Neisseria meningitis, MERS-CoV, monkeypox, Nipah virus infection, novel coronavirus (COVID-19), plague, Rift Valley fever, SARS, smallpox, tularemia, yellow fever, and Zika virus disease. The associated pathogens are increasingly important on the global stage. The majority of these diseases have neurological manifestations. Those with less frequent neurological manifestations may also have important consequences. This is highlighted now in particular through the ongoing COVID-19 pandemic and reinforces that pathogens with the potential to spread rapidly and widely, in spite of concerted global efforts, may affect the nervous system. We searched the scientific literature, dating from 1934 to August 2020, to compile data on the cause, epidemiology, clinical presentation, neuroimaging features, and treatment of each of the diseases of epidemic or pandemic potential as viewed through a neurologist's lens. We included articles with an abstract or full text in English in this topical and scoping review. Diseases with epidemic and pandemic potential can be spread directly from human to human, animal to human, via mosquitoes or other insects, or via environmental contamination. Manifestations include central neurologic conditions , peripheral and cranial nerve syndromes , post-infectious syndromes (acute inflammatory polyneuropathy), and congenital syndromes , among others. Some diseases have not been well-characterized from a neurological standpoint, but all have at least scattered case reports of neurological features. Some of the diseases have curative treatments available while in other cases, supportive care remains the only management option. Regardless of the pathogen, prompt, and aggressive measures to control the spread of these agents are the most important factors in lowering the overall morbidity and mortality they can cause.The World Health Organization (WHO) monitors the spread of diseases globally and maintains a list of diseases with epidemic or pandemic potential. Currently listed diseases include Chikungunya, cholera, Crimean-Congo hemorrhagic fever, Ebola virus disease, Hendra virus infection, influenza, Lassa fever, Marburg virus disease,  The World Health Organization (WHO) provides guidance on diseases that are international threats to populations with a focus on diseases of epidemic and pandemic potential . Close oNeisseria meningitidis meningitis until September 2020. Articles in English or with an abstract in English were included, using data sources dating from 1934 until present.Searches were performed using the keywords of the disease and infectious organism (if different) as well as the terms “neurology,” “brain,” “nervous system,” and related terms for each of the listed disease of epidemic or pandemic potential on the WHO website as of April 1, 2020. Search engines included PubMed, Google Scholar, and the reference lists of articles found through those searches. All searches took place in spring 2020 with updates continuing as more information became available, particularly on evolving pandemics, such as SARS-CoV-2, influenza, Ebola Virus disease, and Data sharing is not applicable to this article as no new data were created or analyzed in this study.Aedes mosquitos as a direct infection to humans, although it can rarely pass through maternal-fetal transmission or blood products. Chikungunya presents with a systemic course that is relatively uniform across individuals, but neurologic manifestations can be more heterogeneous. Its systemic course begins with an incubation period from 1 to 12 days and then progresses to fever, malaise, and headache, as well as a characteristic symmetric arthralgia in as many as 90% of patients , myeloradiculitis, facial palsy, sensorineural deafness, optic neuritis, and more . ApproxiThe time course of neurologic signs varies by the syndrome; encephalopathy often appears concurrently with initial systemic symptoms, occasionally lagging by 1 to 2 days. Spinal disease, with weakness and occasional paresthesias of multiple limbs, can appear at symptomatic onset or with a delay of up to 3 weeks. GBS appears weeks after the initial illness, consistent with its usual presentation .The exact proportion of individuals infected with Chikungunya who exhibit neurologic involvement is unclear, but it appears to be the most common “atypical” involvement in severe disease. Case series during epidemics have reported neurologic involvement ranging from 0.1%  to 16.3%Diagnosis of chikungunya is with real-time reverse-transcription polymerase chain reaction (RT-PCR) or enzyme-linked immunosorbent assay (ELISA) of the serum depending on the timing of testing. Within 5 days of initial symptom onset, serum RT-PCR for viral RNA has up to 100% sensitivity and 98% specificity, while ELISA for immunoglobulin M (IgM) is appropriate for patients who have shown clinical symptoms for at least 6–8 days . TreatmeVibrio cholerae is a gram-negative, comma shaped bacterium most commonly found in seawater and known to have a number of serogroups. Specifically, serogroups O1 and O139 have been implicated in the majority of intestinal infections that are most common worldwide  is a hemorrhagic illness caused by infection with the Crimean-Congo hemorrhagic fever virus (CCHFV), a negative-sense RNA virus of the segments . CCHF wasegments . In 1956segments . As the segments . Althougsegments , 27.Hyalomma tick, through the butchering of livestock that act as amplifying hosts for the virus, or via contact with infected bodily fluids  for certain strains of CCHFV along with neuropathological evidence of gliosis and meningoencephalitis. Viral RNA has also been isolated from ocular samples in mice. There is evidence of vascular congestion in the CNS, indicating a route of entry for CCHFV through the blood-brain-barrier related to the virus's known tropism for endothelial cells . Serum RFiloviridae family, whose infamy as one of the most dangerous human pathogens has earned it recognition as a potential bioweapon  typically begin after an incubation period lasting 4 to 10 days. Early disease presents with non-specific symptoms, including high fevers, headaches, fatigue, myalgias, and body aches; there may be associated conjunctival injection, chest pain, and arthralgias. A few days later, infected individuals may develop abdominal pain with nausea, copious vomiting and watery diarrhea, which can lead to severe volume depletion and shock. Hemorrhagic disease is a late complication that affects a minority of infected people, and usually manifests with petechiae, ecchymoses, oozing from sites of venipuncture, hematemesis, or melena .There is better understanding of neurologic complications from EVD in the wake of recent epidemics; however, neurologic evaluation of acutely infected patients poses significant challenges. Strict quarantining and extensive personal protective equipment can limit a comprehensive neurologic exam and has largely prohibited lumbar puncture (LP) for CSF analysis, except in rare cases. Acute neurologic manifestations vary in prevalence from very common, to quite rare. Commonly seen neurologic manifestations include headaches, diffuse weakness, and a varying degree of encephalopathy. Rarely, EVD can be complicated by hemorrhagic or ischemic strokes. Cerebellar signs and myoclonus have also been observed, but are considered rare . SeizureA proportion of people who survive EVD may develop chronic or delayed onset neurologic complications, and cases have surfaced that demonstrate reactivation of virus within immunologically privileged sites. Survivors of a 2007 EVD outbreak in Uganda exhibited headaches, retro-orbital pain, hearing loss, memory problems, and confusion more commonly than controls, 2 years following infection , 46. HeaParamyxoviridae, and are negative-sense, single-stranded RNA viruses in the order Mononegavirales. Given close homology and cross-reactivity on serological tests, HeV is commonly grouped with the Nipah Virus (NiV); together, these are referred to as Henipavirus (Hendra virus (HeV) belongs to a family of viruses called ipavirus . Hendra ipavirus . The veripavirus . PteropiStudies of acute human infection have demonstrated that a wide variety of tissues may be infected by HeV, including lung, liver, blood vessel, and brain . Humans Outbreaks have appeared to cluster in cooler months, a finding hypothesized to be related to the breeding cycles of flying foxes, or favorable survival of the virus in cooler and drier environments . In 2014Influenza are a group of RNA viruses of the Orthomyxoviridae family, which cause worldwide seasonal outbreaks of acute respiratory illness associated with significant morbidity and mortality. Seasonal cycles are occasionally dominated by pandemic strains of the virus, which cause devastating worldwide disease. There are four types of influenza viruses, A through D, which are characterized based on antigenic characteristics of viral surface proteins. Of these, types A and B primarily produce important human disease . ANE usually develops about 5 days after onset of typical viral symptoms, and is characterized by rapid deterioration in mental status, progressing to coma over the span of days. It is most strongly associated with influenza infection, although other viral infections have also been linked. Mild pleocytosis, elevated protein, and xanthochromia can be observed in the CSF , 65. BraA spike in narcolepsy cases followed 1918 H1N1 influenza pandemic , and theThere are many more neurologic syndromes described following influenza infection, though the full list is beyond the scope of this article. Similar to the other presentations described, these syndromes are not singularly associated with influenza. They include acute disseminated encephalomyelitis (ADEM), transverse myelitis, acute inflammatory demyelinating polyneuropathy (AIDP) and the Miller-Fisher variant, as well as ischemic stroke. Of note, rare cases of AIDP and transverse myelitis have been associated with the influenza vaccine, though the relative risk is estimated to be much lower than the benefit of protection against active infection , 76.One study of 34 children with acute necrotizing encephalopathy (including 11 influenza as prodrome) demonstrated a favorable outcome in 7 of 12 children with early steroid administration compared to 0 of 5 children without this treatment, although this was not statistically significant . Some caMastomys natalensis rats and endemic to Western Africa. It initially infects immune cells in the nasopharynx, with subsequent spread to regional lymph nodes followed by multiorgan dissemination. After an incubation period of 1 to 3 weeks, its clinical course is heterogeneous; the majority of patients experience only malaise, headache, and low-grade fever, but more severe cases can include such diverse manifestations as diarrhea, hypotension, and pulmonary edema. Oozing blood from the oropharynx or, less commonly, the rectum or genitals, is seen in fewer than 20% of patients  appear to be the only indicated management. No proven treatment exists for amelioration of sensorineural deafness, and early treatment with ribavirin does not appear to protect against development of this symptom nor improve its chances of resolution.Filoviridae  is an enveloped, single-stranded, positive-sense RNA virus belonging to the diverse family i Arabia . Humans i Arabia –115, alti Arabia , 117. Moi Arabia , 119. Asi Arabia , making i Arabia , 122.Neurologic manifestations in MERS-CoV infection have been cited in small case series. In one examination of 70 MERS-CoV positive patients, 12.9% developed headache, 25.7% confusion, and 8.6% seizures . One serMonkeypox is a double-stranded DNA, zoonotic virus that characteristically causes a disease similar to smallpox, but with milder exanthem and lower mortality. The definitive reservoir has not been established, but it appears transmissible through monkeys as well as terrestrial rodents including rats and squirrels. Person-to-person transmission is possible but appears to occur at relatively low rates and result in a milder disease course , 128.We have been able to find only two cases of neurologic complications of monkeypox in the literature. The individuals affected were both young girls who developed encephalitis, requiring intubation and mechanical ventilation. One 3-year-old girl died on day 2 of hospitalization while a 6-year-old girl survived after a 14-day course in the intensive care unit. The former patient had no CSF diagnostics performed, while the latter patient had detectable orthopoxvirus-reactive IgM in CSF , 130.Of note, monkeypox appears to have a fatal course almost exclusively in infants and young children, specifically those who have not received vaccination against smallpox .Paramyxovirus of the genus Henipavirus, endemic to Southeast Asia and Africa  is an enveloped, negative-sense single-stranded RNA d Africa , 132. Fid Africa . Bats ofast Asia , 135. Niast Asia .NiV infection presents primarily with a fulminant encephalitis , 137. AfThe first recognized outbreak is thought to have occurred on Malaysian pig farms, where asymptomatic bats in tree branches hanging above farms transmitted NiV in their saliva and other secretions to pigs below leading to rampant pig-pig and pig-to-human transmission via respiratory and other secretions . More thSince this, outbreaks have been recognized in Bangladesh and India nearly annually since 2001 from a related and potentially more dangerous strain of NiV, with additional outbreaks in the Philippines stemming from NiV-infected horses , 154. HuNo NiV-protective vaccines are currently commercially available, although host animal and human Nipah virus vaccines are currently in development. Recent small-scale preclinical studies with these vaccines have reported effective immunity after VZV-platform vaccination of non-human primates , 163. RiIn December 2019, a series of viral pneumonia cases of unknown cause appeared in Wuhan, Wubei, China, and deep sequencing analysis from lower respiratory tract samples indicated that the infection was due to 2019 novel coronavirus (2019-nCoV) . The 201It is too early to tell if there is increased prevalence of neurological complications as a result of COVID-19 when compared to other respiratory viruses. Among a subset of patients with COVID-19 in three hospitals in Wuhan, China: 36% of patients had neurologic manifestations with CNS manifestations in 25%, PNS manifestations in 9%, and skeletal muscle injury in 11% . MultiplSome case series and case reports have described peripheral nerve and muscle complications of COVID-19 that can be seen in other viral infections. GBS was reported in both Wuhan  and ItalDiagnosis of systemic COVID-19 is most commonly performed with RT-PCR on nasopharyngeal swabs, serology tests, and antibody-based rapid tests , 177. SoWe anticipate studies focused on the impact of SARS-CoV-2 infection on neurological function and the long-term neurological sequelae will continue to be published and our understanding will deepen over the coming years. At this point, it is obvious that extrapulmonary manifestation of SARS-CoV-2 includes neurological symptoms and disease, and will be crucial for clinicians to recognize and treat.Yersinia pestis is a zoonotic, gram-negative, non-motile organism transmitted by the tropical rat flea (Xenopsylla cheopis). The plague disease it causes has three clinical subtypes: bubonic, septicemic, and pneumonic. The most common bubonic form is characterized by sudden onset of malaise, dizziness, high fever, and lymphadenitis. This can progress to septicemic plague, which includes a heterogeneous phenotype of hypotension plus other systemic manifestations; approximately 80–90% of septicemic plague cases occur with preceding buboes (enlarged lymph nodes) (h nodes) . The mosThe pathogenesis of plague meningitis is presumed due to hematogenous dissemination from buboes, and in light of this some studies have proposed that axillary buboes predispose to meningitis given easier access to lymphatic tracts leading to intracranial compartments . The casY. pestis  is a negative-sense, single-stranded RNA virus that belongs to the genus ebovirus . RVFV islization .Rift Valley Fever (RVF) was first identified in East Africa in 1931 with nine recognized outbreaks between 1999 and 2010 . There hIn humans, most cases of RVF are mild and can cause a non-specific flu-like illness with fever, muscle pain, joint pain and headache. However, severe cases include ocular, meningoencephalitis or hemorrhagic fever forms. Neurologic manifestations are identified in up to 17% of patients in some outbreaks, which can manifest with encephalopathy, ataxia, vertigo, seizures, and coma . RetinitDiagnosis for RFV is through RT-PCR of viral RNA or ELISA for IgM antibodies against RVF virus . TreatmeCoronaviridae (Severe acute respiratory distress syndrome (SARS) is a viral respiratory illness caused by a type of coronavirus called SARS-associated coronavirus (SARS-CoV) . Coronavaviridae . Infectiaviridae . SARS waaviridae .There has been concern that SARS may translocate and infect the CNS, with reports of isolation of the virus in the brain  and specIn murine models, SARS has been correlated to a possible chronic demyelinating condition that resembles multiple sclerosis . Murine The most sensitive modality for SARS diagnosis is ELISA for IgM against the virus, although this can remain negative in infected patients for weeks into illness . RT-PCR Similar to COVID-19, there are neuromuscular complications in patients with SARS, including skeletal muscle injury and rhabdomyolysis, although these manifestations are theorized to be related to a cytokine storm rather than direct infection by the virus .Orthopoxvirus genus. Although it was eradicated in 1980, there is concern that smallpox could reemerge as a bioterrorism agent. Human to human transmission predominantly occurs through airborne droplets, but can also occur through infected vesicles, scabs, and fomites. It is highly contagious. Transmission in a household can be up to 60%  followed by a cutaneous rash (exanthem) starting in the face and spreading to the extremities that becomes vesicular then pustular. Mortality is estimated at 30–50%  has recognized F. tularensis as a potential bioweapon  is a positive-sense, single-stranded RNA virus that belongs in the  are low .YFV incubates for 3–6 days after transmission and can lead to a mild non-specific febrile illness. However, a portion of patients (~14%) can develop severe disease leading to hemorrhagic fever and multi-organ failure . DiagnosSevere neurologic manifestations have been associated with the yellow fever vaccine. While rare, neurotropic disease associated with the vaccine 17D is estimated at 0.8 per 100,000 people vaccinated, especially in individuals >60 years old . NeurotrAedes mosquitoes (Zika Virus (ZIKV) is a positive-sense RNA flavivirus first isolated and identified in Uganda in 1947 . ZIKV issquitoes , althougsquitoes , blood tsquitoes . Symptomsquitoes . Diagnossquitoes , 250. ZINeurologic symptoms of ZIKV infection in adults can be divided into peripheral and central manifestations. Peripherally, ZIKV infection is associated with GBS, which was estimated to affect 2–3 patients per 10,000 ZIKV infections . SymptomFetuses of women infected with ZIKV during pregnancy are at risk of congenital Zika syndrome, and ZIKV-associated microcephaly. Four to seven percentage of infections will result in fetal loss. Twenty to thirty percentage of the fetuses of women infected with ZIKV will develop infection . InfecteDiseases with the potential for epidemic and pandemic spread have a variety of neurological manifestations, ranging from rare presentations involving the CNS to fulminant manifestations that define the disease outbreak. While some diseases represent regional epidemics, several reflect pandemic diseases. The role of the neurologist and neurologically-focused clinician is critical for early identification of outbreaks and characterization of the range of features of systemic disease-causing pathogens.F. tularensis, influenza, MERS, and Zika virus) additionally include a recognized element of PNS symptom burden. The most common CNS manifestation is a non-specific encephalopathy, although a number of diseases cause more syndromic neurologic features that are pivotal in the disease diagnosis such as in the fulminant encephalitis of Nipah virus, the ophthalmoplegia and ataxia of MERS-CoV-2, and the ICH of Crimean-Congo hemorrhagic fever or Marburg virus disease. The most common PNS manifestation of the diseases on this list is post-infectious AIDP.Each disease listed includes CNS involvement although in some cases the situation is atypical  or the data reflect few cases in vulnerable groups . Six of the diseases  are studied in only a handful of individual case reports, all other pathogens include at least moderately sized case series and often large, population-scale reviews indicating a significant burden of neurologic disease.V. cholerae, ebolavirus, influenza virus, Hendra virus, Lassa virus, N. meningitidis, Nipah virus, Y. pestis, SARS, F. tularensis, yellow fever virus, Zika virus). Four pathogens have possible neurotropism as shown either through presence in the brains of animal models but not humans  or by presence of antibodies but not the pathogen itself in CSF of affected individuals (Marburg virus). In four pathogens, the question of direct neurotropism remains unclear . Many of the diseases show evidence of parainfectious neurologic syndromes in addition to any direct neurotropism they may have .The mechanism of neurological involvement is variable among the diseases. Twelve of the associated pathogens have potential evidence of direct neurotropism as shown either through identification in CSF during active infection or neuropathologic findings , have spread to all or nearly all WHO world regions, often despite having well-identified origins in particular locations. Vaccines are presently available for only eight of the diseases , while two others have a vaccine available for animal hosts (Hendra virus) or under development for humans  . Of the Our work has several limitations. We provide a scoping review but not a systematic review. Our summary is limited by the review being performed of English language articles with searches performed through PubMed and related medical search engines online. We did not include historical literature, for example on plague or smallpox, that may be earlier than our search was able to extend. By the nature of this topic, the data reviewed came from a variety of regions with differing availability of neurological expertise, testing techniques, disease surveillance, and access to healthcare which led to a lack of standardization of some results. Although we attempted to be exhaustive in reporting the range of infections, due to the broad scope of our task and high number of manuscripts available on some pathogens, we highlight the most critical information for neurologists in the field.Future efforts at prevention of the spread of these pathogens—and the epidemics and pandemics that they are capable of causing—should span investments in science, education, clinical care, and public health. Directions for the medical community could include increased funding for vaccine research as well as strengthening medical facilities with the ability to address and evaluate disease surges where and when they arise. Pragmatic efforts include public education on the best methods to prevent the spread of disease including face masks, hand hygiene, quarantine, and proper handling of biospecimens, burials, and autopsies. Effective and unified population-level messaging could decrease morbidity and mortality of the individual patient and prevent further spread while reducing the burden on healthcare systems –261.While the diseases on the WHO list have the potential to cause widespread harm, many of them fortunately have distinct clinical and diagnostic features that make it possible to identify outbreaks when they are suspected, and early interventions by providers can significantly impact the trajectory of a pathogen's spread. This review has outlined the neurological features of all of the currently listed diseases with epidemic and pandemic potential by the WHO with the goal of assisting clinicians who see neurological conditions with the prompt identification of diseases. This list may evolve and expand over time as new pathogens are discovered, pathogenicity changes, or hosts and conditions for spread continue to change. One lesson that is crucial to glean from the high morbidity and mortality of the COVID-19 pandemic is that the interconnectedness of the modern world makes the threat of any of these 20 diseases real and imminent, and the medical community must be vigilant in their surveillance to ensure they do not spread.FM was additionally involved with project supervision. All authors contributed to the manuscript conception, drafting, and editing for critical content.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.


Bos indicus) in the Northern Territory of Australia. The animal was suffering from a severe ephemeral fever-like illness with neurological involvement, including recumbency and paralysis, and was euthanised. Histological examination of spinal cord and lung tissue identified extensive haemorrhage in the dura mata with moderate perineuronal oedema and extensive emphysema. HYV displayed cone-shaped morphology, typical of rhabdoviruses, and was found to be most closely related antigenically to Puchong virus (PUCV), isolated in 1965 from mosquitoes in Malaysia. Analysis of complete genome sequences of HYV (15 025 nt) and PUCV (14 932 nt) indicated that each has a complex organisation (3′ N-P-M-G-GNS-α1-α2-β-γ-L 5′) and expression strategy, similar to that of BEFV. Based on an alignment of complete L protein sequences, HYV and PUCV cluster with other rhabdoviruses in the genus Ephemerovirus and appear to represent two new species. Neutralising antibody to HYV was also detected in a retrospective survey of cattle sera collected in the Northern Territory.Bovine ephemeral fever is a vector-borne disease of ruminants that occurs in tropical and sub-tropical regions of Africa, Asia and Australia. The disease is caused by a rhabdovirus, bovine ephemeral fever virus (BEFV), which occurs as a single serotype globally. Although several other closely related ephemeroviruses have been isolated from cattle and/or arthropods, only kotonkan virus from Nigeria and (tentatively) Mavingoni virus from Mayotte Island in the Indian Ocean have been previously associated with febrile disease. Here, we report the isolation of a novel virus  from blood collected in February 2000 from a bull ( Ephemerovirus, family Rhabdoviridae, comprises negative-sense, single-stranded RNA viruses that infect primarily ruminants and are transmitted by haematophagous insects -methionine/cysteine at 5, 10, 17, 25, 35 and 50 h post-infection (hpi) and analysed by SDS-PAGE and autoradiography  also identified protein bands induced at 18 and 24 hpi that appeared to correspond to the PUCV structural proteins L, G, N, P and M  of 3 TCIDy Figure . A markeRhabdoviridae  were determined by virus neutralisation tests Table . As obseSera collected in June 2000 from 41 cattle in a sentinel herd at Beatrice Hill Farm were tested for neutralising antibodies to HYV  throughout and appetite was unaffected. One of the four animals had a mild discharge from the right eye on day 3 and day 4 post-inoculation. Blood collected from each animal pre-inoculation (day 0), on days 1 to 7 post-inoculation and at termination of the experiment all tested negative for PUCV RNA by RT-PCR and negative for antibody to PUCV G protein by ELISA.Four cattle were inoculated intravenously with 10In Australia, East Asia and the Middle-East, bovine ephemeral fever is considered to be an important disease of cattle, causing significant production losses in both dairy and beef herds –9, 43. BUnfortunately, there was a lag of several years between the disease event and identification of HYV and, with no remaining infectious blood and cell culture passage yielding only low titre material, we were unable to conduct experimental infections to fulfil River’s postulates. An experimental infection in cattle with closely related PUCV was conducted and failed to show evidence of either infection or disease; however, it is well established that, even for BEFV, cattle are usually not susceptible to infection with cell culture adapted virus . NeverthEphemerovirus have several of the following characteristics: (A) minimum amino acid sequence divergence of 15% in L; (B) minimum amino acid sequence divergence of 8% in N; (C) can be distinguished in serological tests; and (D) significant differences in genome organisation as evidenced by numbers and locations of ORFs [HYV is quite closely related, both antigenically and phylogenetically, to PUCV which was isolated from mosquitoes in Malaysia in 1965. This raises the question as to whether HYV and PUCV should be assigned to different species or, like KIMV and MALV, should be considered geographic variants of the same virus . Current of ORFs , 45. By  of ORFs .Serological data presented here indicate that several cattle in the sentinel herd at Beatrice Hill Farm had developed neutralising antibody to HYV by June 2000  between the G and L genes. Of these, only the BEFV GNS and α1 proteins have been identified definitively in infected cells [NS protein, several virus-induced protein bands were detected with a relative mobility range of approximately 9–15 kDa. Although we could not identify these specifically as the small PUCV accessory proteins , there was evidence of expression of their corresponding mRNAs. However, assignment of proteins based only on relative migration in polyacrylamide gels is problematic.The organisation of the HYV genome is similar to those of BEFV, BRMV, KIMV and PUCV, featuring genes encoding the same set of accessory proteins , differences in clinical outcomes between pairs of closely related ephemeroviruses are well known. Both known agents of bovine ephemeral fever, BEFV and KOTV, are closely related to viruses that do no cause disease . InAdditional file 1. Primers used in the study. Details of primers used for RACE for HYV and PUCV and details of primers used for transcription profiling on PUCV.Additional file 2. Initial phylogenetic analysis of isolate V4816 . A Neighbour-Joining phylogenetic tree inferred from a Clustal W alignment of the deduced amino acid sequence of the 100 nt amplicon generated from isolate DPP4816  and the corresponding L protein sequences of Puchong virus , Kimberley virus , Malakal virus , Berrimah virus , bovine ephemeral fever virus , Yata virus , kotonkan virus , Koolpinyah virus , Adelaide River virus , Obodhiang virus , Wongabel virus , Fukuoka virus  and Tibrogargan virus . The alignment and tree were generated in MEGA version 7.0.18 using default parameters.Additional file 3. Sequence identities amongst ephemeroviruses. Comparison of nucleotide and amino acid sequence identities of HYV and PUCV to other related ephemeroviruses.Additional file 4. Comparison of ephemerovirus G and GNSproteins. A Clustal W amino acid sequence alignment of the G and GNS proteins of BEFV, HYV and PUCV. The alignment was generated in MEGA version 7.0.18 using default parameters and adjusted following visual inspection. Identical (*), strongly conserved (:) and weakly conserved (.) amino acids are indicated. Predicted signal peptides) in the N-terminal domains and predicted transmembrane domains in the C-terminal domains are shaded in grey. Predicted N-glycosylation sites are underlined. Conserved cysteine residues in the ectodomains are shaded in black. Twelve cysteine residues (CI–CXII) in the BEFV G also occur in the G protein of vesicular stomatitis Indiana virus in which they form six disulphide bridges indicated by dotted lines (see text). Six additional cysteine residues (a–f) occur in the BEFV G protein and have been predicted to form three additional disulphide bridges (see text). The figure illustrated similarities in the structure of the G and GNS proteins of BEFV, HYV and PUCV.Additional file 5. Clustal W amino acid sequence alignments of the small accessory proteins of HYV, PUCV and BEFV. A) α1 proteins. Predicted transmembrane domains are shaded (grey). Large aromatic residues in the N-terminal domains (underlined) and basic residues in the C-terminal domains (bold) are characteristic of class 1a viroporins. B) α2 proteins which are each encoded in a second consecutive ORF within the α gene. C) β proteins. D) γ proteins. Identical (*), strongly conserved (:) and weakly conserved (.) amino acids are indicated.Additional file 6. Sero-neutralisation test results. Neutralising antibody titres to HYV in sera from selected sentinel cattle from the Northern Territory, Australia.


Glycogen storage diseases (GSDs) belong to the group of inborn errors of carbohydrate metabolism. Hepatic GSDs predominantly involve the liver and most present with hepatomegaly. Biochemically they show known disturbances in glucose and fatty acids metabolism, namely fasting hypoglycaemia and increased triglycerides. Additionally, increased biotinidase (BTD) enzyme activity has been shown to be associated with many GSD types, whereas the mechanism by which BTD enzyme activity is altered remains unknown so far. We aimed to delineate changes in gluconeogenesis and fatty acid synthesis, potentially explaining raised BTD enzyme activity, by using liver (specimens from 2 patients) and serum samples of GSD Ia and GSD IV patients. By expression analysis of genes involved in gluconeogenesis, we ascertained increased levels of phosphoenolpyruvate carboxykinase and fructose‐1,6‐biphosphatase, indicating an increased flux through the gluconeogenic pathway. Additionally, we found increased gene expression of the biotin‐dependent pyruvate and acetyl‐CoA carboxylases, providing substrate for gluconeogenesis and increased fatty acid synthesis. We also observed a significant linear correlation between BTD enzyme activity and triglyceride levels in a cohort of GSD Ia patients. The results of this pilot study suggest that enhancement of BTD activity might serve the purpose of providing extra cofactor to the carboxylase enzymes as an adjustment to disturbed glucose and fatty acid metabolism. Future studies involving a higher number of samples should aim at confirming the here proposed mechanism, which extends the application of BTD enzyme activity measurement beyond its diagnostic purpose in suspected GSD, and opens up possibilities for its use as a sensor for increased gluconeogenesis and fatty acid synthesis. Biotinidase enzyme activity can be used as a diagnostic screening tool for glycogen storage disease and might be suitable for detection of increased gluconeogenesis and fatty acid synthesis.1Glucose is the most important source of energy, which must be constantly available, and its level is meticulously controlled to maintain normoglycaemia. Following carbohydrate intake, glucose is directly broken down via glycolysis, while it must be newly generated via gluconeogenesis or glycogenolysis during times of fasting. Glycolysis is a multi‐step process to produce pyruvate, which is oxidized to acetyl‐CoA and further metabolized to malonyl‐CoA by acetyl‐CoA carboxylase  in the process of fatty acid synthesis. In the reverse process, called gluconeogenesis, pyruvate is metabolized to oxaloacetate by pyruvate carboxylase Storage of glucose as energy source is enabled by the build‐up of glycogen, a polysaccharide consisting of linked glucose molecules via linear α(1 → 4) and branched α(1 → 6) glycosidic bonds. Deficiencies of glycogen synthesis and breakdown cause glycogen storage disorders (GSDs), a heterogenous group of inherited disorders of carbohydrate metabolism.In GSD Ia, the accumulating glucose‐6‐phosphate is shunted to glycolysis, leading to an increase of acetyl‐CoA and thereby raised lipogenesis and malonyl‐CoA through the action of ACACA, whereby the latter metabolite inhibits carnitine palmitoyltransferase I, leading to a further increase of triglycerides in the plasma of patients.BTD, PCK1, PCK2, FBP1, ACACA, PC, and assessed BTD enzyme activity in serum of these patients. We complemented these investigations with BTD enzyme activity measurements in two non‐GSD patient cohorts with a normal glycogen storage pathway, aiming to study the contribution of increased gluconeogenesis and an altered fatty acid oxidation pathway to elevated BTD enzyme activity. First, we investigated a cohort of diabetes mellitus type 1 patients, who are known to have upregulated gluconeogenesis.We analyzed liver tissues of a GSD Ia and a GSD IV patient for the expression of We hypothesize, that in both GSD types one of the biotin‐requiring carboxylases PC or ACACA is upregulated, hence the requirement for their cofactor biotin is increased Figure . We furt22.1Liver samples of a GSD Ia and a GSD IV patient (both paediatric) were harvested for diagnostic purposes and then used for the expression analyses outlined in this study. Due to limited availability of other liver samples, the study was restricted to these GSD subtypes. Two anonymised control liver samples from healthy donors without further clinical information were retrieved from the Centralized Tissue‐Biobank of the University Hospital Zurich.Serum samples of the two GSD patients mentioned above and a group of diabetes mellitus type 1 patients , epilepsy patients on ketogenic diet  as well as of a cohort of enzymatically and/or molecularly confirmed GSD Ia patients  were obtained for the study of BTD enzyme activity by routine blood collection during clinic visits. The patients on ketogenic diet were treated with different regimes of antiepileptic drugs at the time of biotinidase enzyme activity determination . The antiepileptic medication remained unchanged for all measurements of biotinidase enzyme activity.2.22.2.1GBE1 mutations c.1883A > G in exon 14,The GSD IV male patient presented neonatally with failure to thrive, hypotonia, and hepatomegaly. On liver biopsy at 15 months of age glycogen was normal Figure S, but GBE2.2.2G6PC gene. The clinical course was complicated by the occurrence of an inflammatory bowel disease (IBD)—a rare observation in GSD Ia patients.The GSD Ia patient is a female who presented with hypoglycaemic episodes early on in life. The diagnosis was based on significantly diminished G6PC enzyme activity and molecular genetic testing revealing the following compound heterozygous mutations: c.113A > T p. and c.562G > C p.(Gly188Arg) in the 2.3Enzymatic activity of BTD was assessed in serum of patient and control samples as previously published2.4BTD (Hs00163760_m1), ACACA (Hs01046047_m1), PC (Hs01085875_g1), PCK1 (Hs00159918_m1), PCK2 (Hs01091129_g), FBP1 (Hs00983323_m1), via the TaqMan Gene Expression Master Mix on a 7900HT Fast Real‐Time PCR System, with normalization to ACTB (Hs01060665_g1) . Experiments were performed in triplicates.For quantitative real time PCR analysis total RNA was extracted from frozen liver tissue sample lysates from the two GSD patients and two controls , amplified to cDNA with the Prime Script II first strand cDNA Synthesis Kit (TaKaRa ClonTech), and analysed for expression using specific probes for 2.5t test and one‐way analysis of variance (ANOVA). Rstudio was used to plot graphs and calculate Pearson's correlation.GraphPad Prism 8.0.2 software was used to calculate 33.1PCK1, which was upregulated 2.4 times in GSD IV and 1.4 times in GSD Ia  and GSD Ia (1.6 times) , encoding fructose‐1, 6‐bisphosphatase 1, which catalyses the hydrolysis of fructose 1,6‐bisphosphate to fructose 6‐phosphate, showed further indication of upregulated gluconeogenesis .To examine whether gluconeogenesis is affected in GSD IV and GSD Ia, we investigated gene expression levels of key enzymes of gluconeogenesis in liver tissue by RT‐qPCR. The cytosolic phosphoenolpyruvate carboxykinase facilitates the initial step of gluconeogenesis and is encoded by a Figure . The iso) Figure . Upreguls Figure . Only th3.2ACACA and PC genes. We hypothesized that the two investigated genes are upregulated as they are required to supply starting substrates for gluconeogenesis  and fatty acid synthesis (acetyl‐CoA). Expression of the ACACA gene, encoding acetyl‐CoA carboxylase, was upregulated 2.9 times in GSD IV and 3.8 times in GSD Ia , who are known to have increased gluconeogenic flux.Of note, the BTD enzyme activity level in the presented GSD IV patient dropped back to reference range after liver transplantation Figure S and his 4Elevated BTD enzyme activity can be used as a biomarker for hepatic GSDs, however, the mechanism of action underlying the increased activity of this enzyme under GSD conditions is unclear so far. In this study, we investigated this phenomenon by analyzing liver and serum samples derived from GSD IV and GSD Ia patients. Together with increased BTD enzyme activity in serum and expression in liver tissue, we detected elevated expression of two biotin‐dependent carboxylases, supporting the hypothesis of increased biotin‐turnover and thus elevated BTD enzyme activity. Similarly, patients with diabetes mellitus type 1 and patients on ketogenic diet, conditions known for increased gluconeogenic flux and activated pyruvate carboxylase, also showed elevated BTD enzyme activity in serum.4.1ACACA transcript levels in the GSD Ia patient liver. (b) In GSD IV the breakdown of pathological glycogen is impaired, in turn requiring an increased baseline gluconeogenic flux to ensure normoglycemia, which is facilitated by increased expression of FBP1, PCK1, and PCK2, as shown in liver tissue of the GSD IV patient. Additionally, PC expression is also elevated, enabling the initial reaction of gluconeogenesis. (c) In diabetes mellitus type 1 insufficient endogenous insulin secretion leads to upregulated gluconeogenesis.We have examined four different conditions in this study, namely GSD Ia, GSD IV, diabetes mellitus type 1, and ketosis due to ketogenic diet. Based on published findings and on our results, the four conditions are either linked to increased gluconeogenic flux or increased fatty acid synthesis as follows: (a) In GSD Ia glucose‐6‐phosphate accumulates and is shunted to glycolysis (and the pentosephosphate pathway). This causes increased flux through glycolysis, which leads to raised acetyl‐CoA and consequently malonyl‐CoA.4.2ACACA gene in the GSD Ia patient liver was also demonstrated in a GSD Ia rat model facilitated by the application of a G6PC inhibitor.de novo lipogenesis might not be the only mechanism for hypertriglyceridemia in GSD Ia patients,The above described increase of the PC transcript level, the ACACA gene also shows higher expression in the GSD IV patient liver, possibly due to an effect termed carboxylic acid cycle cataplerosis.In the situation of increased gluconeogenesis, as demonstrated in GSD IV by our results of raised key gluconeogenic enzymes on transcript level and as reported in diabetes mellitus type 2 patients,BTD transcript levels as well as BTD enzyme activity is increased.Eventually, our data suggests that as a consequence of the increased levels of the two biotin‐dependent carboxylases ACACA and PC, the demand for their cofactor biotin is raised and Future research should address other factors influencing the pathways discussed in this study, such as effects on intermediary metabolite levels and kinetic activity levels of gluconeogenic as well as fatty acid synthesis enzymes. It will also be of interest to evaluate the correlation between BTD enzyme activity, clinical severity, and episodes of hypoglycaemia in GSD patients. In addition, measurement of biotin levels may be of interest and might provide a rationale to consider supplementation of GSD patients with the biotin vitamin. However, whether a potential normalization of serum biotinidase activity is of any clinical relevance needs to be determined.Based on the results of our pilot study, we propose that markedly elevated serum BTD enzyme activity in hepatic GSD Ia and GSD IV reflects the imbalance of hepatic glucose metabolism that is caused by (a) accelerated gluconeogenesis and (b) increased fatty acid synthesis, pathways that involve the biotin‐dependent enzymes pyruvate and acetyl‐CoA carboxylase. Future studies aiming at confirming this hypothesis should involve a higher number of samples and could eventually expand the spectrum of BTD enzyme activity as a biomarker beyond GSDs to detect perturbed gluconeogenesis and lipogenesis, such as in diabetes mellitus.P Forny has no conflicts of interests to declare. P Burda has no conflicts of interests to declare. P Bode has no conflicts of interests to declare. M Rohrbach has no conflicts of interests to declare.P. Forny, P. Burda, M. Rohrbach contributed to the concept and design of the study. P. Forny collected patient information, performed RT‐qPCR experiments, interpreted results, and wrote the manuscript. P. Forny serves as the guarantor of the performed study. P. Burda performed biotinidase measurements and revised the manuscript. P. Bode performed histological studies. M. Rohrbach revised the manuscript and provided patient information.We have obtained written informed consent from the two GSD patients (or their respective caregiver) for their liver samples and blood sera to be analysed and their clinical information to be used. Ethics approval to conduct the study was granted by the Commission for Ethics of the Canton of Zurich (BASEC‐Nr. Req‐2017‐00800). All procedures were in accordance with the Helsinki Declaration of 1975, as revised in 2013. Control liver samples were obtained via the Centralized Tissue‐Biobank of the University Hospital Zurich, Switzerland, for which patients were consented by the biobank team for research usage of their samples. Biotinidase enzyme activity measurements of patients' sera of diabetes mellitus type 1, epilepsy patients on ketogenic diet and GSD Ia patients were performed as part of routine blood testing.Supp. Figure 1A. Glycogen content in liver homogenate assessed as grams per 100 g of liver tissue (ref range: 2.4‐6.4). B. GBE1 enzyme activity assessed in liver homogenate . C. Triglyceride levels pre and post liver transplantation (2 measurements per mean with a reference range of 0.5‐2.2 mmol/L). D. Liver function tests: ALT, alanine transaminase (reference range: <28 U/L); AST, aspartate transaminase (reference range: <35 U/L); GGT, gamma glutamyltransferase (reference range: <23 U/L). E. Urate levels in blood pre (n = 2) and post (n = 4) liver transplantation (reference range: 111‐353 μmol/L). F. INR, international normalised ratio (reference range: <1.2). G. Albumin in plasma (reference range: 35‐50 g/L). H. Biotinidase enzyme activity assessed in serum of case 1 (GSD IV patient) pre and post liver transplantation, ****: P < 0.0001 by one‐way ANOVA with Tukey multiple comparisons test. The vertical line in plots D, F and G signifies the time point of liver transplantation at the age of 21 months (Tpx).Click here for additional data file.


Kd) obtained frommultiplexed SBA assays correlate well with experiments performed on a larger scale,while the antigen consumption was reduced >100-fold compared to the conventional96-well plate format. Protein screening and characterization by SBAs is a rapid andreagent-saving analytical format for combinatorial protein engineering to addressspecificity maturation and cross-reactivity profiling of antibodies.The precise determination of affinity and specificity is a crucial step in thedevelopment of new protein reagents for therapy and diagnostics. Paradoxically, theselection of protein binders, e.g., antibody fragments, from large combinatorialrepertoires is a rapid process compared to the subsequent characterization of selectedclones. Here we demonstrate the use of suspension bead arrays (SBA) in combination withflow cytometry to facilitate the post-selection analysis of binder affinities. The arrayis designed to capture the proteins of interest (POIs) covalently on the surface ofsuperparamagnetic color-coded microbeads directly from expression cell lysate, based onSpyTag-SpyCatcher coupling by isopeptide bond formation. This concept was validated byanalyzing the affinities of a typical phage display output, i.e., clones consisting ofsingle-chain variable fragment antibodies (scFvs), as SpyCatcher fusions in 12- and24-plex SBA formats using a standard three-laser flow cytometer. We demonstrate that theequilibrium dissociation constants ( Typically, after theinitial selection by panning, additional labor-intensive screening of large numbers ofindividual library members is required to identify clones with the desired affinity andspecificity.4Paradoxically, even with a limited number of clones, the post-selection screening consumesmore time and resources than the primary screening of repertoires of billions of members bypanning selection. This post-selection screening is routinely performed in 96-wellmicrotiter plates using enzyme-linked immunosorbent assay (ELISA), either directly withphage display clones or in a format with soluble protein for more precisecharacterization.5 Screening by phage ELISA requires 10–25μg of protein in a 96- or 384-well microtiter plate.7 By contrast, only 500 ng of the target protein(coating a single microtiter well) is sufficient for one round of panning with1012 antibody-displaying phages.7 Thus, approximately20–50-fold more target protein is needed for the characterization of just one librarymember compared to the actual primary selection. For targets that are difficult-to-expressor synthesize, such as G-protein-coupled receptors (GPCRs)8 and marineciguatoxins9 the availability of the antigen may severely limit theextent of screening experiments. Miniaturization and multiplexing the primary screeningeffort would lead to considerable savings in time and material.Display technologies are powerful tools in protein drug discovery and have been widelyapplied to select antibodies and alternative scaffold proteins from syntheticlibraries.10 and allergen testing.11 SBAs typicallyconsist of microbeads that are encoded with different ratios of organic fluorophores,creating a collection of unique spectral bead “addresses”.12 A test panel for multiple biomarkers is obtained by conjugating each batch of addressbeads with a different capture antibody. The beads are subsequently pooled for sampleincubation, followed by the addition of target-specific detection antibodies. The detectionantibodies, in turn, are coupled to a universal fluorophore 13 and, consequently, multiple analytes can be quantitated in asingle flow cytometry experiment; the identity of the bead-bound analyte is determined byreading the fluorescence of the address bead, and the quantity of the analyte by reading thefluorescence of the detection antibody.As a result of their high multiplexing capacity and low sample volume requirements,suspension bead arrays (SBA) have become a popular choice in medical research, e.g., forcytokine14 and forfluorescence-based Western blotting.15 Despite the clear advantages over96-well plate ELISA screening, with respect to material savings and reduced manual liquidhandling, SBAs have not been widely applied to recombinant binder screening due to therequirement of a proprietary analytical multiplexing instrument . In a studyby Ayriss et al., the binding specificity of recombinant single-chain variable fragmentantibodies (scFvs) was analyzed in an 8-plex Luminex bead assay with bead-bound targetantigens using myc-tag for detecting the antigen-bound scFvs.16 In thisarrangement, each clone is individually examined against the eight targets. However,multiplexing binders instead of the antigens would reduce the number of screening samples,and the amount of antigen needed even further.More recently, bead arrays have been applied to proteomics research for profiling plasmaimmunoglobulins against selected target proteins17 making the purification ofproteins for covalent conjugation on sets of beads  superfluous.First, altogether 48 bead populations with unique optical signatures (referred to as theaddress bead set) were created. The optical addresses of unique bead populations provided areadout of the origin of the displayed protein  based on the bead size and the ratio of the two conjugated fluorophores onthe bead. When these beads are decorated covalently with the proteins of interest (POIs) viaSpyCatcher/SpyTag pairs,17 binding titrations can be carried out with astandard analytical three-laser flow cytometer that simultaneously provides quantitativeinsight into the function  and thesource location of the binder in question.In this work, SBAs are explored as a flexible tool for miniaturizing the primary screeningof antibody fragments in a multiplexed, flow cytometry-based analysis format usingparamagnetic microbeads. We designed a novel SBA set-up that obviates the need to purify thebinders prior to bead conjugation. To this end, we take advantage of the isopeptidebond-forming SpyTag-SpyCatcher protein pair,0.1  + 0.1% w/v Tween-20) and resuspendedin PBST0.05 (1 mL of PBS + 0.05% w/v Tween-20).Carboxylate-modified microbeads from three providers with different sizes were tested formultiplexing: (i) superparamagnetic polystyrene particles  from Microparticles GmbH , (ii) paramagnetic beads (meanØ 7.4 and 18.8 μm) from Spherotech , and (iii)superparamagnetic Dynabeads M-270 (Ø 2.8 μm) from Thermo Fisher Scientific. The beads with 5.18 and 10.31 μm diameter were further used for beadcoating . Toprepare the beads for covalent coupling with tamavidin-2-HOT-SpyTag (T2H-SpyTag), thebeads (24 mg) were washed with 4 × 1 mL of water containing Tween-20 (0.01% w/v), andthe carboxyl groups were activated to react with primary amines by resuspending the beadpellet in 0.5 mL of water containing N-(3-dimethylaminopropyl)-N′-ethylcarbodiimidehydrochloride (EDC)  and sulfo-NHS . The beads were incubatedfor 20 min at RT (23 °C) with rotation, interrupted by frequent vortexing, and washedonce with 1 mL of water. The beads were mixed with T2H-SpyTag , vortexed, and incubated overnight at RT with rotation.The reaction was quenched by incubating the beads in Tris-Cl  for 10 min with rotation. The beads were washed with 3 × 1 mL ofPBST0.1 and resuspended in 1 mL of PBST0.05. The observed dynamiclabeling range for FITC in 1 mL of carbonate buffer at pH 9  ranged from 0.02 μg of FITC/3 mg ofbeads to 28 μg of FITC/3 mg of beads.T2H-SpyTag-coated beads were divided into 3 mg batches and resuspended in carbonatebuffer . Fluorescein-5-isothiocyanate (FITC)powder  was freshly dissolved in dimethyl sulfoxide (DMSO),and the stock solution was further diluted in DMSO to create a series of master solutions(20× concentrates). The master solutions were mixed with 950 μL of bead batchesand immediately vortexed, resulting in the final FITC concentrations of 28, 3.2, 0.8, 0.4,and 0.02 μg/mL. The beads were protected from light and incubated overnight at RTwith rotation. The reaction was quenched by incubating the beads in Tris-Cl  for 10 min with rotation. The beads were washed twice with 1 mL ofPBST0.05 containing differentconcentrations of bio-BSA-ATTO-565. The synthesis of bio-BSA-ATTO-565 is described in theSI. To facilitate bead gating in the analysis phase, bio-BSA-ATTO-565 wasadded at different concentrations on every other FITC-bead batch according to increasingFITC intensity. The first series of beads was labeled with 0, 5, 40, and 200 nMbio-BSA-ATTO-565 and the second with 1, 10, 80, and 400 nM bio-BSA-ATTO-565. For labelingØ 10 μm beads, the first series of beads was labeled with 0.1, 1, 4, and 20 nMbio-BSA-ATTO-565 and the second with 0.5, 2, 8, and 40 nM bio-BSA-ATTO-565, respectively.The labeling reactions were protected from light and incubated for 15 min at RT withrotation. The beads were washed with 3 × 1 mL of PBST0.1 and resuspendedin 200 μL of PBST0.05. The dynamic labeling range for bio-ATTO-565 in 50μL of PBST0.05  was from 0.05 pmol of bio-ATTO-565/300 μg of beads to 20 pmol ofbio-ATTO-565/300 μg of beads.To add the second fluorophore gradient on the FITC conjugated beads, the bead suspensionswere split into 100 μL aliquots ,pelleted, and resuspended in 50 μL of PBSTSI for library design and selection). After phage display selection, thescFv genes were extracted from the phage input and output libraries (display was performedwith phagemid pHB32x) and cloned via SfiI sites into the pHBSC screeningvector to create scFv-SpyCatcher fusions. The screening repertoires were transformed intoα-Select Silver Escherichia coli strain (Bioline) for small-scaleexpression in 96-well plates. Colonies were picked, proteins expressed, and cells lysedwith a combination of lysozyme/benzonase  plates .An anti-digoxigenin (anti-DIG) scFv library was constructed on the parental clone 198C9using NNS–NNS site-saturation mutagenesis, and two rounds of phage displayselections were performed to obtain affinity-improved variants , washed once with 200 μL ofPBST0.1, and resuspended in 50 μL PBST0.05. The beads werepooled into one microcentrifuge tube by combining samples from one row of a 96-well platefor 12-plex assay (input and output A) and two rows for 24-plex assay (output B),respectively. The supernatant was removed from the bead collection tube and the beadpellet resuspended in 1 mL of 5 nM antigen solution, DIG-dsDNA-Cy5, inPBST0.05. The mixture was incubated for 1 h at RT with rotation, washed twicewith 1 mL of PBST0.1 and resuspended in 200 μL of PBST0.05 forflow cytometry on a BD FACScan .The scFv-SpyCatcher fusion proteins thus expressed were attached to T2H-SpyTag beads bymixing 50 μL of the lysate supernatant with 5 × 10E.coli cells were released into cell supernatant as described above and mixedwith 2 × 105 T2H-SpyTag beads for bead conjugation. Following theincubation and washing steps as described above, the beads, coated with differentscFv-SpyCatcher variants, were pooled and split into equal aliquots for adding theDIG-dsDNA-Cy5 antigen dilutions provided in 200 μL or 2 mL volumes inPBST0.05. The antigen dilution series was incubated with the beads at leastfor 2 h at RT with rotation. The beads incubated with different antigen concentrationswere processed one by one for flow cytometry. The beads were collected using a magnet, andthe supernatant was removed. The beads were resuspended in 200 μL ofPBST0.05 and immediately analyzed with a FACScan Cytek. The medianfluorescence intensity (MFI) values for each gated bead population at each antigenconcentration was obtained with FlowJo10 software, exported to GraphPad Prism6 (GraphPadsoftware), and the data were fitted to Y is the fluorescence,Bmax is the maximum specific binding in the same units asY, X is the antigen concentration, andKd is the equilibrium dissociation constant. Thefluorescence signal arising from the nonspecific binding of the labeled antigen to thebeads was obtained from intra-assay control beads that were not coated withscFv-SpyCatcher. This nonspecific signal was subtracted from the total signal obtainedfrom scFv-SpyCatcher beads prior to fitting to the one site-specific binding equation.scFv-SpyCatcher fusions (100 μL) expressed in α-Select Silver 19 Our objective was to enablespecific covalent attachment of the recombinant proteins directly from the cell lysate tothe beads, which was achieved by implementing spontaneous covalent coupling viaSpyTag-SpyCatcher technology, which has been shown to perform robustly under differentbuffer conditions.17 To this end, the SpyTag was produced as aC-terminal protein fused to tamavidin-2-HOT, a heat-stabilized protein tetramer bindingbiotin with high-affinity,21 and containing seven surface-exposed lysine residues in each subunit,which provide anchors for efficient chemical conjugation to carboxyl-modified beads viacarbodiimide-mediated amide bond formation. Consequently, recombinant proteins of interest(POI) expressed as POI-SpyCatcher fusions in E. coli were covalentlyattached to microbeads coated with the SpyTag-fused carrier protein directly in theexpression lysate. Optical address signatures for tracking the source locations of theclones were created on the beads with combinations of two fluorophores at discernibleintensities. The first fluorophore gradient was attached via EDC cross-linking chemistryto target the remaining surface-exposed amine groups of tamavidin on the bead, and thebiotin-binding site of tamavidin-2-HOT was taken as the second orthogonal attachment siteto include the second spectral coding dimension.E. coli as the larger scFv-SpyCatcher (38.2 kDa) fusionprotein than as scFv-SpyTag (30.2 kDa) fusion protein , which supported the use of SpyTag as the anchor point on themicrobead. Two spectrally distinguishable dyes, the yellow laser excitable biotinylatedATTO-565  and the blue laser excitablefluorescein isothiocyanate  were used to create the address bead array.In this arrangement, Cy5  was reserved for thedetection of successful antigen-binding function. Schematic workflow for SBA screening isillustrated in Figure S2A) and that anti-DIG ScFv-SpyCatcher was stably bound on the SpyTagbeads, as detected by the fluorescence of the labeled antigen, DIG-dsDNA-Cy5, on the beadsurface .Our model scFv anti-DIG 180B1 used for studying the SBA concept expressed better in theperiplasm of  protein S1, whichFigure S3A), while the smallest bead size of Ø 1.43 μm did notform a focused spot on the scatter plot but rather overlapped with other bead populations. As Ø 7.4 μm beads overlapped with Ø 10 μmbead sizes and the bead count of the Ø 19 μm stock was at least 5-fold lowerthan that of the other stocks. Only Ø 5 and 10 μm beads were employed insubsequent experiments described below.The ability of the bead size to provide an additional dimension for multiplexing wasexplored. Six stocks of carboxyl-activated paramagnetic beads (Ø 1.43–18.8μm) were analyzed by flow cytometry (FACSCAN Cytek). Focused spots for unambiguousgating were observed with bead diameters between 3 and 18.8 μm  of each subpopulation were coated with anti-DIGscFv-SpyCatcher and labeled with DIG-dsDNA-Cy5. Flow cytometry analysis revealed thatfunctional signals were obtained from all bead populations 4. HoweveThe Cy5 signal was also measured of beads that were not coated with scFv-SpyCatcher, yetincubated, with the Cy5 antigen. This background signal on the RedFL1 channel was thehighest on the brightest ATTO-565 beads (60 ± 18 median RedFL1 au) compared to thenon-labeled beads (4.9 ± 1.3 median RedFL1 au). The variation in the backgroundsignal among the address beads was, however, considered insignificant for primaryscreening of clones as at least 240-fold signal-to-background ratios were obtained withall bead populations. The increased background fluorescence at the highest ATTO-565intensity is speculated to be caused by nonspecific binding of the DIG-dsDNA-Cy5 to thefluorophore beads, as there is virtually no spectral overlap between the selectedfluorophores and the chosen detection channels. We speculate that a more uniformfunctional signal could be attained by internally color-coded microbeads.In the assembly construct 1, a stabSI for details of parental clone origin, phage display library construction,and phage display cycles). We were especially interested in obtaining affinity-improvedbinders recognizing digoxigenin as a DNA conjugate, which could be used as a generaldetection tool for DIG-labeled probes. The phage display selection was performed under twodifferent conditions: either at 20 pM (output A) or at 1 pM (output B) antigenconcentration in solution followed by capture on paramagnetic streptavidin beads. Afterselection, the enriched scFv clones and controls were cloned into the screening vectorpHBSC creating scFv-SpyCatcher fusion proteins for periplasmic expression in 96-wellplates  and SBA screening .The suitability of this suspension bead array technology for multiplexed analysis ofrecombinant binders was demonstrated by screening anti-DIG scFv repertoires foraffinity-improved binders. A site-saturation library of scFv clone 198C9 was constructedand enriched by two rounds of phage display against biotin-dsDNA-digoxigenin . The error caused by address bead labeling to the antigen-bindingsignal was minimized (to ≤2-fold) using a limited set of color-coded beads , leaving thehighest fluorescent ATTO-565 bead sets out. Furthermore, stringent gating on the highestdensity of address bead populations  effectively mitigates the possibility of including members of theneighboring bead population in the antigen-binding analysis. The observation that thefrequency of binders increases as a result of the phage display selection (Table S3) suggests that SBA is a suitable platform for discriminating targetbinding clones from nonbinders. However, a precise ranking of the clones by affinityrequires a more quantitative analysis at different antigen concentrations because thesignal level in single-point screening is dependent both on the expression level ofindividual clones and the manual adjustment of the fluorescence intensities in a flowcytometer prior to analysis. Therefore, the eight most positive clones from the primaryscreening of the input and output repertoires were selected for a secondary screeningassay in which the developed SBA platform was applied for obtaining complete antigenresponse curves. These ligand (antigen) saturation binding experiments, in which theextent of the binding reaction was determined as a function of the concentration of theligand (antigen), were also used for the determination of binding dissociation constantsat equilibrium (Kd).The primary SBA screening data of the three expression plates are shown in Table S4). It was also observed that the primary screening results wereadequate to distinguish binders from nonbinding clones but displayed only a roughcorrelation with antigen-binding affinities that were later obtained by biophysicalanalysis. For example, the antigen-binding signals (MFI RedFL1) obtained in the primaryoutput B  by capturing the selected scFv-SpyCatcher clones from thelysate on the address beads, pooling the clones together, and splitting them again in tenaliquots for antigen labeling. The aliquots were supplemented with increasingconcentrations of DIG-dsDNA-Cy5 and incubated for 2 h to allow the binding reaction toreach equilibrium, followed by flow cytometry analysis. In the decoding step, the beadpopulations were gated based on their BluFL1/YelFL2 fluorescence ratio 7A, allowoutput B 6 were ovKd determination was studied in repeated experiments bycoupling the binders from the lysate each time on spectrally different address beadpopulations. Similar response curves and Kd values wereobtained for each scFv-SpyCatcher clone tested, irrespective of the particular addressbead population used for coupling . The equilibrium binding assay was also studied by providing aDIG-dsDNA-Cy5 gradient in 200 μL and 2 mL volumes to assess possible antigendepletion effects. Incubating the suspension bead array in 2 mL volumes resulted in higheraffinity values for the parental clones 180B1 and 198C9  and the affinity-improved clones . This indicates that maximizing thevolume of the provided antigen dilution helps to minimize the antigen depletion effects.Depletion is a known factor to be taken into account for saturation bindingexperiments,22 especially if the dissociation constants to beanalyzed are in low nanomolar or picomolar ranges. In a typical primary hit screeningassay, the antigen depletion effect is not a significant factor for assay interpretationand kinetic binding analysis  of a parental clone 198C9 (Kd: 21.4± 9.8 nM) and affinity-improved clone 203A2 (1.2 ± 0.6 nM), and found to be ingood agreement with the saturation binding experiments.Over 5-fold improvement in the digoxigenin binding affinity was observed with the outputclones 203A2, 204C6, 203E12, and 204G12  without significant loss inperformance formance 5A.Kd) at equilibrium by ligand saturationbinding experiments. In this multiplexed SBA format, the ligand binding is simultaneouslyexamined for all clones of interest in single series of antigen dilutions minimizingexperimental errors. Furthermore, differences in the maximum functional signal across thebead array (due to the surface-labeling strategy) do not play a role in saturation bindinganalysis as the antigen-binding signals for each clone are recorded on the same spectraladdress bead batch at different antigen concentrations. Most importantly, in flow cytometry,the fraction of the bound fluorescent analyte at equilibrium is measured directly on thebead surface. Therefore, washing steps (that may disturb the equilibrium) can be minimizedor altogether omitted. In contrast, in ELISA or other methods requiring solid-phaseseparations, the antigen-binding equilibrium may be severely disturbed by numerous washingsteps depending on the dissociation rate of interaction.25Suspension bead technology was applied to provide data for the determination of bindingdissociation constants  and reducingthose for reagents by two orders of magnitude will enable extensive research in syntheticbiology, proteomics, medicine, and therapy, where the lack of well-characterized reagentslimits progress.


Our preliminary results using a close genetic relative of SAR-CoV-2, HCoV-NL63, showed the virucidal efficacy of photoactive TNPs deposited on glass coverslips, as examined by quantitative RT-qPCR and virus infectivity assays. Efforts to extrapolate the underlying concepts described in this study to SARS-CoV-2 are currently underway.The newly identified pathogenic human coronavirus, SARS-CoV-2, led to an atypical pneumonia-like severe acute respiratory syndrome (SARS) outbreak called coronavirus disease 2019 (abbreviated as COVID-19). Currently, nearly 77 million cases have been confirmed worldwide with the highest numbers of COVID-19 cases in the United States. Individuals are getting vaccinated with recently approved vaccines, which are highly protective in suppressing COVID-19 symptoms but there will be a long way before the majority of individuals get vaccinated. In the meantime, safety precautions and effective disease control strategies appear to be vital for preventing the virus spread in public places. Due to the longevity of the virus on smooth surfaces, photocatalytic properties of “self-disinfecting/cleaning” surfaces appear to be a promising tool to help guide disinfection policies for controlling SARS-CoV-2 spread in high-traffic areas such as hospitals, grocery stores, airports, schools, and stadiums. Here, we explored the photocatalytic properties of nanosized TiO The novel coronavirus, 2019-nCoV, SARS-CoV-2 causative agent of the pneumonia-like COVID-19 pandemic, has infected more than 77 million people globally , whereasOne of the main reasons for the observed surge in COVID-19 cases could be attributed to multiple modes of viral dissemination, including direct contact with infected person through respiratory droplets generated during coughing, sneezing, or talking, and indirect contact with contaminated surfaces or objects used by infected persons ,7. AnalyCOVID-19 pandemic brought the importance of environmental hygiene and cleaning practices to prevent viral spread. COVID-19 virus was detected on multiple surfaces in the near proximity to patient’s bed as well as in the sink and the toilet bowl . Investi2-nanoparticles or TNPs) have been proven to act as effective photocatalysts and shown to have both bactericidal and virucidal properties [2 has been demonstrated against influenza virus [2 against human coronavirus remains largely unknown. The best way to control the spread of the COVID-19 pandemic is to reduce the viral spread. Strategies to control the spread include use of hand-sanitizers, facemasks, physical distancing, self-isolation of patients, regular cleaning of shared surfaces, and frequent use of disinfectants. Since fomites are one of the ways of virus transmission, it is crucial to identify methods that could reduce virus survival on the surfaces. In this regard, generation of self-disinfecting surfaces based on the advanced oxidation processes/photocatalysis initiated by ultraviolet (UV) radiation will be of great importance. UV irradiation-based methods are powerful disinfectants due to its germicidal ability and have shown effectiveness in reducing infections caused by pathogens. Additionally, nanosized titanium dioxide  among infected individuals. This virus is a Bio Safety Level 2 pathogen and has high similarity  with SAR2.Vero E6 and HEK293L cells  were maintained in Dulbecco’s modified Eagle medium  supplemented with 10% fetal bovine serum , 2 mM L-glutamine, 25 U/mL penicillin, and 25 μg/mL streptomycin. Cells were grown at 37 °C in a humidified chamber supplemented with 5% COCoronaviridae, genus alphacoronavirus. HCoV-NL63 was obtained from BEI Resources  and propagated in Vero cells by infecting the Vero cell monolayer with HCoV-NL63 for 2 h. Unattached virus was removed by washing followed by addition of fresh medium. Supernatant containing virus was harvested after 7 days, cell debris were removed by centrifugation before storing at −80 °C. TCID50 of the harvested virus was determined indirectly by RT-qPCR using a standard curve generated as described later (2.6. RNA extraction and qPCR). By this method the harvested virus showed to have 2.1 × 106 TCID50/mL. An aliquot (100 µL) of this supernatant (HCoV-NL63) was used for extracting RNA and Reverse Transcriptase qPCR (RT-qPCR) for quantifying viral copies. All the assays were conducted under biosafety level 2+ (BSL-2+) containment.HCoV-NL63 strain is a human coronavirus and belongs to the family 2 was a generous gift from Degussa Corporation . TNPs suspension (300 ug/mL) was prepared in deionized (DI) water and vortexed. An aliquot (600 μL) of TNPs was placed on the clean UV-treated glass coverslip and dried at 60 °C, which resulted into a semi-transparent coating. TNPs-coated coverslips were stored at room temperature (RT) until use.TiO5 TCID50) of HCoV-NL63 virus was placed on TNPs-coated and uncoated coverslips  and exposed to the USHIO Germicidal Lamp , which generates UV-C light  for various time points. The UV light source was placed 76 cm, 50 cm, and 10 cm from the bottom of the wells containing coverslips, which applied, calculated 2900 µW/cm2, 4300 µW/cm2, and 13,000 µW/cm2 energies, respectively (where µW = 10−6 J/s). Virus inactivation was also analyzed under different humid environments, 45%, 65%, and 85% relative humidities (Rh), by applying a known amount of virus on TNPs-coated and uncoated coverslips under indicated humidies. The humidity conditions were generated by using humidifier placed inside the biological safety cabinet (BSL2) and measuring the humidities with AcuRite Indoor Thermometer and Hygrometer with Humidity Gauge . Humidity range (45–85%) was selected based on the average (45%) standard in-door humidity recommendation by the Center for Disease Control and Prevention (CDC) and the outdoor humidity registered in many parts of the world including the coastal areas, which have humidities as high as 85% [An aliquot  before washing the dead and disintegrated virus and adding fresh medium. These cells were incubated for 48 h at 37 °C in a humidified chamber supplemented with 5% CO2. Since an infectious virus enters the cells to replicate and produce viral genomic RNA and proteins, detection of intracellular viral RNA and viral proteins confirms the presence of live virus. Infected cells were harvested for the extraction of total RNA and the detection of intracellular viral genomic RNA through RT-qPCR (2.6. RNA extractions and qPCR). Viral proteins were detected through immunofluorescence assay in which infected cells were fixed using 3:1, methanol: acetone and stored at −80 °C until used. Cells were permeabilized with 0.1% Triton X-100 for 30 min, washed (3×), and blocked  for 1 h at RT. Cell monolayers were washed again (3×) and incubated with polyclonal rabbit anti-CoV spike antibody  for 1 h at RT, followed by incubation with goat anti-rabbit Alexa Fluor 488 , secondary antibody for 1 h at RT in the dark. Finally, the nuclei were stained with TO PRO-3 . Coverslips were mounted on the glass slides using antifade and the slides were examined using Carl Zeiss LSM 780 confocal laser-scanning microscope. Control or the UV-treated viruses were added onto the human embryonic kidney cells for 2 h . Several 10-fold dilutions of this isolated genomic RNA  were used for generating a standard curve. The sensitivity of qPCR assay was 3.2 virions in the sample based on the quantitation of RNA present in HCoV-NL63 virus stock from the BEI Resources.HEK293L cell monolayer infected with control or UV-treated virus were subjected for total RNA extraction extraction using Trizol reagent  by directly adding the reagent onto the cells for lysis according to the manufacturer’s recommendation. An aliquot of RNA (1 μg) was used for synthesizing the cDNA . cDNA (2 μL) was used for the relative quantification of viral genome RNA in a qPCR assay . Primers used in this study are NL63-SF 5′-GTGCCATGACCGCTGTTAAT-3′ and NL63-SR 5′-GCGGACGAACAGGAATCAAA-3′. For generating a standard curve to estimate the viral copies, 100 μL of NL63  stock with known amounts of HCoV-NL63 was used for total RNA extraction and cDNA synthesis. An aliquot (2 μL) of HCoV-NL63 cDNA used for RT-qPCR was equivalent to 320 copies of HCoV-NL63  and the HCoV-NL63 virus (100 μL suspension) was applied onto the coverslip placed inside the wells of 12-well plate. The plate containing HCoV-NL63 viruses was exposed, after removing the lid, to the UV light inside a BSL-2 biological safety cabinet for 0, 1, 5, 10, and 30-min. UV-exposed virus was collected for the evaluation of virus inactivation by direct genomic RNA quantitation (RT-qPCR) and infectivity assay on HEK293L monolayer. Presence of the viral genomic RNA and its integrity was determined using qPCR as it was previously used for the detection of SARS-CoV-2 in environmental samples ,22,23. G2 surface area)  onto the coverslip (2.5 cmce area) . HCoV-NLce area) A. We detected a slight variation in the number of recovered HCoV-NL63 copies from no UV treated samples (0 min UV-exposure), which could be because of the anti-microbial property of TNP even in the absence of UV exposure. Not surprisingly, the number of intact HCoV-NL63 genomic RNA was almost to the background levels at longer UV-exposures on both TNP and control surfaces A. To demWe wanted to determine whether TNP coating is effective in inactivating viruses that have been dried after falling onto the surfaces, to emulate the most common settings where respiratory/sneeze droplets coming out of the infected individuals are deposited on surfaces. To evaluate whether HCoV-NL63 virus fallen on the TNP-coated surface can be inactivated even after drying, we added a small droplets of medium containing a known amounts of HCoV-NL63 on TNP-coated surface as well as on the control surface, without TNP coating. These droplets were allowed to dry, which took approximately 20 min at room temperature and normal humidity (45% RH) to visibly dry, followed by exposing them to the UV light for indicated times . HCoV-NL2 and 4300 µM/cm2, respectively. Expectedly, exposing the surface from a distance of 50 cm (4300 µM/cm2) disintegrated the virus genomic RNA more effectively with only a limited number of detectable viral genome at 1 min exposure. Importantly, TiO2 significantly decreased the exposure time to 0.5 min to achieve almost complete disintegration of the viral genome  amount of HCoV-NL63 virus on a normal or TNP-coated coverslips before exposing them to UV source from a distance of 10 cm or 50 cm, which provided a power of 13,000 µM/cml genome A. Furthesurfaces A. 2) for indicated times. Our results showed that an increase in the relative humidity enhanced HCoV-NL63 viral genome degradation with UV light on both control and TNP-coated surfaces  to cover the range of humidities, from dry to humid environments, to UV treat the virus on TNP-coated or control surfaces. The surfaces with virus were exposed to the UV-light ” [2●‾ and OH● radicals [2O2. The TNPs can be used in suspension, liquids, or immobilized on surfaces, also referred to as a “self-cleaning“ surfaces [2 was proposed to have an antiviral effect, targeting both DNA and RNA viruses, airborne and bloodborne pathogens. The photocatalytic titanium apatite filter was earlier shown to inactivate SARS coronavirus up to 99.99% after 6 h exposure [2 coating to efficiently inactivate human coronavirus with UV light through very brief exposure highlights the importance of TNP coatings on publicly used surfaces. Although, UV by itself is used for viral inactivation including the trains of public transport systems but the efficacies are not well established. Since our assays showed augmentation of virus inactivation, HCoV-NL63 by the TNPs coatings under UV exposure, we propose that TNP coatings can enhance virus inactivation including SARS-CoV-2 on surfaces most frequently exposed with COVID-19 patients. HCoV-NL63 and SARS-CoV-2 both cause acute respiratory diseases and have similar virion structure (HCoV) [Nanosized TiOl growth ,30. The  (e−h+)” . The freradicals . These Rsurfaces . This fesurfaces ,35,36,37exposure . Our asse (HCoV) ,39, and 2 can facilitate virucidal effect of the low intensity UV light, which could have important practical applications. As TiO2 can maintain antiviral efficacy of the UV light at the lower intensity, it will help in reducing the potential harmful effect of radiation exposure [Various factors are shown to affect virus resistance to UV light exposure, including UV dose, humidity, and the surface material ,40. It wexposure .2 nanoparticles effectively inactivated HCoV-NL63 under all three tested relative humidity conditions, which are within the standard for the indoor conditions (30–60%) [2 nanoparticles retain virucidal efficacy even at very high humid condition (85% relative humidity), confirming a broader use of TNPs coatings on outdoor surfaces. The environmental humidity was suggested to play a role in the airborne virus spread [2-coated surfaces have a viral inactivation property even on the virus that has been dried. Therefore, the in-vitro viral inactivation property of the TiO2 could be used for preventing the persistence of virus in dry droplets on surfaces.Interestingly, TiO(30–60%) . We haves spread ,47,48. Is spread ,49. A prs spread . A studys spread . A studys spread  showed ts spread . It appes spread . In addi2 nanoparticles act as photocatalyst due to their specific energy structure. UV-irradiation of nanosized TiO2 results in the excitation of electron leading to the formation of a specific “electron-hole (e−-h+)” pair and generation of reactive oxygen species (ROS). The electron holes (h+) catalyze oxidation processes and convert water/hydroxide molecules to peroxide/hydroxyl radicals, whereas electrons (e−) induce reduction reactions and react with molecular oxygen to generate superoxide radicals.A presumed model to understand the underlying mechanism involved in the photocatalytic inactivation of HCoV-NL63 has been described in These resulting ROS have the ability to inactivate the virus through oxidative damage.2 is non-toxic and used as a food additives [Since TiOdditives , althougdditives . Additiodditives ,57. In fdditives . This isIn conclusion, the photoactive TNPs have strong virucidal effect, which are preserved under different environmental condition. In most cases, the genomic copies of human coronavirus NL63 from TNP-coated surface was undetectable within 1 min of UV exposure, which was also confirmed by cell infectivity assay. In an in-vitro virus inactivation assay, when the detection of viral RNA by the RT-qPCR is negative (or below the detection limit) it can be concluded that viral genome has been completely disintegrated. However, in the case of a positive RT-qPCR results, viral genome in the region of PCR amplification may be intact but fragmentation in other parts of the viral genome as well as inactivation of surface glycoprotein could make the virus non-infectious. Therefore, a virus infectivity assay must be performed to determine the levels of infectious virus. However, lack of a RT-qPCR signals can be construed as total inactivation of the virus though disintegration of viral genomic RNA.The work has been submitted for provisional patent filling consideration.


The recently emerged coronavirus in Wuhan, China has claimed at least six lives as of January 22 and infected hundreds if not thousands of individuals. The situation has drawn international attention, including from the virology community. We applaud the rapid release to the public of the genome sequence of the new virus by Chinese virologists, but we also believe that increased transparency on disease reporting and data sharing with international colleagues are crucial for curbing the spread of this newly emerging virus to other parts of the world. It is now clear that the mysterious respiratory illness in Wuhan is caused by a new type of coronavirus distantly related to the SARS coronavirus (SARS-CoV) ,2. We apViruses emerge and re-emerge globally without consideration for borders. In the recent past, we have witnessed outbreaks of SARS, Ebola, Chikungunya, and Zika . With eaThe international virology community, which includes many Chinese American virologists residing in North America, is concerned about this virus. It is imperative that animal reservoirs that transmitted the virus to humans be identified as quickly as possible to aid in control of this disease. It is thus urgent that the results of animal testing from the seafood market in Wuhan, where the virus was initially isolated, be released as soon as possible. This is particularly crucial given the rapidly approaching Lunar Chinese New Year, which will take place on 25 January 2020, during which tens of millions of people will travel, and large amounts of animal meats, some of which may contain this virus, will be consumed. It is noteworthy that recently the local health administration in Wuhan issued guidance regarding the prevention and treatment of the pneumonia caused by this coronavirus . It is aViruses spread irrespective of borders—they jump from animals to humans, and they move from one country to another. Controlling the spread of emerging and re-emerging viruses requires international efforts and collaboration. We anticipate that scientific data and reagents will be shared publicly and fairly, and most importantly, that the scientific collaborations between the US and China, including the study of emerging viruses and infectious diseases, will continue unabated despite some turmoil in other aspects of the US-China relationship.