of $2 millon per person. Figure 27 and 70% of the bed capacity being available to COVID-19 hospitalizations. However, sensitivity analysis has been done by varying length of hospital stay to 7 days and dedicated beds availability to 80%, 90% and 120%.Figure An analysis of costs under the assumption that hospital beds can be shared with neighboring HRRs shows that the shortage of beds can be completely alleviated by sharing. In almost all mitigation scenarios there are no additional deaths due to shortage of beds and hence no additional medical costs, as shown in Figs. 29, to do the sensitivity analysis. We randomly select 30 cells with different combinations of We analyze the sensitivity of our results to multiple factors i.e. the reproductive number  and other private organizations such as insurance companies ($160k). We use an estimate of $2 million per person based onThis study estimates the medical costs of COVID-19 in the US under different mitigation scenarios and helps understand the tradeoffs between deaths, costs, infections, compliance to social-distancing and the duration of stay-home order. Our scenario-based analysis estimates the medical burden of the disease in monetary terms, and helps rank-order mitigation strategies.It shows that costs are most sensitive to the length of the stay home order and then to the level of compliance. A stay home order of 60 days with 80% compliance, can drop the medical costs from $1 trillion to a meager $35 billion. Stay home duration also mitigates the effect of a high We also show that sharing of hospital beds among neighboring “Hospital referral region” during a demand surge can reduce the additional number of deaths to almost zero. Cooperation in sharing medical resources between neighboring regions can save lives and money. This kind of analysis can also help decide where additional bed capacity and temporary hospitals may need to be built to offset the surge in demand.Supplementary Information.


Adenoviridae, Arbovirus, Arenaviridae, Herpesviridae, Picornaviridae, Paramyxoviridae as well as Togaviridae.Emerging and re-emerging viruses may cause meningitis, encephalitis, meningoencephalomyelitis, encephalitis, Guillian-Barré-like-syndromes as well as strokes. Most important viruses belong to the family of Clinical presentation usually consists of a biphasic presentation. Non-specific febrile illnesses may be accompanied by rash, headache, arthralgia and myalgia. Thereafter focal neurological signs may evolve. Diagnostic strategies for the detection of emerging and re-emerging viruses may be difficult due to the short viraemic period. Pitfalls in serology may be due to antibody crossreactivity.Arboviruses are transmitted by arthropods. Aedes mosquitos are one of the vectors for arboviruses like Chikungunya-virus, Dengue-virus, Japanese-Encephalitis-B-virus and West-Nile-virus. Since the last centuries Aedes mosquitos have spread from their naturally habitat in Africa to America as well as Europe. The arboviruses risk profile depend essentially on the occurrence, the activity of the respective vector, this may be the key to fight the disease and its spread. Due to global shifts in the ecological balance but also as a result of more or less successful control measures, some diseases have become rarer, others are more common. The viruses persist in the respective vector months to years; in ticks they may persist for years and in mosquitoes 1 to 4 months. In order to survive bad climatic conditions unscathed, the viruses partially overwinter in arthropods. The increased trade, travel habits, urbanization and climate change are some of the factors, which favour evolution and spread of new pathogens. Infectious diseases are emerging or re-emerging every year. Neuroinfectious diseases may occur as outbreaks in small localized regions or may spread rapidly over large geographical areas. An epidemic occurs when an infectious disease spreads rapidly to many people like the Zika Virus outbreak. A pandemic is a global disease outbreak as seen e.g. in HIV/AIDS. Recent outbreaks caused by Chikungunya-virus, West – Nile-virus, Enterovirus infection and Ebolavirus have caused severe neurological manifestations and spread rapidly across continents. Endemic diseases are constantly present, usually in low numbers. Neuroinfectious diseases transmitted by viruses may cause a broad spectrum of neurological presentations such as meningitis, encephalitis, meningoencephalitis, Guillian-Barré-like-syndromes as well as strokes. Often these patients are left with severe neurological sequelae. It is obligatory that neurologists are aware of current developments including typical and atypical presentations of neurological infections in travellers, diagnostic and treatment recommendations. To avoid fatal outcomes and long-term sequelae, specialized neurological intensive care medicine is crucial for survival.Neurotropic viruses can access the brain via retrograde axonal transport, haematogenous spread crossing the blood-brain-barrier (BBB) or through spread of infected leukocytes across the BBB into the brain parenchyma. In the pathogenesis of neurotropic pathogens the blood brain barrier plays a vital role by controlling the access of circulation molecules, immune cells or viruses into the central nervous system. The BBB is not impenetrable and neurotropic viruses have evolved to disrupt and evade. But it’s known that some of the viruses are able to invade the brain parenchyma like a “Trojan horse”: retrograde axonal transport of a Flavivirus after a mosquito bite provides such a neuroinvasive pathway. Semaphorins  act as a potential entry receptor. Viral infection increases metalloproteinase and tumour necrosis factor levels such facilitating entry of infected neutrophils into the CNS. Flaviviruses like West-Nile-virus, Dengue-virus, Japanese-Encephalitis-B -virus may use this route to invade the CNS. Paracellular entry of virus includes alteration in expression or phosphorylation of tight junction proteins, disruption of the basal lamina and disruption of the actin cytoskeleton.Adenoviridae, Arboviruses (arthropod-borne-virus), Arenaviridae, Herpesviridae, Picornaviridae, Paramyxoviridae as well as Togaviridae. Since the last few years also Bornaviridae (Bornavirus) have been discussed as possibly associated with psychiatric disorders. In 2015 a new lineage  was confirmed as causative pathogen for encephalitis in humans.Many viruses causing neurological disorders belong to the family of Arboviruses are transmitted by arthropods, about 100 of them are human pathogens, which represent a significant public health problem throughout most parts of the world. The term arbovirus comprises many different genetically inhomogeneous viruses being only defined by the transmission through arthropods.Arboviruses multiply in vertebrates, are picked up by arthropods such as blood-sucking insects or ticks during viremia and transmitted to vertebrates by bite or string. In order to survive bad climatic conditions, the viruses partially overwinter in arthropods. With a few exceptions humans and pets like cats and dogs are no hosts for arboviral infection .Flaviviridae, belongs to the most important developing viruses worldwide and comprises over 70 different virus types. Flaviviruses utilize a single-stranded mRNA and translate into precursor proteins, which includes metalloproteinases, structural as well as non-structural proteins.Flavivirus, family Aedes spp., Culex mosquitos or ticks  as vectors. Insect-borne viruses usually have specific endemic geographical areas where the incidences are mostly related to the seasons. Flaviviruses are present in six different continents and global distribution of flaviviruses is dynamic [Flaviviruses use mosquitos like  dynamic .Aedes aegypti). Among the more than 22 types of viruses that can be transmitted by Aedes albopictus are Chikungunya, Dengue, Yellow Fever and West-Nile-viruses. Figure Dengue-virus (DENV), Zika virus (ZIKV), Chikungunya-virus, Japanese Encephalitis, tick-borne encephalitis, West-Nile-virus and St. Louis Encephalitis are among the most common flaviviral infections in which the CNS is involved. The Asian tiger mosquito is an important transmitter of Arboviruses, the yellow fever mosquito (The name flavivirus is derived from the first virus (yellow fever), for which an insect-bound pathway was detected  , 25.Due to the close degree of relationship as well as co-circulating in the same geographical regions coinfections with several arboviruses can occur. Cases of Chikungunya virus (CHIKV) and Dengue-Virus (DENV) co-infections as well as Zika virus (ZIKV) and CHIKV co-infections have been reported , 23, 27.However, it’s unclear whether the co-infection occurs via single or multiple mosquito bites.Ae. aegypti to transmit both ZIKV and CHIKV in one bite. In their results 12% of mosquitos transmit ZIKV and CHIKV in one bite, the simultaneous oral exposure to both viruses didn’t change infection and transmission rate compared to exposure to single virus. [Goertz et al. have investigated the potential of e virus. . Some cae virus. , 26).Viral infections may cause direct virus mediated cell damage as well as autoimmune mediated disorders. Another mechanism is the so-called immune enhancement - after primary infection of one subtype of Dengue-virus and reinfection with another Dengue-virus subtype causes an aggravation of symptoms and severe neurological manifestation , 21.Flaviviridae and has a single-stranded positive RNA, with four known serotypes, DENV-1 to DENV-4. DENV is transmitted by Aedes spp. , nevertheless not every Aedes spp. transmits every serotype due to their genetic variations.Dengue is a member of the family 8 and 109 infectious particles per ml of blood, and the viremia lasts four to 5 days on average. Endothelial cells and possibly also bone marrow cells are susceptible to infection. A previous infection with DENV 1–4 followed by another of the four DENV serotypes can lead to more serious illnesses, which maybe explained by immunoenhancement [Mosquitos without the receptor protein R67 / R64 in their gut epithelial cells are not capable to transmit DENV 1–4. Through the bite of the infected mosquito, the DENV enters the bloodstream and invades the macrophages. The macrophages transport the virus via the lymph channels to the lymph nodes, where it replicates. The viremic patient has between 10ancement .Comparing the amino acid sequences of their E proteins of the four DENV serotypes, they show 63–68% homology between the different serotypes. The homology between different variants of a DENV serotype is over 90%. Partially, the DENV-specific antibodies cross-react with other serotypes in DHF and DSS. Due to their low affinity, they do not have a neutralizing effect, but confer a preferential, more efficient uptake into the cells of the viruses complexed with the AK through interaction with Fc receptors on monocytes and macrophages (the cross-reacting IgG molecules increase the infection). Osman et al. have conducted a study on the frequency of serotypes in DENV infection in Brunei Darussalam, Southeast Asia. The predominant serotype for Brunei is DENV-2, followed by DENV-1. , patientIn 1992, dengue fever (DF) was still considered by the World Health Organization (WHO) as one of the neglected diseases. Since then, a dramatic change has taken place: the disease is now regarded as one of the most important infectious diseases with pandemic proportions. The overall incidence has increased more than 30 times over the past 50 years. For example in Germany, 957 cases were reported in 2016 and 635 cases in 2017 to the Robert Koch Institute (RKI). In 2018, 367 cases have been submitted to the RKI up to the month of October. The annual number of cases is estimated at around 390 million worldwide. Dengue is endemic in more than 100 countries in Africa, America, Eastern Mediterranean, South-East Asia and Western Pacific regions. Most seriously affected are the Americas, South-East Asia and the Western Pacific regions.The incubation time ranges from 3 to 14 days. Dengue infections are usually clinically mild or even often asymptomatic. The neurological complications include meningitis and encephalitis. Patients initially present with fever, headache, muscle and joint pain and rash. Usually there is a sudden increase in fever. Headache, severe muscle and joint pain, bradycardia, hypotension, lymphadenopathy frequently occur, named breakbone-fever. It may come to a second fever peak with a body exanthema as well as petechiae. In general, the course of the disease is short and abrupt, but long periods of reconvalescence with severe exhaustion are described, which can last for weeks. Patients with concomitant hepatitis with moderate transaminase elevation regularly appear. In about 10% there is an involvement of the nervous system. The most common neurological presentations of DENV infection are encephalitis, meningitis, strokes and cerebellar haemorrhage, Guillain-Barré-Syndrome, myositis can also be caused by DENV.Especially in this group, a pronounced fatigue for several weeks in the sense of a depletion syndrome often appears after expiration of the acute illness. The increased permeability of blood capillary walls as well as thrombocytopenia is the cause of dengue haemorrhagic fever (DHF) and dengue shock syndrome (DSS). The DHF has primarily similar symptoms to the DF, followed by a short remission. Before a sudden worsening characterized by hypotension and circulatory collapse, stomachache, vomiting, restlessness, impaired consciousness or a sudden change from fever to hypothermia are the progenitor of DHF. Petechiae in skin and mucous membranes occur secondary to low platelet count and dysfunctional thrombocytes. Intracranial haemorrhage, haemorrhagic pneumonia and gastrointestinal bleeding may occur. The DSS follows an unfavourable course of DHF with mostly lethal outcome. DHF is triggered by host factors such as a previous DENV infection and an age < 15 years. DHF and DSS are typical secondary diseases after a previous primary infection, through which different serotypes bind the originally dengue-targeted antibodies to the surface of the new serotype without being able to neutralize it. The virus is “masked” for another immune response, propagation and proliferation is favoured, this can lead to a serious disease course with bleeding tendency and vascular damage. DENV has also been detected in organs such as the lungs, liver, kidneys and gastrointestinal tract.Zika Virus Infection, Chikungunya virus fever, West-nile-fever, as well as other arthropod-borne viruses and Malaria or Leptospirosis, Guillain-Barré-Syndrome, Stroke .CSF examination is crucial for diagnosis here ELISA is the gold standard, rt-PCR may also be performed. Typically patients show thrombocytopenia. An increase in transaminases and lymphocytosis may occur. CSF has to be repeated in cases of doubt, if possible due to thrombocytopenia. The trias of rash, thrombocytopenia and severe headaches, muscle and joint pains is characteristics for the diagnosis of Dengue fever.There is no antiviral therapy available. DENV infection is treated symptomatically. Early recognition of severe complications for example secondary to thrombocyte dysfunction and thrombocytopenia is highly important. Treatment on a specialized neurological intensive care unit is recommendable, if myelitis or meningoencephalitis occurs neurologic intensive care is life saving.Patients with DF in general have a good prognosis. Patients with severe DHF often progress to DSS with high mortality.By recommendation of WHO countries have to consider dengue vaccine only where epidemiology suggests a high burden of disease, for use in persons 9–45 years of age, complete recommendations may be found in WHO releases. There is no global vaccination for DENV available, since April 2018 WHO revised recommendations for endemic regions.Nipah-virus is an emerging bat-borne virus, belongs to the Paramyxoviruses, a group of negative sense, single stranded RNA viruses. Paramyxoviruses traditionally have been associated with a group of viruses that have narrow host range and typically cause outbreaks of disease with low mortality rates . Fruit bThe name Nipah comes from the Malayan Kampung Sungai, here the virus was isolated the first time . NiV outThe first recognized outbreak of NiV was 1998 in Malaysia, where humans and pigs were infected, due to transportation of infected pigs the virus was also recognized in other Malaysian regions. The transmission of NiV during this time was from pig to human, with the termination of about 900.000 pigs the epidemic was stemmed [Incubation period ranges from 3 to 31 days with an average of 7 days.Most patients present with acute encephalitis, fever, headaches and alteration of consciousness. During the epidemics in Malaysia patients also presented with a flue-like syndrome. According to the World Health Organization (WHO) the mortality rate is estimated at 40–75%, whereby variations during outbreaks can occur, depending on local capabilities for clinical management. For example, the mortality rate for the last outbreak in Kerala, India was 86%.During the epidemics in Malaysia after an average of 10 days death occured. After several weeks, some of the patients suffered from a relapse.Other encephalitis triggering pathogens such as Japanese encephalitis, herpes-simplex-virus type 1- encephalitis, Dengue-Virus.CSF examination is crucial. For specific diagnosis virus isolation, electron microscopy, immune-electron microscopy, immunohistochemistry, serology and PCR can be used. Notice, only special laboratories (Biosafety Level 4- BSL4) are available to handle NiV. The specific detection can be made with CSF, serum, blood as well as swabs. For surveillance and diagnostic purposes three tests were developed not requiring BSL4: ELISA (enzyme-linked-immunoabsorbant assay), liquid protein array multiplex test and a pseudo type virus (different pseudo types carrying F and G proteins have been develop detecting virus-specific antibodies).Neuroradiology: Magnetic resonance imaging (MRI) doesn’t distinguish other viral encephalitides. Abnormalities seen in MRI are multiple, small, asymmetric, focal lesions in the subcortical and deep white matter without surrounding oedema. Brainstem involvement is common.There is no specific treatment available. Specialized neurological intensive care management is essential for survival and prognosis. Ribavirin, which inhibits replication of Hendra Virus in vitro, was used during the outbreak in Malaysia, empirically. Recent animal models found that Ribavirin inhibits the viral spread in vitro but not in vivo. In the animal model Ribavirin delayed death from viral disease in NiV-infected animals for approximately for 5 days .In vivo studies have shown, that Ribavarin is maybe effective to reduce mortality. Of patients who survive NiV encephalitis, 20% have residual neurological deficits, including cognitive difficulties, tetraparesis, cerebellar signs, nerve palsies and clinical depression.There is no commercial vaccine available.Culex tritaeniorhynchus, which breed in rice paddy fields and other water sources. Normally it’s an enzootic cycle, humans are the dead-end host for the virus. After infection, the virus replicates in the lymphoid tissue. From there a viremic spread and invasion into the CNS takes place. Inflammation occurs with focal necrosis of the nerve cells, neuroglia and perivascular lymphocytic infiltrates.JEV belongs to Flaviviridae with a single stranded RNA. JEV is transmitted by Culex mosquitos, commonly JEV is endemic in South and South-east Asia. Less than 1% of individuals develop clinical features after incubation, about 20% of symptomatic patients develop a fatal encephalitis.The incubation period is about 5–15 days. Clinically patients may present with a flue-like illness. This may be followed by focal neurological signs, acute flaccid paralysis and subsequently severe life threatening encephalitis. Often there are significant neurological sequelae in patients surviving JEV encephalitis or acute flaccid paralysis.Other arthropod-born- virus induced diseases with the same geographic distribution of mosquitos like Chikungunya fever , 18.CSF examination is essential. A rapid test for the detection of IgM antibodies is available, with which serum and plasma can be tested. In the further course, increasing IgG titers can be detected. Neuroradiological examination may show involvement of thalamus and basal ganglia.There is no antiviral medication. Specialized neurological intensive care medicines is crucial.Approximately 50% of the severe cases are lethal, 44% with mild or moderate course have a full recovery, 31% having persistent and severe neurologic sequelae, including chronic seizures, motor dysfunction and neuropsychiatric symptoms.A vaccination against JEV is available, it is recommended for people living in or travelling to endemic regions. For Europe an inactivated vaccine i.m. is approved, primary immunization consists of two doses 28 days apart. The European Medicine Agency recommends a booster for exposed adults within the first year.The inactivated mouse brain-derived vaccine is replaced now by cell culture-based vaccines, four types of cell culture-based vaccines are available. A live attenuated vaccine (SA 14–14-2 strain), mostly common in China, where the 1st dose is given subcutaneously at 8 months, followed by a booster dose at the age of 2 years. An inactivated vero cell-derived alum-adjuvanted vaccine (SA 14–14-2 strain), two intramuscular doses 4 weeks apart, booster is recommended after 1 year. The third one is the inactivated vero cell-derived vaccine (Beijing-1 strain), with primary immunization of three doses 4 weeks apart. The live chimeric vaccine (with yellow fever 17D as backbone), only a single dose is recommended, the need for booster have not been determined yet.Bornaviridae with a negative single-stranded nonsegmented RNA. The most likely natural route of entry is nerve endings in the nasal and pharyngeal nucosa. Infectious dose for natural infection is unknown, as well as transmission from infected animals to humans. Vertical transmission is confirmed, for animals and humans.BDV is a member of the family In the 1700s borna disease was first described in European veterinary textbooks as a disease of farm horses with names like hot-headed disease (German: Hitzige Kopfkrankheit) or epidemic encephalomyelitis (German: Seuchenhafte Gehirn-Rückenmarksentzündung). The name borna disease was given after occurrence of major outbreaks in 1894–1896 in the district around Borna in Saxony, Germany.Bornaviridae in the order Mononegavirales, during the first decade of the twenty-first century a novel BDV, now known as avian bornavirus (ABV), was identified in parrots with proventricular dilatation disease. As known in veterinary medicine since many decades, recently bornavirus has been identified a causative pathogen of fatal encephalitis in humans. Between 2011 and 2013 three breeders of variegated squirrels (Scirus variegatoides) had encephalitis with similar signs and symptoms with lethal outcome two to 4 months after onset. Phylogenetic analysis revealed that the virus forms a lineage separate from the known bornavirus species, tentatively it’s named variegated squirrel 1 bornavirus (VSBV-1) [BDV was the first member of the new family For humans with Borna virus infection no exact incubation time is specified. Animal studies have shown symptoms of disease after two to 3 weeks.Neurological signs can be complex and variable, the variety of CNS infection can result in comparable clinical picture.The recent published human cases had similar symptoms. All presented with an encephalitis, fever, qualitative and quantitative unconsciousness with initial cerebellar symptoms, myoclonic twitches and cranial nerve paresis .Tick-borne encephalitis, rabies, botulism, in regions with arthropod-borne flavivirus must be considered.CSF examination is crucial. Diagnosis can be confirmed with BDV-specific antibodies in serum and CSF or BDV-specific antigens, RNA detection in blood or CSF. Western blot analysis, ELISA and indirect immunofluorescence assay (IFA) have been established as serological test. IFA is considered as the most reliable method, with high sensitivity and specificity.There is no specific treatment available. For survival special neurological intensive care medicine is essential.There is no vaccination available. As the vaccination for animals is not reliable, no further investigations for human vaccines are done at the moment.WNV belongs to the genus Flavivirus and is transmitted by culex mosquitos in an enzootic cycle. WNV contains ss-RNA that encodes the capsid(C), envelope (E), premembrane (prM) proteins as well as seven non-structural proteins. Several genetic lineages from WNV exist, lineage 1 and 2 are responsible for the major epidemics in humans. Replication takes place at the site of inoculation, thereafter the virus spreads to lymph nodes and bloodstream. CNS viral penetration appears to follow stimulation of toll-like receptors and increased levels of tumour necrosis factor- α (TNF- α), TNF- α increases permeability of the blood-brain-barrier. WNV directly infects neurons, particular in deep nuclei and grey matter of the brainstem and spinal cord.The first isolation of WNV was in 1937 in the West Nile district of Uganda. Historically there were sporadic outbreaks worldwide. Since the 1999 there were large number of outbreaks in North America, Southern and Eastern Europe, where smaller outbreaks occur annually.Most WNV infections are subclinical, apparently asymptomatic in about 80% .Incubation time is 2–14 days, often West-Nile fever is self-limited. Classical symptoms of West-Nile fever are acute onset of fever, headache, fatigue, malaise, muscle pain and weakness. Rare occasions are gastrointestinal symptoms and a transient macular rash on the trunk and extremities. Some patients develop hepatitis, pancreatitis, myocarditis, rhabdomolysis, orchitis and ocular manifestations. Approximately < 1% develop neurological complications such as meningitis or encephalitis. WNV infection of spinal motor neurons (anterior horn cells) causes acute, asymmetric flaccid paralysis (AFP) similar to poliomyelitis. AFP occurs in about 13% of patients with neurological impairments . The incOther arthropod-born- virus induced diseases with the same geographic distribution of mosquitos like chikungunya fever, dengue fever.CSF examination is crucial. Patients with neuroinvasive diseases generally have lymphocytic pleocytosis in CSF, neutrophils could be predominate in the early stage of disease. Laboratory diagnostics from CSF and blood samples include RT-PCR and IgM/ IgG analysis.MRI is frequently normal but signal abnormalities in the basal ganglia, thalamus and brain stem can be seen in encephalitis patients, signal abnormalities in the anterior spinal cord in patients with poliomyelitis like syndrome.There is no specific treatment for WNV infection. Specialized neurological intensive care medicine is crucial for survival. Patients with severe meningitis often require pain control, antiemetic therapy and rehydration for associated nausea and vomiting. With severe encephalitis syndromes patients should be observed for development of elevated intracranial pressure and seizures. With paralysis or encephalitis patients should be monitored for inability to protect airway. Acute neuromuscular respiratory failure may develop rapidly, particularly ventilatory support may be required.Individual patients have been reported where Ribavarin, interferon-α, WNV-specific immunoglobulin and antisense gene-targeted compounds have been considered but no clinical trails have been completed.No human vaccination is available. Four veterinary vaccinations are used.Aedes mosquitos transmit ZIKV, mainly Aedes aegypti, it can also be transmitted from mother to foetus, through sexual contact, transfusion of blood and blood products and organ transplantation [Zika virus belongs to the flaviviruses, it’s a positive single-stranded-enveloped RNA virus. The genome of ZIKV includes two noncoding regions and one region encoding a polyprotein that is cleaved into ten proteins including capsid, membrane precursor and envelope and seven non-structural proteins. ZIKV gains entry into host cells through endocytosis initiated upon interaction of virus envelope glycoprotein with cell surface receptors. antation , 11, 16.In 1947 ZIKV was discovered in the Zika forest in Uganda, Africa. The first larger outbreak was reported in 2007 on Yap island, Micronesia after which the virus quickly spread to countries in Southeast Asia , between 2015 and 2017 there was a dramatic increase of ZIKV cases in South America, that the World Health Organization announced a health emergency between 02/2016–11/2016.Estimated incubation time is three to 14 days. Generally the symptoms are mild with fever, rash, conjunctivitis, muscle and joint pain, malaise or headache. Most infected people do not develop symptoms. Neurological complications such as Guillian-Barré-like syndrome, neuropathy and myelitis can occur in adults as well as in children.ZIKV can cause congenital syndromes in pregnancy. Meanwhile a causal context between ZIKV infection in pregnant women and subsequent malformations, such as microcephaly and severe brain damage has been confirmed. The virus passes through the placental barrier to infect foetal neural cells, to replicate and to induce severe neurological complications .Most flaviviruses with the same geographic region like DENV and CHIKV.Several diagnostic tools are available including Real-Time RT-PCR (rRT-PCR) assay and MAC-ELISA. RNA NAT should be performed with serum and urine specimens.During the first week of illness ZIKV-specific IgM and neutralizing antibodies develop, IgM levels are variable, but generally positive near the 4th day of onset of symptoms.IgM Antibody Capture Enzyme-linked immunosorbent Assay (MAC-ELISA) is used for qualitative detection of IgM antibodies in serum or CSF.As there is no specific antiviral therapy available, treatment is symptomatic.No vaccine is available, protection against mosquito bites is the key prevention.Togaviridae, genus Alphavirus), which causes endemic infections. CHIKV is transmitted by Ae. aegypti and Ae. albopictus, despite this CHIKV can be transmitted from the viremic mother to child during birth. Worldwide three phylogenetic CHIKV lineages with distinct antigenic characteristics are known, two major lineages circulate in Africa, the third one is present in Asia. Transmitting a specific variant of the African lineage Ae. albopictus is considered to have a high competence. The virus has a single amino acid change from alanine to valine, which leads to a better adaption of the virus in Ae. albopictus [.Chikungunya Virus also belongs to arthropod-borne viruses .CHIKV circulated in France in 2010, 2014 and 2017, in Italy with two major outbreaks in 2007 and 2017. Isolated CHIKV strains in Europa belong to the African lineage. By now CHIKV manifests high fever, headache, maculopapular rash and painful arthralgia with a typical incubation time of 3–7 days. Other symptoms could be retro-orbital pain. Myocarditis and haemorrhage, mostly the patients recover within 1–2 weeks, but joint pain may persist for several months to years.During the outbreak on Réunion Island, 2005–2006, 12% of patients had neurological signs including quantitative and qualitative alteration of consciousness, focal neurological signs such as cranial nerve deficits, seizures, hemi/paraparesis and involuntary movements .Diagnosis is established by detecting CHIKV RNA with RT-PCR or virus serology during the first 5 days following onset of systems with high sensivity and specifity. A negative PCR initiate a prompt serological testing with enzyme-linked immunosorbent assay (ELISA) or indirect fluorescent antibody (IFA). CHIKV can be isolated from CSF, plasma or detected by nucleic acid amplification during the initial fever. IgM can persist for months, therefore detection of IgM antibodies can be useful. For differential diagnosis one should keep other arthropod-borne-diseases with the same geographic distribution in mind.There is no antiviral treatment for CHIKV, treatment is symptomatic, including specialized neurological intensive care medicine.Usually, the prognosis for Chikungunya infection is good. Mortality is higher in elderly patients and immunosuppressed patients.There is no vaccination available.Neurologist have to be aware of the possible occurrence of the emerging and re-emerging viruses such as Dengue-virus, Nipah virus, Japanese-Encephalitis-B-virus, Borna Disease virus, West-Nile-virus, Zika virus and Chikungunya virus.In some of those vaccination is available, in all severe cases of central nervous system involvement specialized neurological intensive care medicine is crucial for survival and prognosis.


Culicoides biting midges in Japan. Several novel arboviruses that may infect ruminants  were recently reported in mainland China based on extensive surveillance. It is noteworthy that some are suspected of being associated with cattle diseases. Malformed calves exposed to an intrauterine infection with orthobunyaviruses  have been observed. Epizootic hemorrhagic disease virus serotype 6 caused a sudden outbreak of hemorrhagic disease in cattle in Japan. Unfortunately, the pathogenicity of many other viruses in ruminants has been uncertain, although these viruses potentially affect livestock production. As global transportation grows, the risk of an accidental incursion of arboviruses is likely to increase in previously non-endemic areas. Global warming will also certainly affect the distribution and active period of vectors, and thus the range of virus spreads will expand to higher-latitude regions. To prevent anticipated damages to the livestock industry, the monitoring system for arboviral circulation and incursion should be strengthened; moreover, the sharing of information and preventive strategies will be essential in East Asia.Epizootic congenital abnormalities caused by Akabane, Aino, and Chuzan viruses have damaged the reproduction of domestic ruminants in East Asia for many years. In the past, large outbreaks of febrile illness related to bovine ephemeral fever and Ibaraki viruses severely affected the cattle industry in that region. In recent years, vaccines against these viruses have reduced the occurrence of diseases, although the viruses are still circulating and have occasionally caused sporadic and small-scaled epidemics. Over a long-term monitoring period, many arboviruses other than the above-mentioned viruses have been isolated from cattle and  Rhabdoviridae, Nairoviridae, Peribunyaviridae, Phenuiviridae, Orthomyxoviridae, Flaviviridae, Togaviridae, Reoviridae, and Asfarviridae (Arthropod-borne (arbo-) viruses are transmitted by hematophagous arthropods. The arboviruses include various taxonomic groups, such as rviridae , 2. TheyOrbivirus, the family Reoviridae) is probably the most well-known arbovirus affecting livestock ruminant productions in the world (Orbivirus), also has been regarded as a threat to European counties in recent years, because its outbreaks has been reported in the countries bordering the region  are still problematic in East Asia, despite the widespread use of vaccines against them. Outbreaks of other endemic arbovirus infections such as Aino virus infection and Ibaraki disease have occasionally been reported. In addition, recent progress in the research tools for virus hunting have enhanced the detection of the circulation of arboviruses that were previously unrecognized in East Asia . NotablyBovine fever ephemerovirus, genus Ephemerovirus, family Rhabdoviridae). The virus was widely distributed through Africa, the Middle East, Asia, and Australia. Phylogenetic analysis based on the surface glycoprotein (G) gene demonstrated that BEFV isolates are grouped into four lineages with different geographical origins: Africa, the Middle East, Asia, and Australia  is generally used to detect rising titers of neutralization antibody in paired acute and convalescent sera from affected cattle which show the typical clinical signs of BEF . For serVaccination is now the current gold standard to prevent BEF. Commercial attenuated and killed vaccines have been developed and widely used in Japan, Korea, China and Taiwan , 37–39. During a large epidemic of an acute febrile disease in 1959 and 1960 in Japan, deglutitive difficulty based in the esophageal and laryngopharyngeal musculature was observed among affected cattle . A totalAn occurrence of Ibaraki disease was recognized in Korea in 1982, when the epidemic was observed in southern Japan . In TaiwAt laboratory diagnosis, detection of neutralization antibody to IBAV and virus isolation from diseased cattle are usually tested , 43–46. Akabane orthobunyavirus, genus Orthobunyavirus, family Peribunyaviridae) was a causative agent of the disease , and a goat in the southern part of mainland China  was first isolated from mosquitoes in Japan in 1964   syndrome occurred in southern Japan . Unlike s (CHUV) . The etis (CHUV) . In addis (CHUV) . It was am virus . Recent am virus , 121. AfThe isolation of the virus and its seroprevalence (91%) among dairy cattle in Taiwan in 1993 were reported . In KorePresence of antibody to CHUV in precolostral serum from abnormal calves indicates its association with lesions in central nervous system . For serOrbivirus and consists of at least 27 serotypes  test were obtained from cattle in southern Japan  which were new in East Asia was identified. In 1999, Peaton virus , which was initially isolated in Australia in 1976  was isolated from mosquitoes in India in 1957, and its prevalence in Nigeria in the 1960s was identified  was initially isolated in Nigeria in 1960s , which is a member of the species  in 1968 . The ana in 1968 . DAGV sp in 1968 , 180.Palyam virus, was isolated from cattle and Culicoides biting midges in Japan in 2008 and 2009 (Bunyip Creek virus (BCV), which is another serotype of species and 2009 , 180. Thand 2009 .Yunnan orbivirus, in 2005 (Yunnan orbivirus (YOUV-2), which is also called Middle Point virus, was obtained from healthy cattle in Australia in 1998 (Yunnan orbivirus (YUOV) was first isolated from mosquitoes collected in the southern part of mainland China in 1997, and it was identified as a member of a new orbivirus species,  in 2005 . A secon in 1998 , 184. In in 1998 . Interes in 1998 . The infSeveral zoonotic arboviruses of importance to human health have been detected from domestic ruminants in East Asia . Some ofJapanese encephalitis virus, genus Flavivirus, family Flaviviridae) caused severe encephalitis not only in humans but also in horses and cattle  infection have occurred across the Mediterranean region, Eastern Europe, Africa, Asia, and North America , which belongs to the species phalitis . Since tnd China , 201. Thnd China . At presBatai orthobunyavirus of the genus Orthobunyavirus, has been isolated in Japan and mainland China since 1994 (Batai virus (BATV), which belongs to the species nce 1994 –205. BATnce 1994 , 207. BAnce 1994 , 209. InCrimean-Congo hemorrhagic fever orthonairovirus, genus Orthonairovirus, family Nairoviridae) was involved in human clinical cases in the northwestern part of mainland China  is widely distributed in mainland China, Japan, and Korea  was recognized in mainland China, Japan, and Korea  is an etiological agent of fever, rash, and leg edema in horses  is assigned to the species  in 1982 . The vir in 1982 , 237. FU in 1982 . Althoug in 1982 . Nishimu in 1982  is also Orbivirus and was initially isolated in 1963 from starlings (Brahminy myna) collected in southern India (Sathuvachari virus (SVIV) is a member of the genus rn India . The virrn India . The genrn India . An unasrn India . No illnAn ephemerovirus tentatively called KSB-1/P/03 was isolated from sentinel cattle in southern Japan in 2003 . The phyOrbivirus (Tibet Orbivirus (TIBOV), the virus has been obtained from mosquitoes and Culicoides biting midges in other regions of mainland China  . TogetheCulicoides biting midges  and orthobunyaviruses  that are pathogenic to domestic ruminants were reported to be transmitted by g midges , 251. Looxystoma   in mainland China and Japan  technologies will support the detection and characterization of such viruses . The finCulicoides biting midges, and ticks should be conducted. The knowledge obtained from vector studies will contribute to increased accuracy of epidemiological analyses based on landscape and meteorological data. Risk assessments based on such analyses will enable the development of better surveillance and preventive measures for arbovirus infections which threaten the production of ruminant livestock, as suggested in earlier investigations (Clearly, knowledge gaps remain regarding the transmission cycles of arboviruses and vector ecology in East Asia. To the best of our knowledge, there is no detailed distribution map of vectors in the East Asia region. The vector capacity for arboviruses of veterinary importance has been evaluated for only a limited number of species. Further studies of the systematics, biology, and vector competence of mosquitoes, igations , 295.Finally, the construction of a network in East Asia and its neighboring countries would be essential to promote research on arbovirus infections of importance to livestock. The sharing of epidemiological information, diagnostic tools, and control/preventive strategies are highly important because neighboring countries are likely to experience the same problems due to arboviruses. Language barriers may present a challenge to information exchanging and sharing. Many scientific reports and public records from East Asian countries have been written in national languages such as Chinese, Japanese, and Korean, but a limited portion of them might have been translated in English. Unfortunately, the status of arbovirus infections in domestic ruminants in Mongolia and North Korea remains unclear. Regional cooperation would therefore be a crucial key to clarify the current status of arbovirus infections and minimize their impacts on the livestock industries in East Asia.TY executed a first draft. YH and KM helped draft the manuscript. All authors contributed equally to the final draft and editing of the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Lutzomyia longipalpis is the main vector of Leishmania chagasi in the New World, but its role as a host of viruses is poorly understood. In this work, Lu. longipalpis RNA libraries were subjected to progressive assembly using viral profile HMMs as seeds. A sequence phylogenetically related to fungal viruses of the genus Mitovirus was identified and this novel virus was named Lul-MV-1. The 2697-base genome presents a single gene coding for an RNA-directed RNA polymerase with an organellar genetic code. To determine the possible host of Lul-MV-1, we analyzed the molecular characteristics of the viral genome. Dinucleotide composition and codon usage showed profiles similar to mitochondrial DNA of invertebrate hosts. Also, the virus-derived small RNA profile was consistent with the activation of the siRNA pathway, with size distribution and 5′ base enrichment analogous to those observed in viruses of sand flies, reinforcing Lu. longipalpis as a putative host. Finally, RT-PCR of different insect pools and sequences of public Lu. longipalpis RNA libraries confirmed the high prevalence of Lul-MV-1. This is the first report of a mitovirus infecting an insect host.Hematophagous insects act as the major reservoirs of infectious agents due to their intimate contact with a large variety of vertebrate hosts.  Viruses are the most abundant biological entities in the biosphere, being found in every environment and infecting a wide range of organisms, such as plants, insects, mammals, and microorganisms ,2,3. SurThe relationship between viruses and their hosts is considered a coevolutionary process since viruses are obligate intracellular parasites and require the host’s cellular machinery for protein synthesis. Also, viruses are subject to the same evolutionary pressures that shape the host genome composition and codon usage ,19. In fHosts and their viruses are under constant adaptation and selective pressure, which is led by hosts developing new defense strategies and viruses developing new infection and counter-defense strategies . One of Leishmania spp. with few others involved in the natural transmission of viruses, such as Phlebovirus (Reoviridae family) ), a Perl program to screen the profile HMMs against 6-frame translated versions of the long RNA datasets. HMM-Prospector uses hmmsearch program from HMMER package v. 3.1 [−5) were used as seeds for GenSeed-HMM [−5 for nucleotide comparison or 1 × 10−3 for protein comparison were considered significant. Viral genomic segments were classified as described [Long RNA reads of ibraries  were usee v. 3.1  to run sSeed-HMM , a tool Seed-HMM  against Seed-HMM  and InteSeed-HMM  were useescribed .A dataset composed of public protein sequences  related K-means as the linkage criterion in R using corrplot package [ecdf function built-in R software where the Kolmogorov–Smirnov test was used to determine statistical significance.The pre-processing of sRNA libraries was performed as described . Briefly package . EmpiricDinucleotide frequencies and codon usage were calculated using programs from the EMBOSS package (version 6.6.0) . First, Lu. longipalpis, we used eight pools containing 10 sandflies per pool, collected from a colony originally started from individuals collected in Teresina, Brazil, and maintained at the Laboratory of Physiology of Hematophagous Insects . Total RNA was extracted from pools of 5 insects each using Trizol reagent according to the manufacturer’s protocol . Total RNA (1 μg) was reverse transcribed using 250 ng of random primers specific primers per reaction. The resulting cDNA was used as template for PCR reaction containing primers designed to amplify a product of 509 bp. Primer sequences are listed in Taq DNA polymerase . PCR products were cleaned up with EDTA 125 mM precipitation protocol and sequenced using Sanger technology.To confirm the presence of the virus found in DNA and long and small RNA libraries were obtained from the NCBI SRA repository, listed with accession numbers in Lu. longipalpis, we tested a set of 506 profile HMMs selected from the vFam database against two sequencing datasets of lRNA data, totaling 48 million reads. This strategy allowed us to select 18 different profile HMMs  genome. BLASTN searches against public databases showed no significant similarity. Using the universal genetic code, only short open reading frames (ORFs) were observed. BLASTX searches against the nr database revealed some similarity to RdRp of narna-like viruses such as the Wenling narna-like virus 9 (accession code YP_009337200) , which uMitoviridae and Narnaviridae [Ourmiavirus (Botourmiaviridae family) and some unclassified viruses. Finally, two enterobacteria phages of the Leviviridae family were used as an outgroup, since these viruses are close relatives to Mitovirus and Narnavirus [Leviviridae-ancestors that infected bacterial endosymbionts, some of which may have generated mitochondria [Plasmopara viticola lesion-associated mitovirus 56 (QIR30279), a fungal mitovirus found in grapevine [Wenling narna-like virus 9 (YP_009337200), identified in crustaceans [Mitovirus and Narnavirus was clearly supported by our analysis. Some viruses such as the Grapevine associated narnavirus 1 (accession code CEZ26304), Wenling narna-like virus 9 (YP_009337200), and the Shahe narna-like virus 6 (APG77166), originally classified as narnaviruses, are members of the genus Mitovirus according to our analysis , Ourmia melon virus (YP_002019757) and Epirus cherry virus (YP_002019754), some recently described narna-like viruses are in fact ourmia-like viruses. Similarly, the Aspergillus fumigatus mitovirus 1 (AXE72932), originally classified as a mitovirus [Ourmiavirus/ourmia-like clade. This group presents a high divergence among their members and it is possible that larger taxa samplings may indicate in the future that it is polyphyletic indeed.We performed a phylogenetic analysis with representatives of the current families aviridae , who weraviridae , includirnavirus . In addichondria ,45,75. Arapevine , and to staceans . The naranalysis . Finallyrus 6 APG166, origMitovirus and Narnavirus genera according to the size of the viral genome and protein sequences (Mitovirus presents an average size of 2000–2500 nt and an ORF (Open Reading Frame) coding for an RdRp with a maximum size of 900 aa residues. Conversely, the Narnavirus genus shows an average genome size of 2500–3000 nt and an RdRp ORF with a maximum length of up to 1200 aa residues. Some narnaviruses also present ambigrammatic sequences, characterized by an additional large ORF coded in the reverse strand of the genome [In addition to the phylogenetic reconstruction, we performed a comparative analysis of the equences . The code genome ,79.To characterize and propose a probable host of Lul-MV-1, we analyzed some intrinsic features of the viral genome. First, we compared the dinucleotide frequencies of Lul-MV-1 with those of mitochondrial genomes of fungi, mosquitoes (Culicidae) and phlebotomines (Phlebotominae). Lul-MV-1 presents a dinucleotide usage profile similar to the profiles observed in insects, but not to those of fungi . In factLu. longipalpis. This result suggests that Lul-MV-1 is highly adapted to the mitochondrial codon usage of its putative host. The Shahe narna-like virus 6 [Plasmopara viticola. Nevertheless, the Plasmopara viticola associated mitovirus 56 presented a profile distantly related to its host, but closely related to other fungal mitoviruses. Finally, the Wenling narna-like virus 7, another virus detected in a metagenomic sample derived from crustaceans [Since di- and trinucleotides patterns can shape codon usage frequency in viruses , we also virus 6 , charactstaceans , showed To better understand these results, we decided to restrict and deepen the analysis to UGA/UGG codons. Mitochondrial genomes often use a genetic code with UGA coding for tryptophan (Trp) rather than acting as stop codon. Thus, we chose a set of viral and mitochondrial genomes and determined the absolute counts and relative usage frequencies of these codons . In all Cryphonectria mitovirus 1 and Cryphonectria parasitica mitochondrion used UGA in 52.9 and 82.1% of the tryptophan codons. In another case, Sclerotinia sclerotiorum mitovirus 1 and Sclerotinia sclerotiorum mitovirus 1-A2 used UGA(Trp) in 76.9 and 66.7%, respectively, with the host’s mitochondrion showing a usage of 81.2%. In Ophiostoma novo-ulmi, another ascomycete fungus, the mitochondrion and Ophiostoma mitovirus 4 used UGA(Trp) in 94.1 and 84.6%, respectively. A remarkable distinct result was obtained for Plasmopara viticola, an oomycete whose mitochondrion exclusively used UGG to encode tryptophan, while the Plasmopara viticola associated mitovirus 56 showed a 50% use of UGA and UGG codons. In the case of Lul-MV-1, the mitovirus presented 73.3% of UGA(Trp), with the Lutzomiya longipalpis mitochondrion using 99%. Two other mitoviruses, found in crustacean metagenomic samples, the UGA(Trp) utilization showed an extreme variation, with the Wenling narna-like virus 9 using 99% of UGA(Trp) and the Shahe narna-like virus exclusively using UGG. When analyzing the AT content, unlike narnaviruses, mitoviruses showed a relatively high correlation with their hosts’ mitochondria.Mitoviruses, which are known to locate exclusively in the mitochondria of their hosts, showed a variable ratio of UGA/UGG codons , with a Aspergillus fumigatus’ mitochondrion used UGA(Trp) in 93.2% of the tryptophan codons, the Aspergillus fumigatus mitovirus 1  at all. While Vesicular stomatitis virus (VSV), another virus that naturally infects Lu. longipalpis [Previous works from our group have shown that the virus-derived small RNAs (vsRNAs) present some features that are host-virus specific and can be used to classify viral sequences . Therefogipalpis . We obseLutzomyia and Drosophila [The sequencing depth and coverage of sRNAs derived from the putative virus were also analyzed . We obseosophila ,34,66.Lu. longipalpis, we performed amplification by RT-PCR using pool samples derived from the same laboratory colonies used to obtain the small RNA libraries. We observed amplification of the Lul-MV-1 in seven out of the eight tested pools of Lu. longipalpis, indicating that this virus is present in high prevalence in Lu. longipalpis individuals  are ubiquitous crepuscular-nocturn insects, found in all continents in both rural and urban areas. In the New World, families ,82 can rfamilies ,83. SomeLu. longipalpis RNA samples. We used profile HMMs together with GenSeed-HMM [Mitovirus. Interestingly, our phylogenetic analysis (Wenling narna-like virus 9 (YP_009337200) and the Shahe narna-like virus 6 (APG77166), both found in crustacean samples [In this work, we described the genome of Lul-MV-1, a novel virus found in Seed-HMM  to selecanalysis  also sho samples . UnfortuNarnaviridae family  many organism hosts, such as invertebrates, fungi, plants and mammals [In recent years, the increase of environmental metagenomic studies has provided the description and identification of virus sequences of the former  mammals ,84,85,86 mammals ,79,84,87Narnavirus were originally described infecting the cytoplasm of the yeast Saccharomyces cerevisiae [Aedes japonicus narnavirus 1 presents a genome of 2069 bases containing two ORFs, one in the positive-sense strand coding for RdRp, and an additional negative-frame ORF. Ambisense coding strategy has been studied in narnaviruses of insects, suggesting that both ORFs could enable replication in the hosts. Reverse-frame ORFs are characterized by the avoidance of CUA, UUA, and UCA codons, which are the reverse complements of stop codons [Mitovirus genus. Sequences related to narnaviruses were also found in two samples of the mosquito Culex pipiens and phylogenetic analyses revealed that these sequences are closer to other narnaviruses associated to mosquitoes than to fungal narnaviruses [Narnaviruses have been reported to infect fungi, plants and dipteran insects ,89. The revisiae . In addip codons , a findip codons . Neverthp codons . In the aviruses .Lu. longipalpis or, alternatively, that the source was a fungal contaminant. We used different analyses to confirm that Lul-MV-1 infects the insect’s mitochondria, rather than the mitochondria of a fungal host. Dinucleotide composition represents one of the host adaptation mechanisms that influence virus codon usage [Lu. longipalpis. This result shows a remarkable virus–host adaptation and points to this dipteran as the putative host of Lul-MV-1, especially considering the high prevalence and abundance of its viral sequence in public datasets derived from different sources of Lu. longipalpis.Mitoviruses have been identified only in fungal and plant mitochondria. Mitovirus-like sequences closely related to fungal viruses, derived from a specific branch, were detected as endogenous elements integrated in plant mitochondrial genomes, and pathogenic fungi were raised as potential source of horizontal transfer . A surveon usage . As parton usage . In facton usage ,90. AccoAspergillus spp. [A biological correlation of codon usage fitting by mycoviruses and fungal host virulence was reported in lus spp. . Mycovirlus spp. , where vPlasmopara viticola associated mitovirus 56, which uses UGA/UGG in a fifty-fifty basis, while the host’s mitochondria does not use UGA(Trp) at all and, in addition, shows a distinct AT content when compared to the host mitochondrial genome . A tempting hypothesis is that this virus has been originated from a fungal host that does use UGA(Trp) in relatively high levels, then switched to Plasmopara viticola and is still fitting its genome composition and codon usage to the new host. However, such an adaptation process would require protein synthesis to occur under a rare use of UGA(Trp) codons in the host, an aspect that should be better elucidated in the future.Many fungal mitoviruses show a large utilization of UGA(Trp) codons, resembling the codon usage of the respective mitochondria ,90. A coPlasmopara viticola associated mitovirus 56, Lul-MV-1, Wenling narna-like virus 9 and Shahe narna-like virus are relatively close to each other but show very discrepant usage rates for UGA/UGG codons.Mitochondrial genomes are often biased toward a preferential use of UGA rather than UGG to encode tryptophan. This may be explained by the fact that organellar genomes, including dipteran mitochondria, usually present high AT content , which cThe fact that a viral genome presents a codon usage that resembles that of the mitochondria is a strong indication that the virus has evolved as to fit the organelle codon usage and, therefore, to use its protein synthesis machinery with high efficiency. On the other hand, dinucleotide frequency and overall AT content seem to be shaped by selective pressures that occur in the site of replication. Thus, viruses that are located and replicate within mitochondria would be more subject to compositional biases imposed in the organelle. Conversely, viruses able to colonize and replicate in the cytosol, would be less affected by selective compositional pressures, which could explain why narnaviruses and ourmiaviruses differ so much from their hosts’ mitochondria not only in terms of codon usage, but also in AT content. To conclude, although an AT content bias can certainly influence the codon usage, both parameters are not totally interdependent and are probably shaped by distinct evolutionary pressures.Vesicular stomatitis virus that infects Lu. longipalpis  have been used in many studies as an evidence of viral infection in an organism, since they are produced through recognition of dsRNA molecules produced during the viral replication cycle ,27,34. Agipalpis C. NevertLu. longipalpis populations. Primers designed to a segment of the RdRp gene were able to positively detect seven out of eight sand fly pools, indicating that this virus is persistent in the population  the viral genome presents a high AT content (69.74%), similarly to what is observed in organellar genomes, including the mtDNA of Lu. longipalpis (78.07%); (3) codon usage is closer to mitochondria of invertebrate hosts than to fungal hosts; (4) the virus uses mainly the codon UGA to code tryptophan, in consonance with the codon usage of of Lu. longipalpis mitochondria; (5) dinucleotide composition of Lul-MV-1 genome resembles the composition of insect genomes rather than fungal genomes; (6) virus-derived sRNAs suggests activation of siRNA pathway in insects rather than fungal hosts; (7) RT-PCR followed by Sanger sequencing, confirmed the presence of the viral genome and the prevalence of Lul-MV-1 in a sand fly laboratory colony; and lastly (8) reads from different RNA and DNA public libraries of Lu. longipalpis were successfully mapped on the viral genome, confirming the high prevalence of the virus in Lu. longipalpis populations.It still a matter of speculation whether mitoviruses found in arthropods are infecting the mitochondria of a fungus or protist belonging to the arthropod microbiota or the mitochondria of the arthropod itself. We believe that Lul-MV-1 is a mitovirus that infects phlebotomine mitochondria based on a body of evidence: (1) sequence similarity and close phylogenetic relationship to mitochondrial viruses of the genus Lu. longipalpis. In addition to compositional and phylogenetic analyses, the utilization of vsRNA profiles represent a valuable approach to properly ascribe the respective hosts of viruses detected in metagenomic datasets. According to our knowledge, this is the first report of a mitovirus infecting an insect host, and the results presented herein highlight the large diversity of the virosphere and the possibility that mitoviruses may infect a much wider range of hosts than initially supposed.Altogether, the experimental and bioinformatic methods applied in this study allowed us to detect, classify, and characterize a novel mitovirus infecting the mitochondria of the sand fly


Mops condylurus bats were recently found »750 km away in western Kenya. This finding supports the role of M. condylurus bats as hosts and the potential for Bombali virus circulation across the bats’ range in sub-Saharan Africa.Previously identified only in Sierra Leone, Guinea, and southeastern Kenya, Bombali virus–infected  Ebolavirus  is the sixth and most recently identified virus of the genus M. condylurus and 18 C. pumilus (M. condylurus and 219 C. pumilus (https://www.thermofisher.com) as described previously  for virus inactivation, and RNA was extracted according to the manufacturer’s instructions. Because previous studies have identified the highest BOMV viral loads in bat lungs (https://www.neb.com) (https://www.beckmancoulter.com), and sequencing libraries were prepared by using a Nextera XT kit (Illumina) according to the manufacturer’s instructions. Sequencing was conducted with the Illumina MiSeq Reagent Kit v2, a sequencing kit with 150-bp paired-end reads. The raw sequence reads were trimmed by using Trimmomatic  and assembled to the reference sequence (MK340750) by using the Burrows-Wheeler Aligner–Maximal Exact Match algorithm implemented in SAMTools version 1 ([https://arxiv.org/abs/1303.3997?upload=1).We conducted next-generation sequencing on positive lung samples. Before sequencing, we applied a multiplex PCR protocol for amplification. The primers for the entire BOMV coding region were designed by using the PrimalScheme tool . All other bats were negative. Two of the BOMV-positive bats were captured in Busia  in 2 distinct trapping locations (both house roosts) »7 km apart  and 1 partial genome . Phylogenetic analysis showed 99% nt identity between the complete sequences from Busia and Taita (200-nt difference) and 97% nt identity with the prototype strain from Sierra Leone . The virM. condylurus bats from separate roost sites in western Kenya, expanding the known virus geographic range in East Africa. This new location in Busia County, on the Kenya–Uganda border, is »750 km from the previously reported site. We also identified a second BOMV-positive M. condylurus bat at the original site near the Taita Hills in southeastern Kenya, indicating sustained local persistence but limited prevalence.We identified 2 BOMV-positive C. pumilus, indicates a dominant host role for M. condylurus compared with other species. Although no evidence of human infection with BOMV has yet been reported , however, is below that for reservoir hosts in other bat pathogen systems (e.g., Marburg virus [Our results expand the known distribution of BOMV and increase support for a role of


Flaviviruses are significant human pathogens that cause frequent emerging and reemerging epidemics around the world. Better molecular tools for studying, diagnosing, and treating these diseases are needed. Reporter viruses represent potent tools to fill this gap but have been hindered by genetic instability. Recent advances have overcome these hurdles, opening the way for increased use of stable reporter flaviviruses to diagnose infections, screen and study antiviral compounds, and serve as potential vaccine vectors. The past two decades have been a lesson in the potential of unknown or unrecognized RNA viruses to emerge from animal reservoirs in new locations to cause local epidemics or widespread pandemics. In 1999, the United States saw the introduction of West Nile virus, which has caused tens of thousands of cases of neurological disease . The yeaTwo of the above examples from the past twenty years, West Nile (WNV) and Zika (ZIKV) viruses, are part of the insect-vectored pathogens from the Flavivirus family. This family also includes the mosquito-transmitted dengue virus (DENV), Japanese encephalitis virus (JEV), and yellow fever virus (YFV), as well as the tick-transmitted tick-borne encephalitis virus (TBEV), Langat virus (LGTV), and Powassan virus (POWV). Collectively, these viruses cause greater than 390 million infections yearly, with the majority of those being from the four serotypes of dengue virus . Along wThe Flavivirus family consists of positive sense, single-stranded RNA viruses. Their ~11 kb genome is translated as one large polyprotein that is co- and post-translationally cleaved into three structural proteins  and seven non-structural proteins  by cellular and viral proteases A.The genome’s single open reading frame is flanked, on both the 5′ and 3′ ends, by an untranslated region (UTR) that, among other functions, contain signals, such as a cyclization sequence, that are critical for RNA replication  were first engineered with a chloramphenicol acetyltransferase (CAT) gene , followeEscherichia. coli is to use low copy number plasmid vectors.Full-length infectious viruses can be more difficult to clone due to instability in their bacterial plasmids, but they do not have the shortcomings of replicons, which do not cover virus entry/fusion and virion assembly/release. The bacterial instability challenge was first overcome by in vitro ligation of cDNA fragments that cover the complete YFV genomic RNA sequence . SimilarThe first report of a full-length reporter flavivirus came in 2003 using JEV , which wSuccessively, two reports of infectious reporter WNV were published in 2005, one with green fluorescent protein (GFP) , and oneAnother less-used strategy for constructing reporter flaviviruses was first published in 2007 , which hThe most robust scheme for reporter flavivirus construction to date was published in 2007, using YFV . In thisThe capsid duplication strategy has been successfully applied to many other flaviviruses, with slight variations giving different results in stability and utility, though addition of a reporter gene causes general attenuation in all cases ,35,36,39As seen throughout the decade following the first reporter flavivirus, better design methods have resulted in more stable and more robust reporter viruses. Despite these improvements, interest still remains in engineering increased stability. Viruses carrying reporter genes with long-standing stability can be used in long-term pathology experiments, such as experiments involving transmission among multiple hosts, in diagnostic applications where reliability of the reporter signal is crucial, and in industrial applications where large viral batches are needed from small seed stocks. Therefore, recent work has shown multiple strategies that can be applied to ensure engineered reporter genes are maintained.349, with no duplication of NS1 , JEV (C34), and WNV (C33)) were stable to ten passages, while those with C25 (ZIKV and YFV), were only stable to five passages. We hypothesized that an extended capsid length, in itself, would be enough to stabilize reporter flaviviruses when codon homology was scrambled. This could be due to RNA signals present beyond C25 that may not be mandatory, but still assist in genome replication. Indeed, ZIKV and YFV with 38 amino acid capsid duplications D were maReporter genes, including fluorescent and bioluminescent genes, allow for easier quantification of virus levels. This simple concept has seen many applications that advance virus and disease understanding, as well as countermeasure development. One of the earliest reports of reporter flaviviruses was in conjunction with antiviral compound testing. Traditionally, these tests are done by adding virus and putative antiviral compounds to relevant cells. Following multiple rounds of viral replication, supernatant samples are assayed by plaque- or focus-forming assay, which take up to five days or more to obtain results and are not amenable to high-throughput. Using reporter viruses, results can be obtained using reporter output in place of an infectious virus, increasing throughput, turnaround, and ease of quantification ,29,40,57Similar to antiviral testing, conventional testing for neutralizing antibodies by plaque reduction neutralization tests (PRNT) is a long and labor-intensive process. Several methods for using reporter gene output, instead of viral titers, have been described to shorten this assay from a week to as little as 4 h and increase throughput by using either a flow cytometer or a plate reader ,58,59,60Another powerful application of reporter flaviviruses comes in the use of luciferase-carrying viruses to be quantified in living animals. Carefully engineered cameras can detect luciferase signals in infected mice, allowing for temporal quantification and tracking of infection in the same mice. This real-time view of infection, known as bioluminescent imaging (BLI), also reduces the number of mice needed and can represent viable models for both antiviral and vaccine studies. Such studies have already been done with DENV2  and JEV The possibility of approved flavivirus vaccine strains, specifically the 17D strain of YFV, being used to deliver heterologous antigens as a vaccine has long been considered and studied. YF17D was initially restricted to small T and B cell epitopes engineered either in a space in the envelope protein ,65 or beSince first creation, reporter flaviviruses have found uses in molecular virology ,23, anti


The throughput of HSA was also dependent on applied transmembrane pressure as was seen by Vmax values of 110 ± 2.5 L m−2 and 63.6 ± 5.8 L m−2 at 3 bar and 5 bar, respectively. Protein loss was low, i.e., recovery > 90%. A distribution of pore sizes between 40 nm and 60 nm, which was present in the prefilter and absent in the virus filter, played a crucial part in removing the HSA aggregates and minimizing the risk of virus filter fouling. The presented results enable the application of virus removal nanofiltration of HSA in bioprocessing as an alternative to virus inactivation methods based, e.g., on heat treatment.This study is dedicated to the rapid removal of protein aggregates and viruses from plasma-derived human serum albumin (HSA) product to reduce the risk of viral contamination and increase biosafety. A two-step filtration approach was implemented to first remove HSA aggregates and then achieve high model virus clearance using a nanocellulose-based filter paper of different thicknesses, i.e., 11 μm (prefilter) and 22 μm (virus filter) at pH 7.4 and room temperature. The pore size distribution of these filters was characterized by nitrogen gas sorption analysis. Dynamic light scattering (DLS) and size-exclusion high performance liquid chromatography (SE-HPLC) were performed to analyze the presence of HSA aggregates in process intermediates. The virus filter showed high clearance of a small-size model virus, i.e., log Human serum albumin (HSA) is one of the most important products derived from human plasma. It has a multifunctional role as an osmotic pressure regulator, transport shuttle and redox modulator . HSA is In recent years, there has been a sustained interest in developing recombinant HSA, which is commercially available in limited quantities for applications as (i) a cryoprotectant for other recombinant biologicals; (ii) a component in serum-free cell culture media for cell therapies; and (iii) a specialty component in diagnostic imaging agents . The mai2. [Because most available HSA is still derived from human plasma, this infers the risk of virus contamination. Donor screening is the first step to mitigate the risk of viral contamination. The next step of assuring HSA’s biosafety is pasteurization, which is typically performed at 60 °C for 10 h in the presence of stabilizers, such as N-acetyl tryptophan and/or caprylate (octanoate) . Another2. . However2. ,23. Furt2. . Moreove2. . Capryla2. . In all,Virus removal filtration is a well-established method of filtering biologicals thanks to its inertness and proven removal performance. However, virus removal filtration of HSA is rarely used due to cost-efficiency issues. Another technical challenge with HSA filtration is its tendency to form aggregates during pasteurization, which leads to rapid clogging of filters. It has been shown that when fatty acids are bound to HSA, its structure does not unfold upon heating even upon extended heating . Since s10 reduction value (LRV) ≥ 5.25 [In recent years a new type of virus removal filter paper has been developed at Uppsala University, which combines desirable pathogen removal properties with high cost-efficiency. Using naturally-sourced cellulose nanofibers, a nonwoven filter paper was produced via conventional hot-pressing of wet pulp . The res) ≥ 5.25 . The pot) ≥ 5.25 .The produced filter paper consists of numerous stacked nanosheets formed via self-assembly of cellulose nanofibers during drainage, wet-cake formation and then hot-press drying, giving rise to -called mille-feuille structure . By contPossible applications of mille-feuille filter paper in downstream and upstream bioprocessing purification have been studied. For upstream bioprocessing, filtration of virus spiked basal media, e.g., Dulbecco’s modified Eagle’s medium (DMEM), LRV ≥ 5 was shown for small-size ΦX174 phages . The filIn this article the virus removal properties for filtration of plasma-derived HSA are explored. We present a two-step sequential filtration of HSA solution through nanocellulose filters of different thicknesses, i.e., first 11 μm (prefilter) and then 22 μm , which enable size-exclusion based removal of undesirable protein aggregates and achieve high protein throughput as well as high virus clearance. The filters were made of identical material and showed only slightly different pore size distributions.−1; ≥96% albumin) was purchased from a local apothecary store and contained the following excipients: sodium chloride, N-acetyl-tryptophan and caprylic acid. Phosphate buffer saline (PBS) was purchased from Sigma-Aldrich . Total protein biuret reagent and sodium chloride were obtained from Sigma-Aldrich . Escherichia coli bacteriophage ΦX174 (ATCC® 13706) and Escherichia coli (Migula) Castellani and Chalmers (E. coli) (ATCC® 13706-B1) were obtained from American Type Culture Collection . Yeast extract, tryptone and agar were obtained from Becton, Dickinson and Company . Cellulose from Cladophora sp. algae, collected from Qingdao, China, was provided by Dr. Jun Liu, Jiangsu University.Human serum albumin (HSA)  was predispersed in deionized water using an IKA T25 high-shear mixer  prior to high pressure-homogenization. The final nanocellulose dispersion was prepared by passing the mixed cellulose suspension through a high-pressure LM20 microfluidizer . The dispersion was passed 3 times through a 200-µm grid chamber and 1 time through a 100-µm grid chamber under a pressure of 1800 bar.The nanocellulose-based filters were prepared as previously described . BrieflyPVDF; Merck Millipore, MA, USA) using a vacuum filtration setup  until a cellulose cake was formed. The wet cake was then dried in a Carver 4122CE press . For the preparation of 11 μm thick prefilters, the nanocellulose wet-cake was dried at 140 °C using a hot-press for 40 min. For the preparation of the 22 μm thick virus removal filter, the nanocellulose wet cake was dried at 80 °C using a hot-press for 24 h. The dry filters were removed, cut into 47 mm diameter disc, and stored at ambient conditions until further use.An Advantec KST-47  filter holder was used. A general-purpose filter paper disc 47 mm in diameter  was placed beneath the nanocellulose filter as a support. The rate of flow was monitored gravimetrically by collecting the outflowing liquid on an MS1602TS analytic balance  connected to LabX software .−1 HSA diluted in PBS (10 mM) adjusted to pH 7.4. The filters were wetted with PBS prior to prefiltration. Prefiltrations were carried out at 1 bar. Due to rapid clogging, for each prefiltration around 25 mL was passed through each filter, corresponding to a 14.4 L m−2 load volume. The permeate fractions were collected, mixed together and stored at 4 °C before use.Eleven µm filters were used for prefiltration. Feed solution was 10 mg mL−1 HSA in PBS adjusted to pH 7.4. Virus stability was controlled by a hold sample taken from the prefiltered spiked feed solution. The filters were wetted with PBS prior to filtration. Filtrations were carried out at 3 different overhead pressures, i.e., 1, 3, and 5 bar. For each pressure, around 50 mL of the entire loaded feed solution was passed through the filters, corresponding to a 28.8 L m−2 load volume. The permeate was collected in two equal fractions. The average flux during filtration was recorded as described above. Permeate samples and hold samples were collected and stored at 4 °C before plaque forming units (PFU) assay. The virus removal efficiency was expressed in log10 reduction values (LRVs) as described below.ΦX174 bacteriophage (28 nm) was used as a model small-size virus. Twenty-two µm filters were used for virus removal studies. A stock solution of ΦX174 was spiked at 0.1% into prefiltered 10 mg mLx-axis and the reciprocal of the relative flux (L m−2 h−1) as the y-axis for linear fitting. When the resulting slope is positive, the reciprocal of the slope is Vmax (L m−2) [To quantify the throughput of down-scale virus filtering, Vmax analysis was performed on the resulting flux curve. An intermediate fouling model was selected according to a procedure described in Badmington et al. . Vmax wa (L m−2) .To obtain a neat cross-section, the filter was first immersed in liquid nitrogen then broken with tweezers. After being sputtered with Au/Pd prior , the samples were observed by a high resolution FEG Zeiss 1550 SEM  system. The selected acceleration voltage was set between 1.5 kV and 3 kV and In Lens detector was used for imaging.TM Silica-Alumina . The deviation between the pore-size mode of the calibration data from the nominal standard values was 0 nm. Measurement on experimental samples was performed in triplicate, and the results were averaged.Prior to analysis, the samples were degassed for 8 h in a vacuum at 95 °C. Nitrogen gas sorption was performed using an ASAP 2020  instrument. Pore size distribution profiles were obtained using the Barret-Joyner-Halenda (BJH) method , based o−1 HSA in PBS solution (pH 7.4) with a Zetasizer Nano ZS DLS instrument . The feed solution, prefiltrate and final permeate were used for analysis. The test angle was 173° (backscatter). About 1.2 mL of the test sample was taken each time and equilibrated in a DLS instrument for 10 min at 25 °C before testing. Three parallel samples of each group were used for testing and the results were averaged.Dynamic light scattering (DLS) was used to assess the particle size distribution of 10 mg mL−1 and run time was 20 min. UV detection at λ = 280 nm was used.An HPLC-UV system was used for size-exclusion chromatography. A Hitachi 5160 pump  was used with a 5280 autosampler, and a bioZen 1.8 μm SEC-3 analytical column Mw 10–1500 kDa . The column temperature was 25 °C, and injection temperature was 20 °C. The mobile phase was 100 mM sodium phosphate pH 6.8. The flow rate was 0.3 mL minpermeate and Absfeed are the absorbance values at 540 nm for permeate and feed, respectively. It was ascertained that the concentration-absorbance relationship was linear in the studied range of HSA concentration (r2 = 0.99968).Total protein biuret reagent was used to assess protein recovery post filtration of HSA solutions. Six replicates were made for each sample; 50 μL of sample was mixed with 150 μL total protein reagent and the reaction was protected from light for 10 to 30 min then absorbance was measured at 540 nm using a Tecan M200 microplate reader. The recovery rate was calculated using Equation (1) as follows:E. coli stock. The resulting suspension was mixed with 1 mL of melted soft agar and poured on the surface of a prepared hard agar plate (55 × 15 mm) and incubated at 37 °C for 5 h. Bacteriophage titer was calculated using Equation (2):5 to 106 bacteriophages mL−1. The limit of detection, i.e., ≤0.7 PFU mL−1, of the current experimental design refers to ≤5 bacteriophages mL−1, corresponding to a single detectable plaque in one of the plates for nondiluted duplicate samples, assuming that at the detection limit each plaque is produced by one bacteriophage. Virus retention was expressed as LRV :The titer of ΦΧ174 bacteriophage was determined by a plaque forming units (PFU) assay. The feed and permeate samples were serially diluted in Luria-Bertani medium (LBM) , and 100 μL of diluted bacteriophage was mixed with 200 μL of −2 the flow rate quickly decreased from the initial 121.6 L m−2 h−1 to about 16.7 L m−2 h−1 and then slowly decreased to 6.7 L m−2 h−1. It should be noted that protein recovery after prefiltration was 94.3 ± 1.5%, suggesting that the fraction of protein causing fouling was relatively small. It should be noted that for practical applications in order to avoid rapid fouling of the prefilter and to maintain high throughput, the sizing of the prefilter can be adjusted, as is normally done in industry, given the high cost-efficiency of the prefilter.−2 h−1. After filtering at 28.8 L m−2, it only decreased to 96.7 L m−2 h−1 and the protein recovery rate was 90.7 ± 6.6% from the feed HSA content, i.e., prior to prefiltration, calculated Vmax ≥ 1000. Under a constant pressure of 3 bar, the filtration flow rate of 28.8 L m−2 slowly decreased from the initial 203.3 L m−2 h−1 to 161.7 L m−2 h−1 and the protein recovery rate was 92.3 ± 0.6% with a calculated Vmax 110.0 ± 2.5. At a constant pressure of 5 bar, the flow rate slowly decreased from the initial 252.5 L m−2 h−1 to 165.8 L m−2 h−1, and the protein recovery was 91.3 ± 3.5% with a calculated Vmax of 63.6 ± 5.8. The protein loss in the second filtration step was within the standard deviation range between the different groups and the main loss of about 5.6 ± 1.5% occurred during prefiltration.For filtration with the 22 μm filter, the flow rate varied depending on the applied overhead pressure. Under a constant pressure of 1 bar, the entire filtration flow rate was relatively stable at about 100 L mTo understand the observed improvement in flow properties after two-step filtration, DLS and SE-HPLC analyses were performed, as discussed below. −2 to 28.8 L m−2, virus clearance decreased from 3.75 LRV to 2.65 LRV when operated at 1 bar. Furthermore, filtration at 1 bar was more time consuming than that at 3 or 5 bar. At 3 bar and 5 bar, constant pressure virus clearance was >5 LRV and stable for increasing load volume fractions. The results of To verify the virus clearance of the 22 μm filter, HSA prefiltered solution was spiked with ΦX174 phage. The removal of HSA aggregates dramatically improved the flow properties of the filter, enabling high protein throughput and virus clearance. A distribution of pore sizes between 40 nm and 60 nm, present in the 11 μm prefilter and absent in the 22 μm virus filter, played a crucial part in removing HSA aggregates. With respect to virus filtration, 1 bar constant pressure filtration showed poor removal ability of ΦX174 bacteriophage (28 nm), i.e., LRV ≤ 3.75, while constant pressure filtration at 3 bar and 5 bar achieved LRV > 5 and overall fast filtration. It is possible to more safely and quickly eliminate virus risk of plasma products while maintaining the biological activity of its various components.