Zika virus (ZIKV) is an arthropod-borne virus that was first isolated in 1947. Since then, multiple outbreaks of ZIKV have been reported in different countries. It is transmitted mostly by Aedes mosquitoes, and the symptoms of fever, joint pain, red eyes, headache, and maculopapular rash closely resemble chikungunya and dengue. The most severe complications of ZIKV infection include the risk of microcephaly and other congenital brain anomalies in infected pregnant women. It is a condition that can be easily prevented with proper education. In this review, we focussed on different aspects of the recent outbreaks of Zika virus disease, including its epidemiological background, transmission routes, presentation, as well as diagnosis and prevention. Zika virus (ZIKV) is an arbovirus that was first isolated from a rhesus sentinel monkey in Kampala, Uganda, in 1947 . In 2007The main complication of Zika virus infection is the risk of microcephaly and other congenital brain anomalies in infected pregnant women. The World Health Organization (WHO) raised the alarm for this condition, as most cases of microcephaly and defects in newborns from Zika virus disease were noted in economically deprived countries. The organization emphasized that it is vital to properly educate the public on preventive measures to avoid a similar occurrence in the future.MethodWe conducted a review of the different aspects of the recent outbreaks of Zika virus disease, including an epidemiological study, an investigation of the transmission routes, the signs and symptoms of the disease, as well as the diagnosis and prevention of Zika virus disease. A search was executed through PubMed and Google Scholar. The keywords used were: “Zika virus disease,” “Epidemiology of Zika virus disease,” “mortality and morbidity of Zika virus disease,” “transmission of Zika virus disease,” “Prevention of Zika virus disease,” and “Pregnancy and Zika virus disease.” The timeline was set from January 1950 to January 2018.What is Zika virus?ZIKV is described as an arthropod-borne virus that belongs to the Flaviviridae family consisting of the Flavivirus genus and Hepacivirus genus. The former consists of Zika virus, yellow fever virus, West Nile virus, and dengue virus, while hepatitis C virus forms part of the Hepacivirus genus. It is a positive, single-stranded ribonucleic acid (RNA) virus with an envelope making it closely related to the Spondweni virus . Two maiHow is Zika virus transmitted?Zika virus has different modes of transmission. The Aedes mosquito bite is the most common one, and it involves different families of Aedes: A. aegypti, A. albopictus, A. polynesiensis, A. vittatus, A. unilineatus, and A. hensilli among many other previously reported subtypes . VerticaWhat is the pathogenesis of Zika virus?Zika virus most commonly follows the sylvatic transmission cycle. It is classified as an arbovirus that is transmitted through one vertebrate to another by a mosquito bite. The virions exist as immature (non-infectious), mature (infectious), and fusogenic (host membrane binding) states. Humans are incidental hosts in the lifecycle of the virions. Monkeys and apes can also serve as host to the virions, while some studies have found that sheep, elephants, and goats had antibodies against Zika, suggesting possible host states as well .The cycle starts when an Aedes mosquito ingests blood containing Zika virus after biting an infected person. The virus starts replicating in the epithelial cells of midgut and goes to the salivary glands of the mosquito. After an incubation period of 10 days, the saliva becomes infected, making the mosquito a vector for infecting a human. Upon entry into the human skin, the virus infects the dermal fibroblasts that serve as receptors for attachment of Zika virus . UpregulWhat are the clinical symptoms of Zika virus disease?Misdiagnosis of Zika virus disease is very common because signs and symptoms in previously healthy patients resemble chikungunya and dengue. After an incubation of three to 12 days, Zika fever appears . The mosThe complications of Zika virus in pregnant women include microcephaly and other brain malformations -19. InfeHow to diagnose and treat Zika virus disease?Diagnosis of Zika virus disease relies mostly on the resources available in a country. The classic presentations and the exposure via either residence in or travel to a high-risk area or even contact with an infected person should raise suspicions. The Centers for Disease Control and Prevention (CDC) highly recommends testing for Zika virus in patients who had recent exposure and have presented with recent symptoms of Zika, pregnant women with possible Zika virus exposure, asymptomatic pregnant women currently exposed to Zika virus, pregnant woman with possible Zika virus exposure having been diagnosed with possible congenital Zika virus infection via prenatal ultrasound and for asymptomatic pregnant woman with no ongoing but recent exposure to Zika. However, Zika virus testing is not recommended as preconception screening or in non-pregnant asymptomatic individuals.Other recommendations by the CDC are as below:Any non-pregnant symptomatic patient should undergo a serum and urine testing for Zika virus ribonucleic acid (RNA), nucleic acid testing (NAT) and Zika virus and dengue virus immunoglobulin M (IgM) testing in serum. NAT is most useful when samples are collected within two weeks of symptom onset. Zika virus and dengue virus IgM should also be tested when the NAT sample collection is done. The IgM levels can be tested as well after 14 days of symptom onset while NAT is not recommended after two weeks of symptom onset.For a symptomatic pregnant woman, it is advised to test for serum and urine NAT and Zika virus IgM in serum up to 12 weeks of symptom onset. If the patient’s serum and urine NAT are positive, then it should be considered as an acute maternal Zika virus infection even if the IgM result is negative. If either serum or urine NAT is positive but not both, and Zika IgM is positive, it should be diagnosed as acute Zika virus infection. If both serum and urine NAT are negative and IgM is positive, a plaque reduction neutralization test (PRNT) should be performed on the patient.Any asymptomatic pregnant woman with current Zika virus exposure has been tested during pregnancy. NAT testing ought to be done during her first initial prenatal visit, followed by two more testing during pregnancy with two non-consecutive visits. IgM serology is not useful and not routinely recommended especially if the patient has previously been confirmed for Zika virus infection. If any of the NAT tests is positive during pregnancy, no further NAT testing should be done.If a woman is asymptomatic and pregnant and was recently exposed to Zika virus but currently not in direct exposure, testing should be considered only on a case-by-case basis and the gap between the last exposures should be considered. Any pregnant woman with possible exposure to Zika and diagnosed with congenital Zika virus infection via prenatal ultrasound should be tested for NAT and IgM on serum and urine as described for symptomatic pregnant women. More invasive procedures and testing such as amniocentesis, placental, and fetal tissues testing can be considered if required .The current treatment protocol for Zika virus infection involves mostly symptomatic care. Patients are advised to have proper bed rest and maintain hydration. Any pain or fever can be treated via acetaminophen. It is also recommended to avoid aspirin or other non-steroidal anti-inflammatory drugs before dengue is properly ruled out due to increased risk of bleeding associated with the dengue .How to prevent the spread of Zika virus?Since Zika virus is spread mostly by the Aedes mosquitoes, preventive measures should focus on eliminating them. The mosquitoes bite during both the day and night, therefore, the use of mosquito repellents that are safe and effective for pregnant, breastfeeding women, or even the elderly should be considered. If someone is using a sunscreen, it is advised to first apply the sunscreen and then apply the mosquito repellent. The application of mosquito repellents in babies younger than two months of age is not advised. It is also advised to avoid applying any repellent close to any child’s eyes or mouth. Furthermore, the children should be taught to avoid touching their eyes or mouth after being sprayed on a particular body part. The use of oil of lemon eucalyptus (OLE) or para-menthane-diol (PMD) is not advised for children younger than three. Other preventive measures include the proper use of mosquito nettings and long-sleeved shirts and long pants whenever possible. At home, it is advised to prevent mosquitoes from breeding by getting rid of any items that can be a source of stagnant water such as flowerpots, trash containers, and tires. It is also recommended to avoid sleeping outdoors and limit outdoor activities ,23.Any plans to travel to high-risk areas during pregnancy should be postponed . Since ZThe key to the prevention of Zika virus infection in underdeveloped countries involves mostly the proper education of the public. At present, there are no vaccines or prophylactic treatments available for the disease. While several mice studies have shown promising results, it will take a few more years before one is available safely. Until then, most countries should focus on prevention, early detection in pregnant woman and proper management, as well as contact tracing to lower contamination risk, and prevent an outbreak in the future.While Zika virus is not deadly, the complications associated with it, especially in pregnant women and children, can compromise quality of life. It can easily be prevented with proper measures and health management. With yearly outbreaks globally, all health groups should devise a proper plan ahead of peak season.


Offering a financial incentive (‘Money for Medication’) is effective in improving adherence to treatment with depot antipsychotic medications. We investigated the cost-effectiveness in terms of medical costs and judicial expenses of using financial incentives to improve adherence. The effects of financial incentives on depot medication adherence were evaluated in a randomised controlled trial. Patients in the intervention group received €30 a month over 12 months if antipsychotic depot medication was accepted. The control group received mental health care as usual. For 133 patients outcomes were calculated based on self-reported service use and delinquent behaviour and expressed as standard unit costs to value resource use.The financial incentive resulted in higher average costs related to mental health care (€449.6 versus €355.7). and lower medical costs related to other healthcare services (€52.0 versus €78.4). Relevant differences in social costs related to delinquent behaviour were not found. Although wide confidence intervals indicate uncertainty, incremental cost-effectiveness ratio’s (ICER) indicate that it costs €2080 for achieving a 20% increase in adherence or €3332 for achieving over 80% adherence. In sum, offering money as financial incentive for increasing compliance did not lead to an overall cost reduction as compared to care as usual.Trial registration NTR2350, 01 June 2010The online version of this article (10.1186/s13104-018-3747-1) contains supplementary material, which is available to authorized users. Adherence to treatment with antipsychotic depot medication is associated with remission from symptoms and improved social outcomes . Yet 25%Therefore, we investigated the medical and judicial costs after offering financial incentives to achieve better adherence. In a trial studying the effects of financial incentives on depot-adherence and psychosocial outcomes , we founThe effect of financial incentives on depot medication adherence was evaluated in a randomised controlled trial: 169 patients with a psychotic disorder were randomised to intervention or control groups, stratified by treatment site, sex, comorbid substance-use disorder, and medication compliance . PatientThe Treatment Inventory Cost in psychiatric patients (TiC-P)  is a freThe Self-Reported Delinquency questionnaire (SRD) provides an account of a wide range of illegal acts and has been widely used, although item difficulty varies across subgroups . We copiMedical costs are typically characterized by an asymmetry of the distribution because some patients have minimal costs or specific standard cost amounts and other patients may have disproportionately high costs. Generalized linear models using a log-gamma distribution, have been suggested to account for this kind of highly skewed data . We usedAt baseline, between groups differences were negligible. For a detailed account of baseline patient characteristics see Noordraven et al. . An adheIn the intervention group average medical costs related to other healthcare services were somewhat higher compared to the control group (€529.6 versus €484.0), but lower after excluding hospitalisation (€52.0 versus €78.4). Fewer patients in the Money-for-Medication program visited their GP, a medical specialist, or social worker. This effect was in the expected direction but small .Additional file On average, the maximum of 30 euro extra cost item as financial incentive per patient per month constitutes about 3% of average total mental healthcare costs (€1062) and less than 7% of outpatient medical costs (€449). Extrapolating costs, excluding hospitalisation, in the previous 4 weeks at 12 months follow-up to total costs per patient per year, averaged to €9273 (SD 13512) in the Money-for-Medication group and to €7900 (SD 19089) in the care-as-usual group. Incremental total costs were €2080 (95% CI − 37972 to 34811) for achieving a 20% increase in adherence and €3332 (95% CI − 22675 to 28128) for taking at least 80% of the prescribed depot medications over the 12-month intervention period.Providing a financial incentive to improve adherence to depot medication in psychotic patients resulted in higher average costs directly related to mental health care and lower costs related to other health care services. Relevant differences in social costs related to delinquent behaviour were not found.An increase in medication compliance was reflected in mental healthcare costs, which were higher in the Money-for-Medication group compared to the control group. In contrast, medical costs related to other health care services were somewhat lower in the intervention group. Effects in terms of medical costs were in the expected direction but differences between the intervention and control group were not statistically significant. Social costs related to delinquency concerned few patients and only minor and non-significant differences were found comparing the intervention and control group.Intervention costs are low considering a maximum financial incentive of 360 euro per patient per year. Currently no threshold values are available for the ICER-values in the range of €2000 for achieving a 20% increase in adherence, and just over €3000 for ‘good’ (80% or higher) medication adherence. Interestingly, these figures are in line with the results of Henderson et al. [Financial incentives are effective in improving treatment adherence in patients with psychotic disorder. However, offering money as financial incentive for increasing compliance did not lead to an overall cost reduction as compared to care as usual. Perhaps that financial benefits of M4M in terms of reductions in medical costs might become manifest only after a longer period of time. Therefore, future studies using longer intervention and follow-up periods are needed to investigate cost-effectiveness also with respect to quality of life.Medical and judicial cost items were patient reported over a 4 weeks’ time span which may reduce memory bias, but may not adequately reflect variability in the level of health service use or delinquent behaviour in our 12-month study period. Also, the national reference costs per health care contact or type of delinquency were crude estimates of the true mental healthcare cost, medical costs related to other health care services, and social costs.The Self-Reported Delinquency questionnaire originally was aimed at adolescents and may be less suited for mapping delinquent behaviour in psychiatric patients, which may explain the low frequency of reported delinquent behaviour. In addition, we maybe overestimated the amount of criminal activities within a non-forensic patient setting; patients with chronic psychiatric diseases are not necessarily involved in criminal activities.During this study, frequency of other social parameters (e.g. participation in volunteer work) were not assessed and the invested time per patient to arrange appointments for proving depot medication was not monitored, so it remains unclear whether implementing M4M did actually save or cost extra time. Furthermore, our study was underpowered for the analysis of highly skewed cost data, resulting in wide bootstrapped confidence intervals for incremental cost-effectiveness ratios.Additional file 1: Appendix S1. Judicial unit prices and baseline costs. The file includes a table with the judicial unit prices and costs at baseline for all patients.Additional file 2: Appendix S2. Service costs 18 months. The file includes a table with the follow-up data for the service costs at 18 months.Additional file 3: Appendix S3. Judicial costs 18 months. The file includes a table with the follow-up data for the service costs at 18 months.


Varroa destructor is an ectoparasitic mite of Asian or Eastern honeybees Apis cerana(A. cerana) which has become a serious threat to European subspecies of Western honeybees Apis mellifera (A. mellifera) within the last century. V.destructor and its vectored honeybee viruses became serious threats for colony survival. This is a short period for pathogen- and host-populations to adapt. To look for possible variation in the composition of viral populations we performed RNA metagenomic analysis of the Western honeybee subspecies A. m. ligustica, A. m.syriaca, A. m. intermissa, and A. cerana and their respective V. destructor mites. The analysis revealed two novel viruses: Varroa orthomyxovirus-1 (VOV-1) in A. mellifera and V. destructor and a Hubei like-virga virus-14 homolog in V. destructor. VOV-1 was more prevalent in V. destructor than in A. mellifera and we found evidence for viral replication in both hosts. Interestingly, we found differences in viral loads of A. cerana and their V. destructor, A. m. intermissa, and its V. destructor showed partial similarity, while A. m.ligustica and A. m.syriaca and their varroa where very similar. Deformed wing virus exhibited 82.20%, 99.20%, 97.90%, and 0.76% of total viral reads in A. m. ligustica, A. m. syriaca, A. m. intermissa, and A. cerana, respectively. This is the first report of a complete segmented-single-stranded negative-sense RNA virus genome in honeybees and V. destructor mites. Varroa destructor is an obligatory ectoparasite of the Eastern honeybee Apis cerana [V. destructor mites spend most of their life cycle inside the colony, reproducing on the honeybee brood and feeding on the pupa. Mites transfer from one host pupa to another on nurse bees that take care of pupa [V. destructor made a host shift to the Western honeybee A. mellifera at the beginning of the 20th century and spread to Europe, USA, New Zealand, Africa, and the Middle East from southern and southeastern Asia during the last century [The mite s cerana . V. dest of pupa . During  of pupa ,3,4,5. V century .Viruses are important pathogens of the honeybee. Different studies reported that Acute bee paralysis virus (ABPV), Deformed wing virus (DWV), Israel acute paralysis virus (IAPV), Kashmir bee virus (KBV), Sacbrood virus (SBV), Slow bee paralysis virus (SBPV), Chronic bee paralysis virus (CBPV), Lake Sinai virus (LSV), Apis mellifera filamentous virus (AmFV), and Black queen cell virus (BQCV) are pathogenic to honeybees ,9,10,11.V. destructor, which has become a growing threat to the existence of A. mellifera [V. destructor and viral pathogens play a key role in the loss of A. mellifera colonies, especially because both the mite and the viruses produce a double effect in colony infestation: V. destructor being tightly linked to honeybee life cycle causes a considerable impact on the brood by feeding on it and transmitting viruses [Some of the above viruses DWV, SBPV, ABPV, IAPV, and KBV—are transmitted by ellifera ,13,14,15 viruses ,17,18. V viruses ,20. Toge viruses ,21,22.V. destructor from A. cerana to A. mellifera is a relatively short period of time for the populations of the pathogen and the host to adapt and thus, A. mellifera is a new host for V. destructor. This is valid for its viral content as well. To look for possible variations in virus composition we investigated the viral populations in samples of three A. mellifera subspecies from the Middle East and North Africa (MENA) regions and their parasitic V. destructor mites as well as from A. cerana and their V. destructor mites. For this purpose we used next-generation sequencing (NGS) and performed metagenomics analysis of viral populations from samples of A. m. ligustica colonies from Israel [V. destructor mites, from Western honeybee subspecies from the MENA region—A. m. intermissa and A. m. syriaca [V. destructor mites, and A. cerana and their V. destructor mites from Thailand. In this study, the viral composition and loads from samples of each bee species and its mite were determined, viral loads and viruses of interest were validated and characterized using molecular biology tools. This analysis indicated differences in viral composition and load in the samples of the above honeybee subspecies and their parasitizing varroa mites. We found that in these samples A. cerana and their parasite V. destructor differed considerably in their viral load, A. m. intermissa and its varroa mites showed partial similarity in their viral load, while A. m. ligustica and A. m. syriaca and their corresponding varroa mites’ viral loads were more similar. Moreover, besides identifying known and recently discovered viruses, we found two novel viruses—an Orthomyxovirus common to A. mellifera and V. destructor—that we named Varroa orthomyxovirus-1 (VOV-1) and a homolog of the Hubei like-Virga virus 14 that we named VDV-4. VOV-1 showed 23–58% homology to the Orhomyxoviruses Thogoto and Dhori (THOV and DHOV), which bear negative-sense single stranded RNA genomes of six segments [From an evolutionary point of view, the shift of m Israel  and thei syriaca —and theisegments ,26,27,28segments . Using tA. m. ligustica , 14 (6 bees), 81 (9 bees), and 401 (1 bee); mites N = 606, from the same hives , collected between October to February 2016, were described before [A. m. syriaca  and A. m. intermissa  were described previously [A. cerana drones (N = 6) and V. destructor mites (N = 20) were sampled during spring 2016 (December) in Phrae, Thailand from two colonies 3c and 4c untreated against mites . The honeybees and corresponding mites were transported in RNA later™ and stored immediately at −80 °C until RNA extraction.The experimental colonies of eviously . A. ceraA. mellifera and mite samples was carried out using TRI Reagent®  according to the manufacturer’s instructions as published before [A. cerana drones and the corresponding V. destructor mites was individually extracted using a GenJet RNA purification Kit  according to the manufacturer’s instructions.RNA extraction from all d before ,30. RNA A. cerana and its corresponding V. destructor mites-RNA samples was performed at the Technion Genome Center on a HiSEq 2000 platform . Paired-end reads were assembled de novo using Trinity [A. m. ligustica, A. m. intermissa, and A. m. syriaca honeybees and corresponding V. destructor mites was described previously [A. m. ligustica, A. m. intermissa, and A. m. syriaca subspecies and A. cerana bees, and of V. destructor mites samples were carried out as described previously and in Construction and paired-end sequencing of the libraries from  Trinity . The obt Trinity . Next-geeviously ,24. MetaV. destructor and honeybee samples, respectively. RT-conditions: incubation of RNA and primers at 65 °C for 5 min., followed by addition of buffer containing 50 mM Tris-HCl (pH 8.3), 75 mM KCl, 2 mM MgCl2, 5 mM DTT, 4 units of RNase inhibitor Ribolock® (Thermo Scientific), and the RT enzyme (200 units) in a 25 μL volume, and further incubation at 55 °C for 30 min. The reaction was terminated by heating at 85 °C for 5 min. PCR-validations were performed with GoTaq®  using 1 μL cDNA template and 0.2 μM of each forward and reverse primer in a 20 μL reaction with the following conditions; 95 °C for 4 min, 32 cycles at 94 °C for 30 s, then 56 °C  or 57 °C  for 50 s, 72 °C for 2 min.  or 1 min. , and a final extension step of 72 °C for 10 min. For VDV-4 the PCR conditions were identical to those used in segments 1, 2, 4, 5 of VOV-1 with the corresponding specific primers. Specific primers used for validations are described in the cDNA was prepared using RevertAid Reverse Transcriptase (Thermo Scientific) with oligo-dT and random primers according to the manufacturer’s instructions. One-hundred nanogram and 2000 ng RNA templates were used from V. destructor mites and honeybees in apiaries located at the North , the Center  and the South (Kibbutz Yad Mordechai) of Israel.VOV-1 prevalence was determined by using RT-PCR to detect the presence of the segment 6 of the virus genome in samples of Viral genome copy number was quantified on a PikoReal 96 machine (Thermo Scientific) using a standard protocol . Each quantitative PCR analysis was performed in triplicate. Nontemplate controls (water) were included in triplicates in each assay. The KAPA SYBR FAST qPCR Master Mix (2×) Universal (Kapa Bio-systems) was used, in a 10 μL final volume. For each analysis 2 μL of the diluted cDNA was used (dilution factor of 4) and specific primers VOV-1-qRT-F1 and VOV-1-qRT-R1 at a concentration of 0.25 μM each . The speTesting for viral replication (presence of the positive strand-sense RNA) was performed by synthetizing the negative-strand cDNA of fragment 6 from the RNA samples using the tagged primer VOV6-46F-TAG, as we described before to analyze replication of BRV-1 . SubsequA. m. ligustica [A. m. intermissa and A. m. syriaca and their varroa from the MENA region [Each RNAseq library was de novo-assembled using Trinity assembler version 2.2.0 . The assigustica  and realA region  and werePhylogenetic analysis was done using MEGA 6 . AlignmeA. m. ligustica, A. m. syriaca, A. m. intermissa, A. cerana, and their V. destructor counterparts . This revealed variation in composition of the viral loads of A. m. ligustica, A. m. syriaca, A. m. intermissa, A. cerana, and their V. destructor mite counterparts . Mapping of the libraries’ reads to viral contigs of honeybees and V. destructor mites resulted inidentification of the most common honeybee viruses: Acute bee paralysis virus (ABPV), Israeli acute paralysis virus (IAPV) in IB1, IV4, and SB2; Apis mellifera filamentous virus (AmFV) in AB3; Bee Macula-like virus/Varroa Macula-like virus (BeeMLV/VdMLV) and Black queen cell virus (BQCV) in all the libraries except for BCER and VCER; Deformed wing virus (DWV) in all the libraries; Lake Sinai virus (LSV) in IB1; and Sacbrood virus (SBV) in IB1, IV4, SV5, and AV6  , Varroa discovery of two new viruses that we designed Varroa orthomyxovirus-1 (VOV-1), an orthomyxovirus with low homology to other viruses from the Orthomyxoviridae family, and Varroa destructor virus-4 VDV-4  showed a similar percentage of viral reads of DWV, as did SB2 and SV5 (A. m. syriaca) and A. m. intermissa, and their V. destructor libraries—AB3 and AV6—showed large differences  and its varroa (VCER) displayed extremely low percentage of viral reads for DWV . As can be seen, our samples BCER and VCER differed in their load of other viruses as well; while the BCER main viral component was ARV-2 [A. cerana only with 38% homology to Hubei picorna-like virus 29 [The presence of viruses varied across ferences . Also, Aectively . The cDNas ARV-2 , the maivirus 29  and 36% A. m. ligustica [A. m. intermissa and A. m. syriaca and their varroa mites from the Middle East and North African (MENA) honeybees and varroa mites that were published recently but did not focus on finding new viruses [We reanalyzed the data obtained before in igustica  and real viruses . This ad viruses ,26,27,28Phylogenetic analysis of open reading frames (ORFs) coding for polymerase subunits PB2, PB1, and PA showed that the polymerase was closely related to negative-sense ssRNA viruses belonging to the Orthomyxoviridae viral family: Thogoto virus (THOV); Aransas Bay virus (ABV); Upolu virus (UPOV) A–C; and A. mellifera ligustica and their counterpart V. destructor parasites , and in none of 32 individual honeybees sampled from the same colonies. We also analyzed its prevalence in colonies located in the Center, North, and South of Israel by testing pools of the honeybees and V. destructor mites with the same PCR method. The virus was identified in 78.57% of V. destructor pools (11 of 14 pools) and only in 8.33% of honeybee pools (5 of 60 pools).Furthermore, we tested the presence of segment 6 of the viral genome to estimate the prevalence of VOV-1 by RT-PCR in individual mites and honeybees. We detected VOV-1 in 35.56% of 2–1.22×106, 2.33×103–4.88×105, and 4.91×102–1.38×105, respectively . A predicted size fragment of ~840 nucleotides corresponding to the VOV-1 positive-sense-strand RNA between nucleotides 46 and 870 was found in tested samples from individual We confirmed by Sanger DNA sequencing that the above specific-primer-tagged amplicons were identical to the VOV-1 sequence comprising nucleotides 46 and 870 of segment 6 of the viral genome.From the two undescribed viruses that we found in VCER we further investigated VDV-4. Phylogenetic analysis using the putative large ORF protein of the virus showed that it is 36% homologous to the hypothetical protein gene of spider viruses Hubei virga- like virus 14 and Hubei virga-like virus 13 as well as to the spider putative protein of the virus Nephila clavipes virus 4 .A. cerana drones and V. destructor individuals suggested that this virus was predominant in the latter  [V. destructor like ARV-1/BRV-1 and VOV-1, while others appeared to be restricted to mites, such as VDV-2 and VDV-3. Interestingly, VOV-1’s presence was limited to A. m. ligustica and A. m. syriaca as well as to V. destructor mites parasitizing them. VDV-2 was detected in all V. destructor libraries and VDV-3 was absent in Varroa destructor mites parasitizing A. cerana. ARV-1/BRV-1 was found previously in A. mellifera, V. destructor, and in Bombus impatiens, but our finding that it is present in A. cerana suggests that it may have a broader host range. V. destructor mite parasites of A. cerana appear to bear two novel viruses with low homology to Hubei picorna-like virus-29 [V. destructor mites from A. mellifera. We validated the presence of a Hubei virga-like virus-14 and designed it VDV-4.Furthermore, we identified recently characterized viruses in 1/BRV-1) ,38, Varr1/BRV-1) , and a nvirus-29  and to Hvirus-29 , which wV. destructor invasion there is high selection on DWV strains such that only a single strain seems to dominate, though which strain dominates varies across colonies and studies [V. destructor-infested colonies [Varroa destructor on A. mellifera [A. mellifera subspecies and their V. destructor counterparts. V. destructor from A. m. intermissa showed lower DWV levels compared to its parasitized host . Interestingly, it was reported that A. m. intermissa was more resistant to V. destructor parasitization than other A. mellifera subspecies [n = 1 repetitions of the transcriptomic data per subspecies.Data suggest that following  studies ,49,50,51colonies ,51,52. Mellifera . In our bspecies ,54. AgaiA. mellifera and V. destructor and VDV-4, a novel virus of V. destructor from A. cerana. VOV-1 possess a single-stranded negative-sense RNA genome and belongs to the Orthomyxoviridae family that among others includes the genus Thogotovirus. Most of the Thogotoviruses have been associated with ticks [A. m. ligustica. Interestingly, it showed greater prevalence in V. destructor mites compared with honeybees of 78.57% and 8.33%, respectively, in samples from apiaries located at the North , the Center , and the South (Kibbutz Yad Mordechai) of Israel. This is why, taking together the above data, we decided to name it Varroa orthomyxovirus-1, VOV-1.We characterized VOV-1, a novel virus common to th ticks  and relath ticks ,57. The Apis mellifera and its recently acquired obligatory parasite Varroa destructor opens a new venue for investigation of viral interactions in honeybee colonies. A number of questions emerge concerning this new host–pathogen relationship that could interfere the preexisting balance. What is the pathology associated with VOV-1? Do varroa mites transmit VOV-1 and VDV-4 directly, e.g., transovarially, or via their host bee? Where do the viruses accumulate in varroa? Is VOV-1 infectious to A. mellifera?The discovery of these novel viruses in Furthermore, there is another issue concerning those viruses crucial for colony health and survival—DWV, ABPV, IAPV, and CBPV—and their interaction with newly discovered ARV1/ BRV-1, VOV-1, and VDV-2 and -3: Do they affect one another on a mutual base? If they do, on what level and what are the factors that may be involved .Our findings and the tools that we have developed in this study pave the way to investigate these questions and extend our knowledge and understanding of the role played by viral pathogens in honeybee colonies.


C) to a pathogenic prion protein form (PrPSc). This pathogenic form, PrPSc, accumulates in the brain and lymphoid tissues. The presence of PrPSc can be detected by an in vitro conversion assay known as real-time quaking induced conversion (RT-QuIC). RT-QuIC has been used to detect PrPSc in a variety of biological tissues from brains to fluids. While this technique is both rapid and sensitive, enhancing the detection of prions would be valuable in the diagnostic laboratories.Scrapie is a transmissible spongiform encephalopathy (TSE) that naturally occurs in sheep and goats. This fatal neurodegenerative disease results from misfolding of the normal cellular prion protein . Coupling of RT-QuIC to PAD-Beads based cleanup allowed detection of PrPSc rapidly and without dilution of scrapie sheep brain homogenates prior to RT-QuIC. The PAD-Beads sample pretreatment step prior to RT-QuIC is a useful enhancement in the diagnosis of TSEs.In this study, we assessed whether PrP C) into a pathogenic form (PrPSc) that accumulates primarily in the central nervous system [Mammalian prion diseases include human Creutzfeldt–Jakob disease (CJD), bovine spongiform encephalopathy (BSE), sheep scrapie, and cervid chronic wasting disease (CWD). These diseases are fatal neurologic diseases known as transmissible spongiform encephalopathies (TSEs), and they result from the misfolding of the normal cellular prion protein  are used as substrates for RT-QuIC reactions and RT-QuIC monitors the fibril formation in real-time [Rodent bioassays can be considered a definitive technique for specific detection of prion infectivity. Two in vitro detection techniques such as protein misfolding cyclic amplification (PMCA) and real-time quaking-induced conversion (RT-QuIC) have demonstrated improved prion detection in PrP animals –8, but ieal-time –12. RT-Qeal-time , 12–15, eal-time , saliva eal-time , 17. WhiSc detection sensitivity. Several studies have reported enhanced detection using PMCA and RT-QuIC assays by enrichment of PrPSc containing samples of various means such as immunoprecipitation [Sc prior to detection and applied the approach to detection of PrPSc from scrapie-infected sheep brain samples by RT-QuIC.Protocols for prion purification allows us to detect and characterize prion in a complex mixture with inhibitors and/or in a diluted samples to improve PrPpitation , detergepitation , or non-pitation , 16. In All tissues samples used in this manuscript are from previously published sheep scrapie pathogenesis studies , 18 condA kit of PAD-Beads was purchased from Microsens Biotechnologies, London, UK , 20. SamE. coli (BL21(λDE3)) was transformed with the pET28a vector containing the Met109 variant of bank vole PrP gene  and the recombinant bank vole prion proteins were expressed and purified as described by Vrentas et al. [−1 cm−1 as calculated for the bank vole prion protein.s et al. . The conRT-QuIC reactions were performed and analyzed as previously described –25. The C) were seeded with different dilutions of PAD-Beads eluate for comparison with that of the directly diluted sheep brain homogenate and monitored for increased ThT fluorescence. RT-QuIC reactions containing bank vole substrate and 300 mM NaCl were seeded with brain stock solution  positive for scrapie. Both reactions seeded with brain stock and PAD-Beads eluted brain showed fibril formations as monitored by ThT fluorescence. Assays seeded with negative sheep brain homogenate did not produce ThT fluorescence under the conditions of the experiment. Assays seeded with high concentrations brain homogenate (non-diluted or 10−1 dilution) of brain homogenate also did not show ThT increase a result typically interpreted as indicative of inhibitors in the sample. In contrast, non-diluted PAD-Beads eluate seeded reactions exhibited positive ThT fluorescence suggesting PAD-Beads enrichment removes these unidentified inhibitors. Reactions seeded with PAD-Beads eluted brain samples showed a shorter lag time compared to the reactions seeded with non-PAD-Beads treated samples. A 20 h lag time was observed for non-PAD-Beads enriched brain homogenates dilutions from 10−2 to 10−4, but that lag time is reduced to only 10 h following enrichment with PAD-Beads for dilutions from 10−1 to 10−4. The undiluted PAD-Beads eluate without dilution showed the ThT fluorescence lag time of around 30 h. As can be seen in Fig. 0 to 10−4 for animal # 1 and between 100 to 10−2 for animal #2 show higher rate constants compared to the reactions seeded with brain homogenate dilutions.To evaluate the efficacy of PAD-Beads based enrichment for the purposes of RT-QuIC, reactions containing recombinant bank vole prion protein  volume. The use of a larger starting volume (200 µl) of PAD-Beads resulted in a reduced lag time  coated magnetic beads could bind infectious prions in brain and blood samples and prions bound to the beads could be detected by RT-QuIC [Sc relative to other components in the complex mixture that is present in a tissue homogenate enhance the detection of PrPSc by RT-QuIC, most likely due to the removal an undetermined contaminant that is inhibitory to the in vitro conversion process.Other approaches exist for enrichment of PrPss steel , 31–33,  RT-QuIC , 34–37.  RT-QuIC . Later, antibody B3 coatedSc from a complex mixture such as tissue homogenate or biological fluids. For the detection of scrapie prion using PAD-Beads, we have applied RT-QuIC and confirmed the presence of PrPSc by high seeding activity. PAD-Beads enrichment takes about 1 h, and the enriched samples resulted in a shorter lag time by approximately 10 h relative to unenriched samples for the detection of scrapie prions in brain samples. In addition, PAD-Beads enriched samples eluted with lower volume allowed higher detection sensitivity of RT-QuIC reactions. In a similar way, use of a larger starting volume (200 µl) of PAD-Beads shortened the lag time for detection of scrapie PrPSc by RT-QuIC.Commercially available PAD-Beads are a practical technique for separating misfolded proteins such as PrPGiven the rapid-simple protocol and improved sensitivity shown here for detection of scrapie, the established ability of PAD-Beads to bind many different proteins in an amyloid conformation as well as the applicability of RT-QuIC to other amyloid diseases, this method could provide a useful enhancement to RT-QuIC for other diseases such as Parkinson’s Disease since RT-QuIC has now been applied successfully to amplify α-synuclein aggregates , 39.The primary limitation of this work is that we did not specifically address additional TSEs, but we would anticipate that the method is applicable to other TSEs from other host species based upon the broad applicability of both PAD-Beads and RT-QuIC to various TSE agents. However, we cannot comment as to the degree of enhancement in sensitivity or the time savings. Despite these limitations, PAD-Beads enrichment does provide a useful method to enhance RT-QuIC diagnosis of scrapie.


Tupaia belangeri, family Tupaiidae), which is regarded as a “low-level primate”, has α2,3- and α2,6-linked sialic acid receptor distributions similar to those of humans and is potentially a useful mammalian model for studying mild human influenza (H1N1) virus infection. In this study, we used the tree shrew experimental model to investigate the pathogenesis of avian influenza A (H9N2) virus infection and the effect of the E627K mutation in the PB2 gene, an adaptation to mammalian hosts. Evidence of disease, virus titers in the upper and lower respiratory tract, histopathology and induction of proinflammatory cytokines are described. We also established ex vivo culture models of tree shrew respiratory tissues to study the tropism and replication of the H9N2 virus. Our results demonstrated that the tree shrew is a viable new in vivo experimental model for avian influenza research that provides results comparable to those observed in ferrets. The disease spectrum and pathogenesis in tree shrews correlate well with what is observed in humans.Outbreaks of avian influenza virus continue to pose threats to human health. Animal models such as the mouse, ferret, and macaque are used to understand the pathogenesis of avian influenza virus infection in humans. We previously reported that the tree shrew ( Moreover, reassortment between H9N2 and other avian viruses has generated new zoonotic subtypes, such as H5N1, H7N9, and H10N8, which have caused lethal zoonotic disease7. The H9N2 viruses that are circulating in poultry in China have evolved into two major phylogenetic lineages: the H9N2/G1-like lineage, represented by A/Quail/Hong Kong/G1/97 (H9N2/G1), and the H9N2/Y280-like lineage, represented by A/Duck/Hong Kong/Y280/97 (H9N2/Y280)8. Although H9N2 virus can cause severe disease in immunocompromised patients, most human infections in immunocompetent persons are associated only with mild respiratory disease4. In vivo studies have shown that a mutation from glutamic acid (E) to lysine (K) at amino acid residue 627 of the PB2 gene is an important adaptation marker of avian influenza viruses that contributes to pathogenesis in mammalian hosts11.Influenza A (H9N2) virus is the most widespread subtype of avian influenza found in poultry in Asia. In addition to the economic impact on the poultry industry, interspecies transmission of H9N2 viruses from poultry to humans has occurred repeatedly14. On the other hand, H9N2 viruses cause mild respiratory symptoms in the ferret, approximating human infection16. However, the use of ferrets is limited by the relatively high cost and by the requirement for specially designed facilities and husbandry. Cynomolgus macaques have been used as experimental models for influenza18. However, the SA receptor distribution in macaques does not parallel what is found in humans, and the facilities needed for macaques are not widely available.Mouse and ferret have been commonly used to study the pathogenesis of H9N2 viruses in mammalian hosts. However, the distribution of sialic acids (SA)—which act as receptors for influenza viruses—differs notably between mice and humans. Many influenza viruses that infect humans do not infect mice without prior adaptation. Meanwhile, H9N2 infection in mice causes severe inflammation in the lower part of the lung, which is not what is usually observed in humansTupaia belangeri, family Tupaiidae), indigenous to South Asia, Southeast Asia, and Southwest China, is classified as a separate taxonomic group of mammals that diverged from the primate order approximately 85 million years ago but still be considered as a “low-level primate” based on their close relationship19. Tree shrew is phylogenetically more closely related to humans than ferret and mice. We have previously shown that the tree shrew supports the replication of human influenza viruses without prior adaptation by serial passages and that the resulting infections bring mild disease symptoms resembling those in humans20. Importantly, we showed that the distribution of SA receptors in the respiratory tract of tree shrews is similar to that of humans. Recently, the genome of the tree shrew has been fully sequenced, which will help to establish research tools for use in a tree shrew model21. In this study, we investigated whether the tree shrew is susceptible to infection with avian influenza (H9N2) virus, and we evaluated the virologic and immunological features of the H9N2-infected tree shrews through in vivo and ex vivo models.The tree shrew  by reverse genetics and tested in parallel with the Y280-wt virus , and the viral titer from the supernatant of the homogenized tissues was determined by TCIDdpi Fig. . Replicadpi Fig. . Virus rdpi Fig. .Fig. 2ViInflammatory cells (lymphocytes and neutrophils) and focal edema on the submucosal layer of nasal turbinate were found in the tree shrews infected with Y280-wt virus at 2 dpi Fig. . The tistumor necrosis factor α (TNF-α), interferon β (IFN-β), IL-4, IL-6, CXCL8 (IL-8), IL-10, IL-13, CXCL10 (IP-10), CCL5 (RANTES), CXCL9 (MIG), and CCL2 (MCP-1), were measured from nasal turbinate, throat, trachea, and lung tissues obtained from tree shrews infected with the H9N2 viruses. The results are expressed as fold change compared to mock-infected tissue. In general, tree shrews infected with either wild-type or PB2 mutant H9N2 virus showed higher induction of cytokines in the examined tissues than the uninfected controls. In general, the expression of all cytokines peaked at 2 dpi  and IL-8, RANTES, and IFN-β in the lower respiratory tract (trachea or lung) were significantly higher in the tree shrews infected with Y280-PB2-E627K than in those infected with Y280-wt virus. On the other hand, Y280-wt virus triggered significantly higher mRNA expression of MIG , IL-13 , MCP-1 , and IL-10  than Y280-PB2-E627K virus.The mRNA expression profiles of selected cytokines, including p < 0.05)  to compare the replication of the H9N2 virus and its PB2-E627K mutant. Under comparable infection conditions, we found that the Y280-PB2-E627K virus replicated more efficiently in the nasal turbinate, trachea, and lung tissues than the Y280-wt virus did (05) Fig. .Fig. 6ViTNF-α (24 h and 48 h), IFN-β (24 h), IL-8 (48 h), IL-10 (72 h), IL-4 (24 h), and IL-13 (72 h) were significantly higher in the ex vivo lung tissues infected with the Y280-PB2-E627K virus than in those infected with Y280-wt virus. In contrast, higher levels of IL-8 (24 h), RANTES (24 h), IP-10 (24 h), and IL-10 (48 h) in the tracheas as well as IL-6 (24 h) and IP-10 (72 h) in the lungs were found in the Y280-w-infected group than in the Y280-PB2-E627K-infected group.The mRNA expression profiles of the selected cytokines were also measured from the ex vivo cultures after infection with H9N2 viruses Fig. . The cyt23. We found that infection with the wild-type avian H9N2 virus (Y280-wt) showed transient virus shedding without an obvious increase in body temperature or weight loss. However, our histological findings clearly identified evidence of infection, with inflammatory cells and focal edema observed in the submucosal layer of the nasal turbinates at the early stage of infection. Although one tree shrew showed no virus shedding after infection with the H9N2 wild-type virus, the high antibody titer against the H9N2/Y280 strain in the blood of this tree shrew suggested that there was asymptomatic infection.Our study aimed to characterize the susceptibility of tree shrews to avian H9N2 virus infection as part of an investigation to determine whether this experimental animal model is useful for studying the pathogenesis of avian influenza viruses in mammals. Unlike those with H5N1 or H7N9 viruses, patients with H9N2 infection mainly manifest mild upper respiratory symptoms, including fever, coryza, sore throat, and inflamed oropharynx, analogous to the effects of seasonal influenza virusesPB2 gene contribute to the pathogenicity of avian influenza virus in mammalian hosts11. Specifically, the PB2-E627K mutation is associated with an increase in the virulence and replication of the avian influenza virus in both in vitro and in vivo models. We also found that H9N2 virus with the E627K mutation (Y280-PB2-E627K) was shed by tree shrews in higher quantities and for a longer period than its wild-type control. The virus was disseminated more widely in the respiratory tract, and there was evidence of inflammation in the lower lung in some but not all tree shrews. Taken together, our results showed that the tree shrew is highly susceptible to avian influenza H9N2 virus infection.Mutations in the 14. It was consistently found that H9N2 virus with this mammalian adaptation mutation usually caused more severe disease and greater lethality and weight loss in mice than in humans. This more pronounced phenotype in mice may be due to the availability of α2,3-linked SA receptors in the upper respiratory tract of mice, which would facilitate lower lung infection even by intranasal inoculation of the virus24. The human and ferret respiratory tract predominantly has α2,6-linked SA receptors in the upper respiratory tract, with α2,3-linked SA receptors being present only in the lower respiratory tract. In our study, both ferrets and tree shrews supported the replication of the H9N2 virus, and this replication was further enhanced by the PB2-E627K mutation. However, the overall severity of infection in these two animal models remained mild, which is comparable to what is observed in humans. The ferret has been used as a model to assess the pathogenicity and transmissibility of the H9N2 virus because it has a distribution of α2,3 and α2,6 SA receptors that resembles the distribution seen in the human respiratory tract25. In our experiments, the wild-type H9N2/Y280 virus replicated efficiently in ferrets even without the PB2-E627K mutation, while the difference between the wild type and the PB2 mutant was more significant in tree shrews, supporting the previous finding that tree shrews are susceptible to avian H9N2 virus infection. Moreover, our newly established tree shrew ex vivo cultures demonstrated that the PB2-E627K mutation enhanced the early cycles of H9N2 virus replication in the nasal turbinate and trachea, in which the kinetics were consistent with our findings from in vivo infection. Interestingly, the PB2-E627K mutant clearly replicated more efficiently than the wild-type virus in the ex vivo study, while virus replication was observed only in the lungs of two tree shrews that were infected with the mutant. This result suggests that the PB2-E627K mutant has adapted by gaining the ability to replicate in the lower lung, but replication in this site may not occur in every infected individual and may be caused by other factors, which is similar to the situation in humans.Different experimental animal models have been used to simulate the outcomes of avian influenza virus infection in humans. Mice and ferrets are the two commonly used experimental animal models used for the in vivo study of avian influenza virus. Macaques have occasionally been used as an experimental model but are extremely costly; additionally, there are ethical issues with their use, and the outcomes of infection in macaques do not closely mimic those observed in humans. We and others have previously studied the pathogenesis of H9N2 virus infection and the contribution of the PB2-E627K mutation in mice26. We established a set of primers that can detect the mRNA level of 11 cytokines in the tree shrew. Elevated levels of cytokines were detected in the tissues isolated from the respiratory tract after infection with either the wild type or the PB2 mutant compared to the levels in the uninfected control, suggesting that infection with H9N2 virus can trigger innate immune responses in tree shrews. We found that the increase in proinflammatory cytokines in the in vivo experiments generally peaked at day 2 and decreased at day 4, matching the viral titers detected in nasal wash at the corresponding time point, which indicates a strong correlation between cytokine expression and virus replication in the upper respiratory tract. Transient cytokine expression during early infection was also reported in ferrets28. However, we did not find similar cytokine profiles between the in vivo and ex vivo experiments. This result is understandable, as the results from these two experiments were based on different infection conditions. While the former showed the regulation of cytokines in tree shrews under pathological conditions, the latter reflected the cytokine expression phenotype of wild-type H9N2 and its PB2 mutant in different tissues. Thus, the results from the two experimental settings provide different angles from which to characterize the pathogenesis of the virus.The induction of proinflammatory cytokine and chemokine expression has been shown to contribute to the pathogenesis of avian influenza virus infection21.In conclusion, our study further demonstrates the potential of the tree shrew as an additional experimental animal model for investigating the pathogenesis of influenza. The virus replication patterns and phenotype mimic what is observed with humans and ferrets. As an experimental model, the tree shrew is smaller in size, easier to handle, less aggressive and less expensive in terms of the cost per animal than ferrets (approximately 1/10 of the price). Importantly, tree shrews are evolutionarily closer to humans and primates than ferrets are2.H9N2 virus A/duck/Hong Kong/Y280/97 (Y280-wt) and its PB2-E627K mutant (Y280-PB2-E627K) were grown in 9- to 11-day-old specific-pathogen-free (SPF) chicken eggs. MDCK cells were maintained in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum at 37 ℃ with 5% CO50 assay in MDCK cells. Seroconversion of the animals was tested by HI assays. All animals used in the experiments were humanely euthanized at day 21 postinfection. At days 2, 4, and 6 postinfection, four tree shrews were randomly selected for euthanasia, and nasal turbinate, throat, trachea, and lung tissues were obtained. The left and right nasal turbinates and lungs, as well as the left and right sides of the trachea, were set aside in equal portions. One portion was used for histopathological analysis and immunostaining with anti-influenza NP antibody. Another portion was homogenized with PBS supplemented with ofloxacin, amphotericin B, penicillin, and streptomycin followed by centrifugation at 13,000 × g, 4℃. The virus titer in the supernatant was determined by a TCID50 assay, and total RNA from the cell debris was extracted to measure the mRNA levels of the selected cytokines and chemokines.Tree shrews  and ferrets  were purchased from the Faculty of Life Science and Technology, Kunming University of Science and Technology, Kunming, China. The animals were confirmed as serologically negative for the H9N2 Y280-wt virus before use. The animals were first anesthetized via intraperitoneal injection of 3% pentobarbital sodium dissolved in PBS and inoculated intranasally with Y280-wt or Y280-PB2-E627K virus (day 0). Body temperature and body weight were recorded daily using subcutaneous transponders , starting 4 days prior to infection. At days 2, 4, and 6 postinfection, 0.25 mL (0.125 mL per nostril) of PBS was instilled into the nostrils of the infected animals and allowed to exit into a Petri dish. The fluid  was collected for virus titration using the TCID29. Briefly, the tissue blocks were first infected with 1 mL of 106 TCID50 influenza viruses and washed with warm PBS five times after 2 h of incubation. The tissue blocks were then cultured with fresh medium  at 37 °C. At 1, 24, 48, and 72 hpi, the supernatant was collected for TCID50 assays. The tissue blocks were collected and homogenized with TRIzol . Total RNA was then extracted, after which the mRNA expression of the cytokines was measured using quantitative PCR.Ex vivo cultures of nasal turbinate, trachea, and lung tissue were carried out as previously describedTotal RNA was converted to cDNA using PrimeScriptTMRT Master Mix (Takara), and quantitative PCR was then performed with 2× H-TECH SYBR qPCR Mix according to the manufacturer’s protocols. The levels of cytokine mRNA expression were reported as fold changes compared to the results from mock-infected tree shrews. The primers were designed using Primer 5.0 and are listed in Supplementary Table 30. Immunohistochemical staining of the influenza nucleoprotein in the tissues was performed as follows. Four-micron-thick sections were treated with 0.1% Pronase  in Tris buffer  at 37 °C for 15 min, transferred to 95 °C for 15 min, and then blocked with 3% H2O2 for 20 min, followed by treatment with a Streptavidin/Biotin Blocking Kit . After being blocked with 10% normal goat serum for 20 min, the sections were incubated with 1/10 HB65  primary antibody for 1 h at room temperature, followed by incubation with biotinylated goat anti-mouse secondary antibody  for 30 min at 37 °C. After incubation with the Vectastain ABC Kit , the sections were developed with the Vector NovaREDTM Substrate Kit .The tissues isolated from the respiratory tract were first fixed with 10% neutral buffered formalin, embedded in paraffin, sectioned at 3 μm and stained with hematoxylin and eosin (H&E) as previously describedt test or the Mann−Whitney U test. P values < 0.05 were considered significant.Statistical analysis was performed using GraphPad Prism 7.0 software. Differences in viral titers and mRNA expression among groups were compared using an unpaired, parametric Sup Fig 1Sup Fig 2Supplementary figure legendsSupplementary tables


Avian avulavirus 1. Here, we describe the molecular epidemiology of Latin American NDVs, current control and prevention methods, including vaccines and vaccination protocols, as well as future strategies for control of ND. Because the productive, cultural, economic, social, and ecological conditions that facilitate poultry endemicity in South America are similar to those in the developing world, most of the problems and control strategies described here are applicable to other continents.Newcastle disease (ND) infects wild birds and poultry species worldwide, severely impacting the economics of the poultry industry. ND is especially problematic in Latin America  where it is either endemic or re-emerging. The disease is caused by infections with one of the different strains of virulent avian Newcastle disease virus (NDV), recently renamed The online version of this article (10.1007/s11250-019-01843-z) contains supplementary material, which is available to authorized users. T 1) Mayo . The vir 1) Mayo , and inf 1) Mayo .Based on genetic characteristics, NDV has been classified into class I and class II viruses. Class I NDV have been isolated predominantly from wild birds, are mostly of low virulence, and their presence is only rarely reported in poultry species. Classification of class II NDV strains has been thoroughly reviewed elsewhere , Nicaragua (2001), and Belize (2008) suggesting that this genotype may have become established in some unknown reservoir  and Brazil in 1953  was obtained in May of 2008 from a commercial pullet flock presenting high mortality rates and clinical signs of ND  employed most commonly as seed strains for live, and inactivated vaccines are applied worldwide  chickens that have no maternal antibodies to NDV under laboratory conditions, they do not prevent replication and shedding of virulent challenge virus into the environment and into eggs , smaller eggs with rough, thin, and whiter “bleached” shells and poor-quality contents (whites and yolks)  vaccine and a recombinant genotype seed strain vaccine are also often used in Mexico. The rP05 vaccine contains a complete recombinant NDV of low virulence expressing the F and HN proteins of a genotype V NDV. Most of the oil emulsion type inactivated vaccines for the prevention of ND are mixed with two to four additional infectious antigens to decrease the number of times the birds have to be handled, resulting in less stress for the birds and decreased costs for vaccine administration. For example, in addition to the NDV genome, most vaccines also contain fowl adenovirus , infectious bronchitis virus, avian influenza virus (H5N2), and infectious bursal disease virus (Gumboro)  expressing one of the genes encoding the antigenic proteins of NDV, mostly the fusion protein gene, are available and reviewed by Dimitrov et al. (Layers have extended vaccination programs to provide protection during their longer (compared with broilers) lifespan. For example, in Mexico during breeding (0 to 18 weeks of age), hens receive at least four vaccines against NDV (2 live/2 inactivated)  are also included. HVT is a persistent apathogenic virus for chickens that induces long-term immunological protection, and is therefore, considered a good viral vector for use in poultry. In addition, HVT also induces protection against Marek’s disease virus, so these vaccines are capable of inducing protection against both viruses  in comparison with 90.5% survival in the experimental farm  Hoerr . ImmunosStressful rearing situations, in addition to live agents, may contribute to immunosuppression Hoerr , and proNDV is capable of increasing the concentration of corticosterone up to sixfold in the serum of infected birds .Prevention and control strategies for Newcastle disease in endemic countries need improvement. Ideally, these would include measures that include culling, active surveillance, improved biosecurity, and stringent vaccination programs. Any strategy needs to be accompanied by measures that ensure strict compliance of policies and procedures, which would include adequate record keeping and control of movement and disposal of infected animals. As with the outbreaks in the USA , the measures might include control of the viruses replicating in backyard and pet species, together with control of viruses in commercial birds. The current reliance on the use of vaccines formulated from seed strains from older genotypes that are less antigenically similar to the virulent challenge viruses may not eliminate the continuing persistence of the virus in farms due to viral excretion from the vaccinated-but-infected birds or prevent egg drops in vaccinated layers


The potential economic benefit in terms of reduced healthcare costs when patients quit smoking after hospital discharge has not been directly measured. The aim of this study was to compare the costs for hospital admission and six-month follow-up for a cohort of patients who self-reported abstinence from cigarettes at 6 months after hospital discharge and a matched group of patients who reported continued smoking.ClinicalTrials.gov ID: NCT01575145), with cohort membership determined by self-reported 7 day point prevalence abstinence at 6 months after the index hospital discharge. Participants were admitted to Mayo Clinic Hospital, Rochester, MN, between May 5, 2012 and August 10, 2014 for any indication and lived in the areas covered by postal codes included in Olmsted County, MN. Propensity score matching was used to control for differences between groups other than smoking status, and any residual imbalance was adjusted through generalized linear model with gamma distribution for cost and log-link transformation.This was a secondary analysis of a recent population-based clinical trial cohort (P = 0.038). However, there was no difference between mean 6-month follow-up costs, number of inpatient hospitalizations, or number of emergency room visits for abstainers versus non-abstainers.Of 600 patients enrolled in the clinical trial, 144 could be contacted and self-reported 7 day point prevalence abstinence at 6 months after hospital discharge. Of these patients, 99 were successfully matched for this analysis. The cost for the index hospitalization was significantly greater in patients who abstained compared to those that did not abstain (mean difference of $3042, higher for abstainers, 95% CI $170 to $5913, There was no evidence to support the hypothesis that abstinence at 6 months after hospital discharge is associated with a decrease in health care costs or utilization over the first 6 months after hospital discharge. An estimated 5 million patients who smoke require admission to US hospitals annually . HospitaClinicalTrials.gov ID: NCT01575145) in which 600 residents of Olmsted County, MN who were hospitalized at Mayo Clinic Rochester hospitals for any indication were randomized to receive an intervention aimed at facilitating telephone counseling (“quitline”) utilization or a standard brief smoking intervention to explore the efficacy of using telephone quitlines in hospitalized patients to achieve smoking cessation [We recently published a controlled trial , were hospitalized in Mayo Clinic, Rochester between May 5, 2012 and August 10, 2014 for any admission indication, and reported current smoking at the time of admission. All indications were included to examine the association of abstinence with post-hospitalization costs within all members of a geographically-defined population. In conformity with Minnesota statutes, patients that did not authorize the use of their medical record for research were excluded from the analysis.Data for the study patients including demographic and clinical characteristics were collected from electronic databases. Admission diagnosis was categorized using Clinical Classification Software (CCS) categories based on the primary diagnosis . The RocPropensity score matching was conducted to select the non-abstinent cohort based on baseline characteristics including age, gender, and clinical indication based on CCS. The propensity score model used logistic regression with a one-to-one matching approach without replacement. Patients were matched using the nearest neighbor method with a 0.001 caliper. Common support was implemented. Pre- and post-match balance was assessed using the t-test for continuous covariates, and chi-square tests for categorical covariates. Any residual imbalance following propensity score matching was adjusted through generalized linear modeling (GLM). Both index hospitalization and 6-month post-discharge costs were reported for the abstinent and non-abstinent cohorts, along with the mean difference in costs. In an additional analysis, 6-month post-discharge costs were adjusted for index hospitalization costs. All GLM models for cost analyses assumed a gamma distribution for cost and a log-link to account for the skewed nature of the cost costs distribution . Predictp-values showed no statistical difference in covariates and thus good balance. Other variables not included in the propensity matching, including marital status, insurance status, receipt of surgery during admission, and comorbidity counts were compared between abstainers and non-abstainers and no significant differences were found  (Table p = 0.433)  and those that assess costs in smokers who do and do not quit. Regarding the former design as applied to hospitalized smokers, a prior analysis that also included smokers who were enrolled in the present clinical trial found no association between smoking status and hospital costs for either medical or surgical patients . In anotTo our knowledge, no prior study has compared post hospitalization costs and utilization in patients who did and did not quit smoking after hospitalization, so there are no data directly comparable to our results. The original intent at the time that the randomized trial was planned was to analyze post hospitalization costs according to treatment assignment, hypothesizing that postoperative abstinence rates would differ between groups, and that the economic impact of these differential rates could be analyzed. However, there was no difference in quit rates according to treatment assignment, thus no rationale to perform this originally-planned economic analysis. Nonetheless, these data provided the opportunity to examine the association between quitting and post hospitalization costs, albeit with the same potential limitations inherent in other observational studies. In particular, it is possible that differences in patient characteristics not accounted for in the analysis, such as severity of illness, contribute to the lack of difference in costs between those who did and did not quit. More severe illness may contribute to both the likelihood of quitting and increased costs, obscuring any potential benefit of abstinence on healthcare utilization and costs. This possibility is supported by the finding that the costs of the index hospitalization of the patients that abstained after discharge were higher, as both groups of patients were abstinent while in hospital. It is also possible that differences might have become apparent if subjects were followed for a longer period of time. Thus, these results cannot be used to evaluate the potential economic benefit of a smoking cessation intervention. Nonetheless, we find no evidence that quitting is associated with a decrease in initial post-hospitalization costs. Thus, although there are certainly many good reasons to treat hospitalized patients for tobacco dependence, there is not yet evidence that such treatment is associated with immediate economic benefit. Rather, based on the results of long-term longitudinal studies of costs showing that economic benefits take some time to accrue, it may also take a considerable period of time before similar benefits would be observed in hospitalized patients.Although there are several strengths of our study design, including the fact that participants were drawn from an area of geographic residence  and a billing resource that allowed for complete ascertainment of costs using a validated methodology, this study has some significant limitations. First, the number of patients available for analysis was relatively small, and, as typical of cost studies, there was large variation in costs among cases. Some of those who abstained were not included in the analysis because a suitable matched patient could not be identified. This limits the ability to detect differences in costs. Second, as with any observational study, there is the potential for confounding factors not captured in our analytic techniques employed to adjust between groups . The analysis relied on self-reported smoking status at 6 months, as only about half of the patients in the clinical trial provided urine samples for biochemical confirmation . FinallyIn conclusion, this study found no evidence to support the hypothesis that maintaining abstinence after hospital discharge is associated with a decrease in health care costs or utilization over the first 6 months after hospital discharge. These results may suggest caution in using the incremental costs of smoking-related illnesses to estimate the immediate potential economic impact of smoking cessation in medical populations. However, because this study had several limitations, including relatively small size and relatively large inter-individual differences in in health care costs, larger studies will be needed to more adequately address this important question.Additional file 1. Pre- and post-matching balance of age, sex and clinical indication propensity score matching of abstainers and non-abstainers.Additional file 2. Frequency of diagnoses by 6-month abstinence.Additional file 3. Mean and median costs of index hospitalization and 6-month follow-up costs by indication for subjects who continued to smoke versus those who were abstinent at 6-month follow-up, according to diagnostic category.


CDC will continue to update recommendations as new information becomes available.Zika virus infection can occur as a result of mosquitoborne or sexual transmission of the virus. Infection during pregnancy is a cause of fetal brain abnormalities and other serious birth defects ( Aedes aegypti mosquito, Zika virus can also be transmitted through unprotected sex  with an infected partner. As of July 3, 2018, 52 cases of confirmed sexual transmission of Zika virus infection have been reported in the United States since 2015 (https://www.cdc.gov/zika/reporting/case-counts.html). Most documented reports of sexual transmission have involved transmission from a man to a woman  of Zika virus appeared to enhance viral dissemination to the female reproductive tract, compared with subcutaneous inoculation  during the periconceptional period have been associated with infection in the fetus and adverse pregnancy outcomes; although in some cases, the timing of infection relative to conception was uncertain  or their last possible Zika virus exposure (if asymptomatic) before trying to conceive with their partner .CDC continues to recommend shared patient-provider decision making, in which couples and health care providers work together to make decisions about timeframes to wait before trying to conceive after possible Zika virus exposure. Some couples might choose to wait shorter or longer periods after possible Zika virus exposure, based on individual circumstances , clinical judgment, and a balanced assessment of risks and expected outcomes. Other guidance for preconception counseling and prevention of sexual transmission of Zika virus after possible Zika virus exposure remains unchanged (Zika virus infection during pregnancy is a cause of serious birth defects. CDC previously released interim guidance on preconception counseling and prevention of sexual transmission of Zika virus in October 2016.CDC now recommends that men with possible Zika virus exposure who are planning to conceive with their partner wait at least 3 months after symptom onset or their last possible Zika virus exposure before engaging in unprotected sex. This updated timeframe also applies to prevent sexual transmission of Zika virus.These recommendations provide couples planning pregnancy with updated timeframes expected to reduce the risk for fetal Zika virus infection.


We studied trends in lung cancer treatment cost over time by phase of care, treatment strategy, age, stage at diagnosis, and histology.Using the Surveillance, Epidemiology, and End Results (SEER)‐Medicare database for years 1998‐2013, we allocated total and patient‐liability costs into the following phases of care for 145 988 lung cancer patients: prediagnosis, staging, surgery, initial, continuing, and terminal. Patients served as self‐controls to determine cancer‐attributable costs based on individual precancer diagnosis healthcare costs. We fit linear regression models to determine cost by age and calendar year for each stage at diagnosis, histology, and treatment strategy and presented all costs in 2017 US dollars.P < 0.001). Surgery in 2017 cost $30 096, decreasing by $257 per year (P = 0.007). Chemotherapy and radiation costs remained stable or increased for most stage and histology groups, ranging from $4242 to $8287 per month during the initial six months of care. Costs during the final six months of life decreased for those who died of lung cancer or other causes.Monthly healthcare costs prior to lung cancer diagnosis were $861 for a 70 years old in 2017 and rose by an average of $17 per year (Cost‐effectiveness analyses of lung cancer control interventions in the United States have been using outdated and incomplete treatment cost estimates. Our cost estimates enable updated cost‐effectiveness analyses to determine the benefit of lung cancer control from a health economics point of view. However, many of the cost‐effectiveness analyses (CEAs) that have been performed to understand the economic value of new lung cancer screening recommendations and treatment options use either incomplete or outdated cost estimates.To ensure accurate cost estimates for future CEA studies of lung cancer control, we estimated lung cancer treatment costs over time by age, stage at diagnosis, histology, and treatment strategy for patients over the age of 65, the eligibility age of Medicare. We calculated costs associated with patient‐level lung cancer treatment using the SEER‐Medicare database for years 1998‐2013 and adjusted costs to 2017 US dollars. Additionally, for patients who enrolled in Medicare Part D, we investigated prescription drug costs before and after lung cancer diagnosis for years 2007‐2013. Using these patient‐level cost data, we assessed trends, by phase of care, in cost of lung cancer treatment for patients enrolled in Medicare in the United States.22.1https://healthcaredelivery.cancer.gov/seermedicare/.Our analysis was performed using the Surveillance, Epidemiology, and End Results (SEER)‐Medicare database for years 1998‐2013. SEER is a resource of the National Cancer Institute that compiles clinical, demographic, and cause of death information from 17 cancer registries across the United States, representing about 28% of the US population.Our cohort included Medicare beneficiaries 65 years or older who were diagnosed with American Joint Committee on Cancer (AJCC) stage I‐IV lung cancer between 2000 and 2011. Patients were excluded if lung cancer stage was not recorded, if they had previous or subsequent cancer diagnoses other than lung cancer, if they were not continuously enrolled in both Medicare Part A and Part B coverage during the period of 15 months prior to cancer diagnosis through death or the end of 2013, if they received Medicare benefits because of disability or end‐stage renal disease, if they enrolled in managed care at any time during the study period, if month of diagnosis was unknown, if diagnosis was made at autopsy, or if the date of death recorded in the Medicare database differed from that of the SEER database by more than three months.2.2Codes used to define chemotherapy, radiation, and surgery can be found in Appendix 2.3The prediagnosis phase—defined as the one‐year period ending three months prior to lung cancer diagnosis—was used to estimate what that patient's healthcare spending would have been had the patient not developed lung cancer. The three months prior to diagnosis were excluded from cost calculations so that the average baseline costs would not be influenced by treatments given to symptomatic patients not yet diagnosed with lung cancer. Monthly costs relative to diagnosis date can be found in Appendix A patient who received only nonsurgical treatment(s) or best supportive care has a one‐month staging phase beginning on the date of lung cancer diagnosis. In practice, staging can occur over a period longer than one month for some patients; the one‐month staging phase is based on clinical practice at our institution. A patient treated with surgery has a one‐month surgery phase beginning on the date of surgery. Since many patients (38.0%) received surgery within one month of diagnosis, the inconsistent amount of time between diagnosis and surgery dates was excluded. The staging or surgery phase is followed by a six‐month initial phase, a continuing phase that varies in length depending on how long the patient lived following diagnosis, and, if a patient died before 31 December 2013, a six‐month terminal phase ending on the date of death. We saw a clear rise in costs around 6 months before death; further explanation of our decision to use of a six‐month terminal phase can be found in Appendix 2.4Costs were defined as the sum of Medicare reimbursements (payments from Medicare to the service provider), coinsurance reimbursements (payments from a coinsurer to the service provider), and deductibles and copayments billed to patients. We determined total and patient‐liability costs, although within SEER‐Medicare data, patient‐liability costs paid out of pocket at the time of service cannot be differentiated from those paid by a purchased Medigap policy—insurance sold by private companies to help cover coinsurance, copayment, and deductible costs.Each patient's cancer‐attributable costs were determined by subtracting the patient's own mean monthly prediagnosis phase costs from the mean monthly costs during treatment phases. The self‐control method enabled us to control for the prior presence of comorbid conditions and health‐related behaviors that may be correlated with healthcare costs, but are not included in SEER‐Medicare data, such as smoking status.Payments were converted to constant 2017 US dollars by adjusting Part A claims using the CMS Prospective Payment System Hospital Price Index and Part B claims using the Medicare Economic Index.2.5We analyzed prescription drug costs, including patient‐liability costs, for the subset of patients who were consistently enrolled in Medicare Part D, also known as the Medicare prescription drug benefit, during 2007‐2013 from lung cancer diagnosis through death or 31 December 2013. We additionally determined average costs for those treated with erlotinib hydrochloride , a drug currently approved for first‐line treatment of metastatic NSCLC patients whose tumors have certain epidermal growth factor receptor (EGFR) mutations.2.6α = 0.05 level. Parameter estimates for all models can be found in Appendix We calculated total, patient‐liability, and cancer‐attributable monthly costs for each patient. Multiple linear regression models were fit to estimate population average total, patient‐liability, and cancer‐attributable monthly costs for each phase. Separate models were fit for each phase, stage at diagnosis, histology, and treatment strategy. Models for monthly prescription drug costs were fit for each phase and treatment strategy. Treatment strategy costs are not shown if less than 10% of patients within a stage/histology group received that treatment; best supportive care costs are shown for all groups  and age of diagnosis (P < 0.0001). Average monthly costs during the three months immediately prior to diagnosis, which was excluded from the prediagnosis phase, were significantly different (P < 0.001) from average monthly costs during the one‐year period ending three months prior to diagnosis.Monthly healthcare costs during the prediagnosis phase were subtracted from mean monthly costs during treatment phases to determine cancer‐attributable costs. For a patient diagnosed at age 70 in 2017, average costs per month during the prediagnosis phase were $861 , of which the patient was responsible for $84   to $13 608  (see Table S5 in Appendix 3.2P = 0.007), patient liability increased by $22 each year (P < 0.0001). Cost of surgery did not change significantly with age at diagnosis.For a patient who received surgery at age 70 in 2017, average costs during the month beginning on the date of surgery were $30 096 ; the patient was liable for $1738 . Although total cost decreased by $257 each calendar year  received erlotinib. The mean monthly cost of erlotinib during 2007‐2013 was highest during the terminal phase, at $1844  per month; the patient was responsible for $252  per month. Monthly costs of erlotinib during other phases of care can be found in Appendix 4Prior to lung cancer diagnosis, our results indicate that baseline healthcare costs increased and patients had greater liability for these costs. Staging costs ranged from $6670 to $13 608 and were relatively stable over time. For early‐stage patients who receive surgery, costs during the month of surgery have been decreasing and costs during the initial and continuing phases have been considerably lower than those of chemotherapy, radiation, or both. Costs of chemotherapy and radiation for late‐stage patients have increased, while costs during a lung cancer patient's six‐month terminal phase decreased over time both for patients who died of lung cancer and for patients who died of other causes, although costs decreased more quickly over the study period for nonlung cancer deaths compared with lung cancer deaths. Monthly prescription drug costs were highest during the terminal phase.Lung cancer treatment costs have been previously published for patients diagnosed between 1992 and 2002 in the United States, using SEER‐Medicare data and a self‐control method with similar phase of care definitions.Cancer‐attributable costs determined by matching cancer patients to noncancer controls have also been published.Our granular, phase‐specific cost estimates will allow cost‐effectiveness analyses (CEA) to more accurately assess the economic value of lung cancer prevention, screening, and treatment options.This analysis is subject to certain limitations. First, we were unable to estimate costs for immunotherapy drugs, which have shown promising gains in survival for some patients, because approvals for lung cancer treatment came after the end of our study period.In this analysis, we assigned lung cancer treatment costs to phases of care by age, stage at diagnosis, histology, and treatment strategy. Baseline healthcare costs prior to lung cancer diagnosis rose, while costs during the final six months of life fell. Costs during the initial and continuing phases varied widely depending on treatment strategy. The cost of best supportive care remained stable for most groups. These cost estimates will be a crucial component of updated, comprehensive cost‐effectiveness analyses, which are essential to our understanding of lung cancer control interventions.The authors declare that there is no conflict of interest. Click here for additional data file. Click here for additional data file. Click here for additional data file.


Immune cell adaptor protein SKAP1 couples the antigen-receptor (TCR/CD3) with the activation of LFA-1 adhesion in T-cells. Previous work by ourselves and others have shown that SKAP1 can directly bind to other adaptors such as ADAP and RapL. However, it has been unclear whether SKAP1 can form homodimers with itself and the regions within SKAP1 that mediated homodimer formation.Here, we show that SKAP1 and SKAP2 form homodimers in cells. Homodimer formation of immune adaptor protein SKAP1 (SKAP-55) are mediated by residues A17 to L21 in the SKAP1 N-terminal region. SKAP1 dimer formation was not needed for its binding to RapL. These data indicate that the pathway linking SKAP1 to RapL is not dependent on the homo-dimerization of SKAP1. T-cells are activated by a combination of protein-tyrosine kinases and adaptor proteins which mediate the formation of multi-protein complexes , 2. ImmuIn this study, we have assessed whether SKAP1 and SKAP2 can form homodimers and the region involved in the dimerization. We show biochemically that SKAP1 and SKAP2 can form homodimers in the generation of signals in T-cells. Homodimer formation of immune adaptor protein SKAP1 (SKAP-55) are mediated by the N-terminal region.l-glutamine, penicillin, and streptomycin.293T cells were grown in DMEM culture medium with 10% fetal calf serum (FCS), 2 mM Antibodies to GFP and GST were from Santa Cruz. Anti-SKAP1 (BD Transduction Laboratories), anti-V5 (Invitrogen), anti-FLAG and anti-β-actin (Sigma) were purchased as assigned. HRP-conjugated secondary antibodies (1:5000) were from Santa Cruz and biotinylated secondary antibodies were from DAKO.The constructs of SKAP1 and SKAP2 were inserted into a pGEX5x-3  and into a 3×Flag-tagged as well as the vectors encoding EGFP-tagged pcDNA3.1-Hygro (Invitrogen). Site-directed mutagenesis was conducted using QuickChange protocol and Pfu Ultra II Fusion HS DNA Polymerase (Stratagene). Transfections were conducted using BTX ECM 830 electroporator as described .Precipitations were conducted by lysis in Triton X-100 lysis buffer followed by the incubation with antibody for 1–2 h at 4 °C and purification of complexes using protein G-Sepharose beads (10% w/v) as described –18. For Escherichia coli BL21 cells at 37 °C for 2 h by the addition of 1 mM IPTG. GST-fused proteins were purified with the Cell Lytic B protocol (Sigma #B7435). Cell lysates were incubated with GST fusion proteins for 3 h followed by analysis in SDS-PAGE and western blotting as described [The expression of recombinant GST-proteins was induced in escribed .To assess whether SKAP1 can interact with itself and SKAP2, each was expressed in 293T cells followed by precipitation with anti-Flag . As a control, the blotting of cell lysates showed the expression of the various Flag and GFP tagged proteins (right panel). These data demonstrate the residues in the region of A17 to L21 of the N-terminal region of SKAP1 mediates dimer formation.We previously showed that SKAP1 binds to RapL and is needed for RapL binding the GTPase Rap1 and the activation of LFA-1 adhesion. N-terminal SKAP1 domain binds to the C-terminal SARAH domain of Rap1. We also show that SKAP1 is needed for RapL binding to membranes in a manner dependent on the PH domain of SKAP1 and the PI3K pathway , 17. OthLastly, we next showed the presence of the SKAP1 dimer by blotting with anti-SKAP1 rather than antibodies to tags on the proteins Fig. . CombinaOverall, our study shows that SKAP1 forms homodimers dependent on residues A17/F20/L21 in the N-terminus of SKAP1. We previously showed that this region has alternating leucine residues and shares homology with the coiled-coil domain of SKAP-2 . Both SKBy contrast, the N-terminal region of SKAP1 binds to RapL such that a RapL mutation (L224A) abrogates SKAP1 binding and arrests T-cells even in the absence of antigen . We now Work restricted to non-lymphoid cells.


In this study, we designed and developed a novel, replication-competent, CHIKV reporter virus (CHIKV-iRFP) encoding a near infrared fluorescent protein (iRFP). In vitro and in vivo characterization demonstrated that CHIKV-iRFP retained similar replication and virulence phenotypes to its parental virus. Neonatal BABL/c mice and IFNAR−/− A129 mice were highly susceptible to CHIKV-iRFP infection. Following intracranial (i.c.) inoculation, CHIKV-iRFP efficiently replicated and disseminated into whole body, resulting in rapid death in an age-dependent manner. Remarkably, upon footpad injection, CHIKV-iRFP readily disseminated from footpad to head and whole skeleton, with a specific tropism for bone marrow. Taken together, this novel reporter virus provides a powerful tool to track real time CHIKV replication and to test the in vivo efficacy of vaccines and antiviral therapeutics.Chikungunya virus (CHIKV), a mosquito-borne alphavirus, has become an important re-emerging pathogen with its rapid spread to many non-endemic areas. The lack of effective vaccines and antiviral agents is largely attributed to the elusive infection and dissemination dynamics  Alphavirus of the family Togaviridae, is an important human pathogen transmitted by Aedes aegypti and Ae. albopictus mosquitoes in vivo v as DENV . Neverthin vivo replication efficiency of CHIKV. This finding is consistent to previous studies [in vivo viral replication, our study provides straightforward evidence that the CHKV infection severity is age- and immune-dependent. In addition, intensive fluorescence signals of CHIKV-iRFP in the muscles and bones of infected A129 mice were detected, further demonstrating CHIKV muscular and arthralgic tropism.Based on the imaging efficiency of CHIKV-iRFP reporter virus in mouse, it was shown that two factors, including age and functionality of type-I IFN signalling, are the key determinant for  studies ,34 as weThe insertion site of reporter gene in alphavirus genome is critical for the stability of reporter virus and the virulence in animals . A commoin vivo imaging in living small animals. Through investigating the imaging efficiencies of reporter virus in mice at different ages or with different immunity status, it was demonstrated that age and immunity levels of mice are the key factors for efficient in vivo replication of CHIKV. This well-established CHIKV-iRFP reporter virus system will facilitate the study of viral replication, pathogenesis and the development of antiviral therapeutics.In summary, it was shown that this novel CHIKV-iRFP reporter virus could be used for