Carcinus maenas is in the top 100 globally invasive species and harbours a wide diversity of pathogens, including viruses. We provide a detailed description for a novel bunyavirus (Carcinus maenas Portunibunyavirus 1) infecting C. maenas from its native range in the Faroe Islands. The virus genome is tripartite, including large (L) (6766 bp), medium (M) (3244 bp) and small (S) (1608 bp) negative sense, single-stranded RNA segments. Individual genomic segments are flanked by 4 bp regions of similarity (CCUG). The segments encode an RNA-dependent RNA-polymerase, glycoprotein, non-structural protein with a Zinc-Finger domain and a nucleoprotein. Most show highest identity to the ‘Wenling Crustacean Virus 9’ from an unidentified crustacean host. Phylogenomics of crustacean-infecting bunyaviruses place them across multiple bunyavirus families. We discuss the diversity of crustacean bunyaviruses and provide an overview of how these viruses may affect the health and survival of crustacean hosts, including those inhabiting niches outside of their native range. Bunyavirales was erected in 2017 to contain a group of viruses with negative sense single-strand ribonucleic acid (-ssRNA) genomes composed of 2–4 segments3. The order contains 10 families comprised of 215 recognised species3, including viruses that infect humans4, other vertebrates5, invertebrates6 and plants7 from a wide range of terrestrial, marine and freshwater ecosystems. To date, a single viral family exists to contain crustacean-infecting bunyaviruses, the Cruliviridae, which contains a single member, the ‘Wenling Lincruvirus’3.The order Carcinus maenas ; Carcinus mediterraneus (“S-virus” and “Y-organ virus”12) and Macropipus (=Necora) depurator (“S-virus”12); the cancrid crab Cancer pagurus (“Cancer pagurus Systemic Bunya-like Virus”13); the varunid crab Eriocheir sinensis (bunya-like virus GenBank accession number KM405247); and Mourilyan virus (MoV) from Penaeus sp. (bunya-like virus GenBank accession number AAY15205). Several viruses detected via broad virome screening programmes on tissue and environmental samples include the “Athtab virus” from the crayfish Cherax quadricarinatus14; “Wenzhou shrimp virus 1” and “Wenzhou shrimp virus 2” from Penaeus sp.15; and Wenling crustacean viruses 7 and 9 from unidentified crustacean hosts contained within pooled samples16. Wenling crustacean virus 9 is currently the only crustacean-infecting bunyavirus recognised by the ICTV, where it represents the sole member within the genus Lincruvirus in the family Cruliviridae3.Although numerous bunya-like viruses have been tentatively proposed to infect crustacean hosts, formal taxonomic descriptions have not been reported for most. These include several bunya-like virus infections in the portunid crabs C. maenas, C. mediterraneus, M. depurator and C. pagurus are lacking, histological and ultrastructural data have provided insight into host-pathogen interactions in these crustacean-infecting bunyaviruses. In C. maenas, infection of the cytoplasm of haemocytes and amoebocytes of the gill, inducing haemocytopoenia and subsequent reduced clotting ability in the blood is noted17. Transmission to uninfected conspecifics following injection of filtered haemolymph has been associated with high rates of mortality in experimental infections of M. depurator12. Finally, the bunya-like virus reported from C. pagurus likely infects haemocytes, with high viral burdens detectable within the haemolymph of infected crabs13. These viruses have been associated with disease when crustaceans are retained in close proximity, while little data exists for prevalence and disease in wild populations17. For those bunya-like viruses detected in crustaceans via nucleotide sequencing methods, no pathology data is available; however, the genomic information associated with these discoveries provides important insights into the potential diversity, taxonomy and evolution within the order. As an example, the Wenling Lincruvirus, sequenced from an unidentified crustacean host, showed the typical bunyavirus genome structure of three genomic segments  encoding between three and six proteins: an RNA-dependent-RNA-polymerase (RdRp); non-structural proteins; a nucleocapsid protein; and either two glycoprotein subunits or a pro-glycoprotein. Additionally, Sakuna et al.14 showed that the RdRp and glycoprotein (G1) amino acid (AA) sequences of crustacean-infecting bunya-like viruses were phylogenetically related14, except for in a few specific isolates (Wenzhou Shrimp virus 1 and Wenling crustacean virus 7).Although genomic data for the viruses infecting C. maenas collected from the Faroe Islands (north Atlantic). Our genetic, phylogenetic and developmental data show that the virus is a novel member in the order Bunyavirales and is a representative of the family Cruliviridae in a newly suggested viral genus (Portunibunyavirus) pending ICTV affirmation and is referred to herein as ‘Carcinus maenas Portunibunyavirus 1’ (CmPBV1). The information presented here updates the original morphological description of CmPBV1 (then described as an “irido-like virus”) from C. maenas18. In addition, we explore the phylogenetic and gene-similarity data for all known crustacean-infecting bunyaviruses and explore their evolutionary relationships and links to ecological effects.In this study we describe a bunyavirus from the portunid crab The development of the virus is predicted from transmission electron micrographs of infected gill tissues. Infection by CmPBV1 began with single membrane bound virions that apparently entered the host cell through endocytosis before acquiring a second membrane with low-level structural integrity, eliciting the appearance of variable shape virions Fig. . InfecteWenling Lincruvirus (accession: YP009329880)   segment, 6676 bp was represented by 3470X sequence coverage. The medium (M) segment, 3244 bp was represented by 2439X sequence coverage. The small (S) segment, 1608 bp was represented by 10274X coverage (~3X greater coverage than the M and L segments). Each genomic segment began and ended in complementary RNA sequences of “3′-CAGG–CCUG-5′”, respectively  and, that CmPBV1 and Wenling Lincruvirus branched adjacent to the Fimoviridae and Peribunyaviridae with low bootstrap confidence (54%). The Wenzhou shrimp virus 1 groups within the Phenuiviridae with high support for the species representing this family (100%)  all showed 33–36% RdRp similarity  and, to CmPBV1 (~65% pairwise similarity)  showed highest identity to the Kaisodi virus; however, the Wenling crustacean virus 7  does not yet have a sequenced glycoprotein for comparison.Glycoprotein sequence comparisons included that derived from a bunya-like virus detected within the crab Bunyavirales (family Cruliviridae), originally reported during a histological screen of the shore crab Carcinus maenas18. We suggest a novel genus (Portunibunyavirus) and species  for this virus, which may represent the bunya-like virus originally identified by Bang8.This study explored the genomic arrangement, phylogeny and putative development of a novel member of the Wenling Lincruvirus’ group (Cruliviridae), the ‘Athtab-Wenzhou shrimp virus 2’ group (higher taxonomy N/A), and the ‘Wenling crustacean virus 7-Wenzhou shrimp virus 1’ group within the Phenuiviridae ; Carcinus mediterraneus (“S-virus” and “Y-organ virus”12); Macropipus (=Necora) depurator (“S-virus”12); Cancer pagurus (“Cancer pagurus Systemic Bunya-like Virus”13); the bunya-like virus from the Chinese mitten crab, Eriocheir sinensis (NCBI accession: KM405247); and MoV in penaeids (NCBI accession: AAY15205). The amino acid sequence identity is 91% similar between the glycoprotein precursor of the E. sinensis bunya-like virus and Wenling Lincruvirus, supporting its inclusion in the Cruliviridae. The partial sequence data for MoV suggests it is a strain of the Wenzhou shrimp virus 1  and another potential crustacean-infecting member of the Phenuiviridae.Using the genome of CmPBV1, the 18. Infection resulted in eosinophilic inclusions within gill epithelia, connective tissues, amebocytes and haemocytes, caused by the development of viral paracrystalline arrays  were detected in the space between infected cells; and apparently exited the cell to enter the intercellular space via a putative developmental cycle similar to other bunyaviruses  in the intertidal area of coastal Nesvík, Faroe Islands, which was the only location that clinical signs of infection were detected in a multi-country  studyays Fig. 18. Virio/181 in tBunyavirales, as relatives to the Peribunyaviridae, Fimoviridae and Tospovirus or, in the case of Wenling crustacean virus 7 and Wenzhou crustacean virus 1, are present within the family Phenuiviridae  and the Cruliviridae as an early branching group before the Fimoviridae and Peribunyaviridae  are some of the earliest hosts for bunyaviruses, further indicating that marine settings may be a potential source of yet-undiscovered bunyavirus diversity, which will greatly benefit further taxonomic understanding of this order and its origin(s).Bunyaviruses isolated from crustaceans are present across the dae Figs , 5. Our ies Fig. . ConcateCarcinus maenas  has negatively impacted biodiversity and aquaculture/fisheries on shoreline habitats and has been identified as a carrier of pathogenic species, from viruses to parasitic Metazoa24. This host has been found to introduce parasites and pathogens to multiple locations and the biocontrol of C. maenas via parasites has been considered26. CmPBV represents a pathogen that could co-invade with C. maenas and interact positively or negatively with local fauna in novel settings. Bunyaviruses can cause mortality in humans, animals and plants but little is known about those infecting Crustacea, meaning that an attempt to predict a co-invasion effect is difficult. In Australia, the native red clawed crayfish Cherax quadricarinatus (a fecund and wide-spread freshwater invader elsewhere) has a bunyavirus that causes host mortality when viral copies exceed 106, resulting in 20–40% population mortality over 3-weeks14. Just as CmPBV1 was identified from C. maenas native to the Faroe Islands, the presence of two crustacean bunyaviruses restricted to their hosts native range suggest they may be commonly left behind during an invasion, likely by dynamics surrounding the enemy release hypothesis27. Experimental data on the host range and pathological effects of CmPBV1 are now required to further understand how it may affect other natural populations and communities, as well as fisheries and aquaculture industries.From the perspective of the host, 8 (despite a different geographic location) and now has corresponding genomic, ultrastructural and pathological information, which has provided insight into its systematics. Confirmation of this re-discovery requires the application of diagnostics to the original material, or recollection of specimens from the same population used by Bang8. The identification of a bunyavirus genome from C. maenas has provided additional taxonomic clarification to the crustacean-infecting bunyaviruses and has revealed that crustacean-infecting bunyaviruses are present across the Bunyavirales. Whether this virus poses certain threats to native fauna across the globe, or could be used to control C. maenas, requires exploration. Invaded areas with high densities of C. maenas provide interesting study populations to determine whether the virus is present outside the host’s native range and whether it may be applicable to manage the invasive species in non-native habitats.In conclusion, CmPBV1 is likely a re-discovered virus based on the initial observations by BangCarcinus maenas (n = 181) were collected by hand from the shoreline at Nesvík, Faroe Islands  in August 2014. Dissection of samples for histology, electron microscopy and molecular diagnostics has previously been detailed by Bojko et al.18. During the study, Bojko et al.18 recorded histopathology consistent with virus infection in the gills of two crabs. Viral aetiology was confirmed using transmission electron microscopy (TEM); however, no genomic data associated with this virus was generated. Bojko et al.18 reported the finding as ‘iridovirus-like’ due to the morphological characteristics of virions and their presence within the cytoplasm of infected cells within paracrystalline array. Samples collected at the time, fixed in 99% EtOH and stored at −20 °C, were utilised in the current study as well as TEM grids from the Cefas Registry of Aquatic Pathology.28 using the C. maenas transcriptome (accession: GBXE00000000.1)29. De novo assembly of the remaining paired-end reads was performed in SPAdes v3.5.0 (default parameters) and provided 73519 contigs  30. BLASTX analysis of the resulting contigs was performed in Blast2GO against the National Centre for Biotechnology Information (NCBI) GenBank non-redundant (nr) protein database. Protein annotation was completed using ExPASy in addition to protein domain assessment32. The integrity of the genome sequence was verified by mapping raw reads using Bowtie 233 and inspecting the alignment in Tablet v1.17.08.1734. The viral genome was annotated using CLC genomics workbench and the functions were predicted based on BLASTP searches against the NCBI GenBank nr protein sequence database, ExPASy and VIRALpro35. The annotated genome is available under Bioproject SUB5372281, Biosample SUB5372296 and accession numbers L (MK861116), M (MK861117), S (MK861118) through NCBI.RNA (86 ng/µl) was extracted from EtOH-fixed gill tissue using a Zymo RNA extraction kit (Quick RNA miniprep kit). A cDNA library was generated using a NEBNext Ultra II RNA Library Prep Kit for Illumina and sequencing was performed using a V3 600 cycle kit on a MiSeq sequencer (Illumina). Raw data were processed to remove host reads by first running KRAKEN v.236 and the phylogenetic tree was constructed using IQ-Tree37 with Bayesian information criterion to determine the best model fit and 1000 non-parametric bootstraps to test the robustness of the clades.Maximum Likelihood phylogenetic analysis was performed on the full-length AA sequence of the L protein (RdRp) for 100 representative bunyaviruses including 6 available crustacean bunyaviruses and a single outgroup glycoprotein sequence from the L and M segments respectively to produce a tree consisting of 31 viral taxa across 13 bunyavirus families. The second tree utilised RdRp, (pro)glycoprotein and nucleocapsid protein from the L, M and S segments respectively to produce a tree consisting of 29 viral taxa. For the concatenated trees, the AA sequence alignments were performed for each gene via MAFFT v5.8 using default parameters40.For genetic analyses, two protein distance matrices were constructed using the RdRp [10 bunyaviruses (6 crustacean-infecting viruses)] and the glycoprotein [11 representatives from closely related families (6 crustacean-infecting viruses)] using the Sequence Demarcation tool v1.2Supplementary Dataset 1Supplementary Figure 1


Congenital Zika virus syndrome consists of a large spectrum of neurologic abnormalities seen in infants infected with Zika virus in utero. However, little is known about the effects of Zika virus intrauterine infection on the neurocognitive development of children born without birth defects. Using a case-control study design, we investigated the temporal association of a cluster of congenital defects with Zika virus infection. In a nested study, we also assessed the early childhood development of children recruited in the initial study as controls who were born without known birth defects,. We found evidence for an association of congenital defects with both maternal Zika virus seropositivity (time of infection unknown) and symptomatic Zika virus infection during pregnancy. Although the early childhood development assessment found no excess burden of developmental delay associated with maternal Zika virus infection, larger, longer-term studies are needed. Zika virus is a mosquito-vectored flavivirus first isolated in 1947 in the Zika forest in Uganda  by Because a rate of ≈50% Zika virus seropositive mothers was expected in the control group of the case-control study, an additional cross-sectional study was designed to assess whether Zika virus infection in the mother was associated with delayed or abnormal early childhood development (ECD) in the child. Two nurses were trained to conduct anthropometric and neurocognitive testing. ECD was then compared between children with seropositive or seronegative mothers.Toxoplasma gondii and rubella virus during pregnancy from medical files; information on cytomegalovirus (CMV) seroconversion was available for case-patients only. We assessed exposure to deltamethrin by spatiotemporally linking outdoor spraying by the vector control teams with the mother’s residence address during pregnancy. We stratified maternal socioeconomic status as low, medium, or high, adapting from the 4-factor Hollingshead scale  during pregnancy. We retrieved information on seroconversion for >20 as seropositive and those with a titer <20 as seronegative.We detected anti–Zika virus and anti–dengue virus (DENV) neutralizing antibodies in serum from mothers of the case-patients and the controls by using seroneutralization tests, as previously described (http://www.ged-cdas.ca), to assess ECD. The Child Development Assessment Scale, which is available in French and is well correlated with the Bayley scale 3rd edition  using WHO Anthro software  based on serology results; and reported Zika virus infection, a categorical variable, which used information reported by the mothers (Zika-like illness during or outside pregnancy), associated with serology results, and divided into symptomatic infection during pregnancy; asymptomatic infection, timing unknown; and no infection (seronegative mothers) and symptomatic infection when not pregnant. Zika-like illness corresponded to a clinical diagnosis of Zika virus disease or a recalled infectious episode characterized by rash, fever, or both associated with 2 test to compare categorical variables. We estimated the association between CNS congenital defects and maternal Zika virus infection by matched crude and adjusted odds ratios (ORs) with 95% CIs. We maximized the study power for the case-control study, for which we aimed to include all eligible cases (n = 25), and we chose a control–case ratio of 5:1 to have 98.5% power to detect a bilateral (α = 5%) significant difference, assuming 50% of controls , as well as parity and history of congenital defects in the family. Variables at p<0.2 in the univariate analysis were further tested by multivariate conditional logistic regression and were retained in the final model if p<0.1. When data sparsity did not allow adjustment for more variables, we retained only those at p<0.05; we grouped maternal middle and high socioeconomic status categories together and compared them with low socioeconomic status. We used a Wilcoxon signed-rank test to compare means between cases and controls and Pearson χd = 0.32 at α = 0.05. The observed effect size (Cohen d) for the cognitive domain is 0.31, and 0.2 for the motor dimension. For the affective dimension, the effect size (ϕ coefficient) is 0.03.The main outcome was a binary variable derived from the results of the Child Development Assessment Scale . We performed a multivariate logistic regression analysis to assess the associations of ECD with maternal Zika virus seropositivity and reported Zika virus infection. We used the same method as described in the preceding section, with breastfeeding as an additional risk factor/confounder. We performed statistical analyses using Stata version 13.0 . For the comparison between the French-speaking Canadian children (n = 269) and the sample of our cross-sectional study (n = 107), we had a power of 80% to detect an effect size The cross-sectional study on ECD among controls was approved on June 3, 2016. We obtained written informed consent from all mothers.We identified 26 case-patients; 1 case-patient was later found to have a congenital defect with genetic etiology and thus was no longer eligible and was excluded from the analysis, along with the related 5 controls. The mother of 1 child with microcephaly, already described by Besnard et al. . Thus, aWe recruited 5 controls per case-patient for all but 2 case-patients; because of the tight matching and the lack of suitable controls, 1 case-patient had 4 matched controls and 1 case-patient had 3. All controls and 17 case-patients were from Tahiti or Moorea . >1 CNS abnormality. For 10 case-patients, pregnancy resulted in termination; of the remaining 11 newborns, 7 were still alive as of July 2017  of the case-patients and 60% (61/102) of the controls; seroconversion occurred during pregnancy for 1 control. Rubella serology was positive for 81% (17/21) of the case-patients and 94% (96/102) of the controls; no seroconversion occurred in any group. We performed maternal CMV serologic testing for 86% (18/21) of the case-patients: 2 were positive without signs of recent seroconversion, but further testing for CMV in amniotic fluid was negative . We classified mothers of 38% of case-patients and 17% of controls as having had symptomatic Zika virus infection during pregnancy and mothers of 57% of case-patients and 60% of controls as having had asymptomatic infection  in a cross-sectional study and assessed them using the Childhood Development Assessment Scale. Of these children, 44 (41%) were girls, 17 (16%) were born prematurely, and 12 (11%) were classified as having low birthweight . Except for 1 low birth length baby, anthropometry at birth was within reference range; at the time of evaluation, none of the children was underweight, had low length for age, had low weight for length, or had microcephaly  Table 1.We noted evidence for a difference between mean scores of the study participants and the reference population (French-speaking Canadian children) only for the cognitive domain (p = 0.001)  Table 2.We report a case-control study assessing the role of Zika virus infection in CNS malformations, including, but not limited to, microcephaly, in newborns and fetuses whose gestation occurred during the Zika virus outbreak in French Polynesia during 2013–2014. We also report a population-based developmental assessment of children born without birth defects after a Zika virus outbreak.At the time of the outbreak in French Polynesia, risks associated with reported Zika virus infection were unknown. Four years later, 1 case-control study from Brazil provided evidence that Zika virus infection during pregnancy is associated with microcephaly, and a few cohort studies described Zika virus–associated adverse pregnancy outcomes , which makes the study underpowered to detect a strong association between maternal Zika virus seropositivity and birth defects, as illustrated by the 95% CI crossing the null value. Another limitation is how the exposure was assessed. We used maternal Zika virus seropositivity as a proxy for Zika virus infection during pregnancy, but it is probable that some women were infected with Zika virus outside the gestational period. However, misclassification of mothers, which is likely to be nondifferential assuming they were infected during pregnancy when, in fact, they were infected outside the gestational period, would weaken any association between fetal CNS abnormalities and Zika virus seroconversion. We adjusted for the rapid variations in exposure over a short time by matching controls to cases by date of conception.Our second measure of exposure, reported Zika virus infection, includes a rough estimate of time of infection (outside or during pregnancy), based on serology data combined with recalled information. This measure may be susceptible to recall bias, because mothers of case-patients are more likely to recall Zika-like illness during pregnancy. However, serology data were compatible with recalled information for both cases and controls (only 1 of 26 mothers who reported Zika-like illness during pregnancy was seronegative). Because of the geographic spread of French Polynesia and the lack of funding, controls could not be residents of any island other than Tahiti or Moorea, which contain >70% of the overall population. We also excluded private hospitals, where 40% of deliveries occur. Therefore, it is likely that socioeconomic status confounded the associations, which is why we adjusted for maternal socioeconomic status in the analysis.Our study confirms the association between maternal Zika virus infection and CNS congenital defects. Among children with no known congenital defects, we found no evidence that congenital Zika virus infection had a major negative effect on the early stages of childhood development. Because the first large Zika virus outbreak occurred in French Polynesia about 2 years before the Zika outbreaks in Latin America, children exposed to Zika in utero in French Polynesia are now older than those in other countries, but it may still be early to detect subtle developmental delays. Although our data are encouraging, systematic in-depth assessment of childhood development in larger cohorts of exposed children, and at older ages, is needed to detect potential developmental and learning delays.Anthropometric scores for case-patients and reference children in study of Zika virus in French Polynesia, 2013–2016.


Glycogen storage disease type I (GSD I), also known as von Gierk disease, is a metabolic disorder leading to the excessive accumulation of glycogen and fat in organs, characterized by hepatomegaly, hypoglycemia, lactic acidemia, hyperlipidemia, hyperuricemia, puberty delay and growth retardation, which can be indicated by height, weight, blood glucose and blood lipids.Here we present a 16-year-old male patient with GSD Ia complicated with hepatic adenoma and combined with hepatitis B. As a chronic hepatitis B patient, the patient was admitted to hospital in order to further clarify the nature of hepatic space occupancy because of suspicion of hepatocellular carcinoma. However, the imaging studies did not support hepatocellular carcinoma certainly. And by tracing his clinical history, we suggested that he might suffer from GSD I. Finally the diagnosis was confirmed by MRI (Gd-EOB-DTPA), liver biopsy and whole exome sequencing (WES).The WES discovered a homozygous point mutation at the exon 5 of G6PC gene at 17th chromosome, c.G648 T . This pathogenic mutation causes CTG changing to CTT at protein 216. Though both codons encode leucine, this silent mutation creates a new splicing site 91 bp downstream of the authentic splice site. According to previous research, this mutation is a disease causal variant for GSD Ia, and has a high frequency among GSD patients in China and Japan.This patient was finally diagnosed as GSD Ia complicated with hepatic adenoma and combined with chronic hepatitis B, and received corn starch therapy immediately after GSD was suspected.After receiving corn starch therapy, the height and weight of the patient were increased, and the secondary sexual characteristics were developed, including beard, pubic hair and seminal emission. Unexpectedly, the liver adenomas were still increasing, and we did not find any cause to explain this phenomenon.This patient was diagnosed as GSD Ia combined with chronic hepatitis B, who responded to corn starch intervention. For childhood patients with hypoglycaemia, hyperlipidemia, puberty delay and growth retardation, GSD should be considered. Gene sequencing is valuable for the quick identification of GSD subtypes. Glycogen storage disease type I (GSD I), also known as von Gierk disease, is an autosomal recessive disorder resulting from the deficiency of glucose-6-phosphatase (G6Pase) activity . G6Pase Some clinical manifestations are similar in forms of GSD associated with liver, including GSD 0, GSD III, GSD IV, GSD VI, GSD IX, GSD XI, but the disease-causing genetic mutations and the treatment of them may be different. To improve the understanding of this disease, we report here a case of GSD Ia hospitalized in Southwest hospital in November, 2016. This study was approved by the ethics committee of Southwest Hospital, Chongqing, China. Informed consents were obtained from his parents. The study protocol conforms to the ethical guidelines of the 1975 Declaration of Helsinki.A 16-year-old male patient was admitted to the Department of Infectious Disease, Southwest Hospital for defining the nature of his space-occupying lesions in liver on November, 2016. He was diagnosed as hepatitis B around 1-year-old . The patient had not received any treatments due to the poor local medical condition, but he had regular visits in several hospitals. On June, 2015, he was found to have hepatomegaly and multiple space-occupying lesions in liver by ultrasonography, and was considered the possibility to suffer hepatocellular carcinoma.Physical examinations showed this 16-year-old boy was 135 cm in height and 29 kg in weight, below the average values of peers. And his secondary sex characteristic was undeveloped. His liver could be palpable 2 cm below the right rib and 4 cm below the xiphoid, with rigidity and blunt edge. The marked percussion tenderness over liver region was present.Imaging studies showed some evidence supporting hepatocellular carcinoma, as well as some evidence didn’t support it. Upper abdominal enhancement CT scan showed chronic liver disease performance and nodular low-density shadows in the left and right posterior lobe of the liver. Contrast-enhanced ultrasonography of the abdomen showed the space-occupying lesions with high central density and low density rings around. Significant enhancement of high central density was seen in arterial phase, persisting in portal phase and equilibrium phase. While low density rings enhancement was insignificant.Laboratory examinations showed decreased blood testosterone , blood estradiol , insulin-like growth factor-1 and increased 8:00 cortisol .Tracing the clinical history of the patient, we found that he was admitted to hospital (the detail is unreachable) for growth retardation on July, 2015. The Bone age test indicated that the left hand development maturity score was 714, which equals to a 11.6-year-old male’s bone age. And the lab examinations were showed in Table According to these evidences, he was suspected of suffering from glycogen storage disease type I instead of hepatocellular carcinoma.To confirming the doubt of GSD I, the patient underwent the Gd-EOB-DTPA tumor specific examination and liver biopsy.The Gd-EOB-DTPA tumor specific examination suggested glycogen accumulation Fig. . As showThere are 12 subtypes of glycogen storage disease, and their genetic variation, treatment, prognosis, diet intervention are different. So it’s necessary to confirm the subtype of glycogen storage disease.After informing the patient and family, and obtaining the signed informed consent, we collected the peripheral blood of the patient and his father, mother and two sisters (one elder sister cannot be collected for marrying to other province), and extracted DNA from white blood cells. Due to various types of glycogen storage disease involving many genes, and nonspecific symptoms easily confused with other liver metabolic disease, so we first sequenced the entire exome of the patient to find the mutant gene, and then used first generation of sequencing to verify the mutation in the patient and his families. The exome sequencing applied Illumina Hi-seq using Agilent Surelect Kit, and the platform for Sanger sequencing is Applied Biosystems® 3730 DNA Analyzer by using BigDyeTM Terminator v3.1 Cycle Sequencing Kit.The result of exome sequencing suggests that there was a homozygous mutation c.G648 T  on exon 5 of G6PC gene rs80356484), which causes CTG changing to CTT at protein 216 and creates a new splicing site 91 bp downstream of the authentic splice site, though both codons encode leucine [56484, whAccording to clinical manifestations, auxiliary examinations, tissue pathology and genetic testing, this patient was diagnosed as a GSD type Ia complicated with hepatic adenoma, and combined with chronic hepatitis B.The patient was treated by corn starch treatment  and practiced low fat diet immediately after GSD was suspected. Since then, he was followed up regularly in Department of Infectious Department of Southwest Hospital. The examination data of the patient were shown in Table During follow-up, the patient stopped corn starch diet for about three months, and the laboratory measures showed deterioration in July, 2017.Unexpectedly, we found that the size and the number of hepatic adenomas were increasing during the follow-up. In November, 2017, the patient had an indication for surgery as MRI imaging showed the largest one of hepatic adenomas had reached 3.1 × 3.4 cm, he was therefore admitted to the Department of Hepatobiliary Surgery, Southwest Hospital for radiofrequency ablation and liver biopsy. The postoperative pathological result confirmed as hepatic adenoma.This case was diagnosed as GSD type Ia complicated with hepatic adenoma and combined with chronic hepatitis B.The patient showed the most significant characteristic of GSD type I, growth retardation, because kidney injury causes the uric acid depositing in the joints, resulting in osteoporosis . AlthougSome of GSD I patients will manifest diarrhea and skin cholesterol deposition (xanthoma) with age . Other cThe genetic variation of the patient reported here is a homozygous point mutation at the exon 5 of G6PC gene at 17th chromosome, c.G648 T . This pathogenic mutation causes CTG changing to CTT at protein 216. Though both codons encode leucine, this silent mutation creates a new splicing site 91 bp downstream of the authentic splice site, and has a high frequency in the population of Chinese and Japanese glycogen storage disease type Ia patients [This case was diagnosed as GSD type Ia complicated with hepatic adenoma, combined with hepatitis B based on: (1) HBsAg positive and HBV-DNA positive; (2) clinical manifestations: growth retardation, facies hepatica, hepatolienomegaly; (3) laboratory examination: fasting hypoglycemia, postprandial hyperglycemia, transaminase abnormality, hyperlipidemia, hyperuricemia, and no neutrophils deficiency; (4) imaging examination: Gd-EOB-DTPA MRI indicated glycogen accumulation in the liver, and ultrasonography considered fatty liver and hepatic adenoma; (5) liver biopsy: PAS staining suggested a large deposition of glycogen in hepatocytes; (6) gene detection: G6PC gene mutation, and related reports existed; (7) no evidence of other types of liver disease.However, the deficiency of our report is that there was no activity detection of related enzymes, especially the enzyme activity detection of liver tissue. After receiving corn starch therapy, the liver adenomas are still increasing, and we did not find any cause to explain this phenomenon.This patient was diagnosed as GSD Ia combined with chronic hepatitis B, who responded to corn starch intervention. For childhood patients with hypoglycaemia, hyperlipidemia and growth retardation, GSD should be considered. Before the cause of growth retardation is identified, the application of growth hormone should be careful. Although gene sequencing is valuable for the quick identification of GSD subtypes, it is usually not required when suspicious clinical and biochemical evidences are apparent. If we encounter adolescent chronic hepatitis B patients with growth retardation, hypoglycaemia and hyperlipidemia, we must consider diagnosis of GSD as soon as possible, to avoid misdiagnosis due to inertial thinking. When GSD I is suspected, treatments should be started without any delay before confirmation tests are applied.


Dear Editor,Mammarenavirus genus, Arenaviridae family. Arenaviruses are classified into two main groups—Old World (OW) and New World (NW)—based on virus genetics, serology, antigenic properties and geographical relationships. The OW LASV and Lujo virus (LUJV), as well as NW Junín virus (JUNV), Machupo virus (MACV), Guanarito virus (GTOV), Sabiá virus (SABV) and Chapare virus (CHAPV), are known to cause severe hemorrhagic fever and are listed as biosafety level 4 (BSL-4) agents. The arenavirus glycoprotein complex (GPC) contains three subunits—the retained stable-signal peptide (SSP), the receptor-binding subunit GP1, and the membrane fusion subunit GP2  belongs to the 50 values < 1 μmol/L are presented in Table S1. Hit compounds 21, 29 and 57 exhibiting robust inhibition of the LASV pseudotype virus  entry with IC50 values lower than 0.2 nmol/L  was used as a control. As a result, two non-synonymous substitutions—L428S and F446L—were obtained in the compound 21 and the compound 29, 57 and 72 treatment groups, respectively  in the presence of 1 μmol/L of any of the compounds 21, 29, and 72, or 10 nmol/L of compound 57, respectively, which approximately corresponded to the ICt-Bu) moiety in compound 57 might broaden the antiviral spectrum of the backbone . Moreover, compound 57 blocked NW GPC-mediated membrane fusion. As shown in Figure Since the parent compound, ST-161, possessed specific antiviral activity against LASV, we investigated whether the four hit compounds extended their antiviral activities to other pathogenic arenaviruses. As shown in Figure S2, compounds 21, 29 and 72 largely maintained LASV specificity. In contrast, compound 57 showed promising inhibitory effects on the entry of NW pathogenic viruses, with a sharp blockage on the entry of GTOVpv and MACVpv in a picomolar range, as well as CHAPVpv, JUNVpv and SABVpv in a nanomolar range, suggesting that the tert-butyl , the National Natural Sciences Foundation of China (Grant No. 31670165), the Open Research Fund Program of CAS Key Laboratory of Special Pathogens and Biosafety, Wuhan Institute of Virology, and the Open Research Fund Program of Wuhan National Bio-Safety Level 4 Lab of CAS (NBL2017008), the Open Research Fund Program of the State Key Laboratory of Virology of China (2018IOV001).Guangshun Zhang, Junyuan Cao, Yan Cai, Yang Liu, Yanli Li, Peilin Wang, Jiao Guo, Xiaoying Jia, Mengmeng Zhang, Gengfu Xiao, Yu Guo and Wei Wang declare that they have no conflict of interest.Supplementary material 1 (PDF 819 kb)Below is the link to the electronic supplementary material.


Experimental studies have shown that the size and infectious-stage of viral inoculum influence disease outcomes in rhesus macaques infected with simian immunodeficiency virus. The possible contribution to disease outcome of antibody developed after transmission and/or present in the inoculum in free or bound form is not understood. In this study, we develop a mathematical model of virus-antibody immune complex formation and use it to predict their role in transmission and protection. The model exhibits a bistable switch between clearance and persistence states. We fitted it to temporal virus data and estimated the parameter values for free virus infectivity rate and antibody carrying capacity for which the model transitions between virus clearance and persistence when the initial conditions (in particular the ratio of immune complexes to free virus) vary. We used these results to quantify the minimum virus amount in the inoculum needed to establish persistent infections in the presence and absence of protective antibodies. Uninfected CD4 T cells are produced at rate s = 104 per ml per day  = 0 molecules per ml. Donor antibodies decay at rate dA = 0.07 per day , we can ignore one of them. For simplicity, we set α = 0. Immune complexes dissociate at rate km = 100 per day +V(0) = D(0)/300 copies per ml, where D(0) is the inoculum vRNA  and XD(0) vary.When an animal is challenged with SIV, the inoculum plasma may contain both free virus or donor's immune complexes. As in Vaidya et al. , we assu1) ml per day per virion, antibody carrying capacity K molecules per ml, and antibody independent expansion rate r per day are unknown and we estimate them through data fitting. All fixed parameters and initial conditions are presented in Table We assume that the free virus infectivity rate β>β ml per dr by simultaneously fitting VT(t) = V(t)+XD(t)+XR(t) given by model (6) with known total virus initial conditions to both chronic and no-infection virus data. Two monkeys (35036 and 33815) were protected when challenged with 2 ramp-up vRNA and chronically infected when challenged with 20 ramp-up vRNA. We use these to get initial concentrations of VT(0) = 2/300 and VT(0) = 20/300 copies per ml, respectively. When a monkey is protected, no vRNA data is collected. We create an artificial data set VTiterlow = {2/300, 1/300} copies per ml at times τj∈{0, 30} days in each animal that did not get infected. When a monkey gets infected, total virus concentrations VTiterhigh(ti) above the limit of detection were collected at ti∈{2, 5, 7, 9, 12, 14, 21, 28} days post infection for each subject.We estimate parameters β and VT(0) = 150/300 and VT(0) = 1, 500/300 copies per ml for VT(0) in model (6). When a monkey is protected, no vRNA data is collected. We create an artificial data set VTiterlow = {150/300, 1/300} copies per ml at times τj∈{0, 30} days in each animal that did not get infected. When a monkey gets infected, total virus concentrations VTiterhigh(ti) above the limit of detection were collected at ti∈{5, 7, 9, 12, 14, 21, 28} days post infection for subjects 33952 and 34373, and Similarly, three monkeys  were protected when challenged with 1.5, 15, and 150 set-point vRNA and chronically infected when challenged with 1500 set-point vRNA. We therefore use initial concentrations We use the “fminsearch” algorithm in MATLAB R2016b  to minimize the functionaln is the number of data points. Finally, VT(ti) and VT(τj) are the theoretical predictions for the total viral concentration as given by model (6) at times ti and τj.where S0 =  and a positive chronic state S1 = . We can show analytically that steady state S0 is locally asymptotically stable when The model exhibits bistable switch between a clearance state Inequality (8) shows that even if viremia occurs in the absence of antibodies, S1 is asymptotically stable as well. That means that given S1 can be reached. We numerically investigate the relationship between the model's initial conditions, animal data and the model's long-term behavior.XD(0) = 0 and V(0) = 20/300 copies per ml in animals 35036 and 33815; V(0) = 2/300 copies per ml in animal 32970. The best estimates for β and r were obtained by minimizing J given by (7) for the above initial conditions . Following challenge they became viremic. In contrast, animal 32970, who was challenged 2 vRNA copies from the same plasma pool, did not develop a persistent infection  = 20/300 copies per ml, the total virus load increases to a maximum of 2 × 108 copies per ml, before decreasing to equilibrium levels of 9 × 106 copies per ml, three months after infection. By contrast, when V(0) = 2/300 copies per ml, the total virus concentration grows to 850 copies per ml four days following infection, before it decays below the limit of detection of 50 vRNA copies per ml.Our model predicts a bistable switch in the virus dynamics between persistence and clearance when the free virus initial concentration changes from 20/300 to 2/300 copies per ml  seven days after infection. When V(0) = 20/300 vRNA copies per ml, free virions bind recipient antibodies to form recipient immune complexes which, at equilibrium, exceed the free virus concentration 7.8 times. When V(0) = 2/300 copies per ml, immune complexes are formed, but they decay below limit of detection 7 days after inoculation. Model (6) assumed that B-cell priming by the virus is followed by an antigen-independent antibody expansion with a maximum per capita growth rate r. We estimated a median per capita growth rate r = 2.14 per day, corresponding to the doubling time of antibody population of 7.8 h.The antibody populations do not depend of the initial viral inoculum  < 17.5/300 copies per ml and persists otherwise.We are interested in determining the largest initial virus inoculum that allows for viral clearance under the ramp-up-stage modeling assumptions. For the median V(0)+XD(0) = 1500/300 copies per ml and clearance for V(0)+XD(0) = 150/300 copies per ml (and consequently for 1.5/300 and 15/300 vRNA copies per ml).Animals 33952, 34846, and 34373, were challenged with vRNA copies titrated from a plasma pool containing virus collected during the set-point-stage of infection  of the seven donor monkeys /V(0) = 7.8, as predicted by the model (6) fitted to ramp-up data and run to equilibrium values. We minimize functional J given by (7) with XD(0)/V(0) = 7.8. The estimated parameters are presented in Table As before, we assume that the donor's antibody concentration is below the limit of detection. However, there are 7.8 times more immune complexes than free virus in the inoculum plasma, r is 1.55 times smaller, corresponding to a median antibody population's doubling time of 12 h. We decided to compare median values, rather than averages, since the set-point plasma results are biased by the estimates in animal 34846 whose virus growth is delayed.We predict a bistable switch between persistent and cleared virus populations based on initial conditions  ≤ 42/300 vRNA copies per ml, XD(0)/V(0) = 7.8 and persists otherwise. We also investigated the relation between viral clearance, VT(0) and the ratio XD(0)/V(0). We note that when the ratio between the immune complexes and free virus in the initial plasma is low, even a small inoculum size can create virus persistence. Conversely, when the immune complexes dominate the initial plasma, a large initial virus inoculum is needed to establish an infection  = 20/300 copies per ml and XD(0) = 0 copies per ml (see Tables AD(0) to account for donor's antibody being present in the inoculum. We find that a minimum of −6 mg per ml) are needed for clearance. This value is above the antibody's limit of detection, but more than three orders of magnitude below the antibody's equilibrium values. This suggests that even low antibody levels may help protect the host.To address this experimental setting we assume the model follows the dynamics given by median parameter values in the ramp-up case with VT given by model (6) with V(0) = 20/300 vRNA copies per ml, XD(0) = 0 vRNA copies per ml and K = 5 × 1012 molecules per ml, 10 times lower than the previous carrying capacity in ramp-up infected animals. The antigen-independent expansion rate r = 1.62 per day is 1.3 times smaller than the median per capita antibody expansion rate in the ramp-up infected animals, but similar to that of set-point infected animals  be the number of viruses and infected cells starting the SIV infection,Let nV virion and nI infected cells, be the probabilities of extinction given an infection that is started by one virus or one infected cell. Then the probability of virus persistence given where N and on the probability that a virus infects a cell, N = 300 virions per infected cell, T = 106 cells per ml, and c = 23 per day, we determine the infectivity values β that can explain the relationship between extinction/persistence and the inoculum size in ramp-up/set-point vRNA cases when free virus establishes the infection, i.e., nI = 0 , a high antibody carrying capacity K = 5 × 1013 molecules per ml and estimated the recipient antibody expansion rate needed for protection. With our model, however, it is difficult to separate out the effects of changing the avidity rate KA = kp/km and changing the antibody carrying capacity K. For example we can preserve the results by decreasing avidity and increasing the carrying capacity. Similarly, the antibody antigen-dependent and antigen-independent growth factors, r and α, have synergistic effects. If we decrease r we can maintain the overall virus-antibody dynamics by increasing α (as detailed in the sensitivity Figure We assumed that the recipient and donor antibody have high avidity of 101 = 0.1 × β ml per day per virion. This was an arbitrary choice, and the estimated infectivity rate β may change when the β to β1 ratio is varied.We have also assumed that the immune complexes infect at rate βAn important variable in our study is the basic reproduction number burst stochastic model developed in Pearson et al. (To address whether stochastic effects alone can explain the data, we computed the probability of virus persistence under the assumption of a n et al. . We findn et al. . FurtherOur study uses data from an intravenous inoculation experiment. It is not clear if a similar relationship exist with mucosal virus inoculation or with non-neutralizing antibody responses. In fact, some antibody responses were associated with enhanced risk of heterosexual HIV acquisition in RV144. Further, immune-complexed virus may be preferentially transported across epithelial surfaces by the neonatal Fc receptor Haynes, , 2015. OIn summary, we have developed a model of virus-host dynamics during SIV infection that gives insight into the relation between the structure of infecting inoculum, virus infectivity and disease outcomes. In particular, we showed that a large set-point-stage inoculum is needed for persistent infection due to the excess of immune complexes over the free virus. Moreover, we have estimated the antibody levels and the free virus infectivity and their relation to the disease outcome when the inoculum size and structure are well understood. Such predictions are important for vaccine design.We investigate whether antiviral factors can explain the change in virus infectivity during acute and chronic stages of simian immunodeficiency virus infections. Our hypothesis is that acute plasma contains mostly free virus while chronic plasma contains antibody-virus immune complexes in addition to free virus. If such immune complexes can still infect, then their infectivity rate is reduced compared to that of the free virus. We test this hypothesis by developing a mathematical model of antibody-virus dynamics that assumes interactions between virus, recipient and donor antibodies, and the corresponding immune complexes. We fit the model to published viral load data from recipient rhesus macaques challenged intravenously with different virus loads collected during donor's acute and chronic stages of SIV infection. The fits give us parameter estimates for long-run antibody concentration, free virus infectivity rate, and the relation between protection and the free virus—immune complexes ratio in the inoculum.SC and JF designed the study and performed numerical experiments. CM provided the data. SC, CM, and JF wrote the paper.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


During a waterborne outbreak of norovirus in Spain, we estimated 50% illness doses for a group of exposed (secretor) persons to be 556 (95% CI 319–957) genome copies/day for norovirus GI and 2,934  genome copies/day for norovirus GII. Use of a propidium monoazide viability assay reduced these values. Human noroviruses are a major agent of acute gastroenteritis, are distributed worldwide, and affect all age groups , determined from volunteer secretor adults challenged with a GI.1 from a stool specimen, range from 18   virus genomes/oyster consumed and a dose causing illness in 50% of exposed persons of 32 (95% CI 1.32 to >106) virus genomes/oyster consumed for norovirus GI and an ID50 of 1.6 (95% CI 0.74 to >106) virus genomes/oyster consumed and a dose causing illness in 50% of exposed persons of 4.86 (1.24 to >106) virus genomes/oyster consumed for norovirus GII . Informed written consent was obtained from all persons.>3 loose stools within 24 hours) and >2 of the following: nausea, abdominal pain, or fever (temperature >37.8°C). We collected saliva samples to determine secretor status by genotyping the FUT2 gene  to 26 persons who had been exposed to drinking water from a water cooler in Spain during 2016. Acute gastroenteritis was defined as vomiting or diarrhea  showed development of acute gastroenteritis and only GI viruses were detected in their stool. Although we could not completely rule out that GII viruses might have also infected these persons but were not detected, it is plausible that gastroenteritis in these persons was related to the GI infection. Although binding of GI.2 to nonsecretor histoblood group antigens has been demonstrated in vitro (3) and GII (5.8 × 103) had been detected. Intact virions, estimated by using a viability real-time quantitative PCR, represented <4.4% to 5.6% of the total number of genomes. We estimated 50% illness doses according to the percentages of persons with gastroenteritis and the median water volumes recorded for each group (We used data for quantification of norovirus in the contaminated water cooler to which the subjects were exposed (ch group . We calc50 and 50% illness dose data obtained by other investigators (,,Our data showed differences in infectivity between norovirus GI and GII, which differs from what was previously observed in oyster-related outbreaks (Because of the interaction of norovirus with specific carbohydrates found in oyster intestinal tissues (Because the proportion of infectious genomes might vary in each particular environmental scenario, infectivity data for norovirus relating to total and infectious genomes should be consistently investigated whenever possible for a better refinement of risk assessment approaches. Outbreaks caused by consumption of contaminated water and shellfish are often caused by multiple virus strains. Although this factor might hamper infectivity estimates, these types of studies are nevertheless valuable to risk managers and regulators for future formulation of guidelines on acceptance levels for norovirus in specific matrices.


Madariaga virus (MADV), also known as South American eastern equine encephalitis virus, has been identified in animals and humans in South and Central America, but not previously in Hispaniola or the northern Caribbean. MADV was isolated from virus cultures of plasma from an 8-year-old child in a school cohort in the Gressier/Leogane region of Haiti, who was seen in April, 2015, with acute febrile illness (AFI). The virus was subsequently cultured from an additional seven AFI case patients from this same cohort in February, April, and May 2016. Symptoms most closely resembled those seen with confirmed dengue virus infection. Sequence data were available for four isolates: all were within the same clade, with phylogenetic and molecular clock data suggesting recent introduction of the virus into Haiti from Panama sometime in the period from October 2012-January 2015. Our data document the movement of MADV into Haiti, and raise questions about the potential for further spread in the Caribbean or North America. Madariaga virus (MADV) is the name given to what used to be called South American eastern equine encephalitis virus (EEEV), based on recent studies suggesting that MADV is distinct genetically from the EEEV circulating in North America. Until now, MADV has been found primarily in animals in South and Central America, with a limited number of human cases reported . Our group has been responsible for a series of studies assessing the etiology of acute febrile illness (AFI) among children in a school cohort in Haiti. Unexpectedly, in April, 2015, we identified MADV on viral culture of plasma from a student with AFI in this cohort; an additional seven cases were identified on culture of samples from children with AFI in this same cohort in February, April, and May 2016. On sequence analysis, all strains were very similar genetically, and appear to have come from a strain introduced into Haiti from Panama sometime in the period from October 2012- January 2015. Symptoms of children were similar to those seen with dengue; none had encephalitis. Our data indicate that this virus, which has the potential for causing serious illness, has been recently introduced into Haiti, and raises the possibility that it might move into other parts of the Caribbean or North America. Togaviridae. Recent ecologic and genetic studies of eastern equine encephalitis virus (EEEV) have demonstrated clear separation between North and South American EEEV strains: North American EEEV cluster in a single genetic lineage , also known as South American eastern equine encephalitis virus, is an alphavirus in the family o et al. ), with So et al. –3. Howevo et al. . In popuo et al. ,3,5, sugo et al. . The virOur group maintains regular surveillance for AFI through a school clinic serving a cohort of approximately 1,250 children who attend schools at the four campuses within the school system operated by the Christianville Foundation (a U.S.-based non-profit organization): this includes the main campus, housing grades K-13 (School A), and three small satellite elementary school campuses . Clinic The University of Florida IRB and the Haitian National IRB have approved all protocols, and written informed consent was obtained from parents or guardians of all study participants.Whole blood was collected into yellow top (Acid Citrate Dextrose/ACD) tubes , and an aliquot used to prepare blood smears for microscopic analyses for malaria parasites. To obtain plasma for virology analyses, a portion of the collected blood was centrifuged to pellet red and white blood cells, and the resulting plasma (ca. 650μL) transferred to sterile screw-top vials and stored at -80°C pending tests.As there was a possibility that viruses such as yellow fever virus or EEEV might be present in samples, RNA extractions and virology work were performed in the Lednicky BSL3 laboratory at the University of Florida’s Emerging Pathogens Institute, Gainesville, FL. RT-PCR tests for the detection of chikungunya virus (CHIKV), dengue virus (DENV), and ZIKV-genomic RNAs (vRNAs) in the plasma were accomplished as previously described ,11. Brieet al. ;,9,10;6,9H0) in favor of the more complex model (HA), is evaluated according to the following guidelines: lnBF<2 no evidence; lnBF = 2–6—weak evidence; lnBF = 6–10—strong evidence, and lnBF>10 very strong evidence ). Six of the children were from school campus A, with one case from each of two of the other campuses. Mean age of case patients was 9 years (range 3–12 years); six were girls and two were boys.When seen in the clinic, four children had temperatures above 37 degrees C; maximum recorded temperature in the clinic for a MADV case patient was 39 degrees C. The other four children were afebrile at the time of the clinic visit, but they or their parents reported subjective fever prior to coming to the clinic. Five reported cough, four reported headache, three reported nonspecific abdominal pain, and one reported myalgia; none reported arthralgias. When compared with previously reported symptom profiles of children with AFI in this cohort , symptomOne patient, who had a prior history of seizure activity, had an episode described as syncope followed by a short period of confusion and amnesia before being brought to the clinic. On exam, one child had a rash; one had conjunctivitis; and one was noted to have swollen tonsils. Physical exams were otherwise unremarkable. All children recovered without apparent sequelae, with six of the eight having remained in the school system through the time of preparation of this manuscript (2018), with continued follow-up by the clinic.−4 nucleotide substitution rate per year, in agreement with previous estimates calculated for this virus [The topology of the ML  and MCC is virus .Reports of human infection with MADV are rare, although cross-sectional serologic studies  in Panama and Peru have reported seropositivity rates in human populations of between 2 and 5% ,5, consiet al, in their study in Panama [While most MADV case reports have involved patients with encephalitis, it is likely that the majority of infections occur in persons who are asymptomatic or who have only relatively mild disease ,3,26. Thn Panama ; as withCulex (Melanoconion) pedroi has been identified as a primary enzootic vector for MADV in the Amazon Basin [Cx. (Melanoconion) taeniopus in an epidemic outbreak in Panama [Aedes fulvus and Psorophora albigenu and Ps. ferox have been shown to be susceptible to and capable of transmitting the virus [Cx. pedroi has not been previously identified in Haiti, but there are four known species in the Melanoconion subgenus including Cx. atrutus, Cx. carcinophilus, Cx. erraticus, and Cx. pilosus that are present [Ps. ferox, which is known to be a very aggressive biter of humans. It remains to be determined if these native Melanoconion subgenus mosquito species and/or Ps. ferox serve as vectors for MADV in Haiti. In an extensive survey by Vittor and colleagues [Zygodontomys brevivauda; 8.7% seroprevalence, with one animal viremic for MADV) and the black rat . While Vittor found no evidence of infection in birds [on Basin . The virn Panama , and in he virus . Cx. ped present , togethelleagues  of possiin birds , there ain birds .In Haiti, no prior data are available on MADV in vectors, animal reservoirs, or humans. While we cannot exclude the possibility that MADV was present in Haiti before the current case cluster, our phylogenetic studies are strongly suggestive of recent introduction of the virus into Haiti from Panama. Recent work by our group –11,31,32S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S3 Table(DOCX)Click here for additional data file.S1 Fig(A) Absence of substitution saturation indicated by plotting pairwise nucleotide transition (s) and transversion (v) substitutions against the Tamura and Nei 1993 (TN93) genetic distance (B) Likelihood mapping triangles indicating presence of phylogenetic signal by reporting over 30% of alternative topologies in the tips, as compared to unresolved quartets in the center, and partly resolved quartets in the edges.(TIFF)Click here for additional data file.S2 FigThe ML tree was obtained based on the GTR+F+G4 nucleotide substitution model chosen as best-fit model according to BIC using IQ-TREE. Black circles at nodes indicate robust bootstrap support (BB> 90). Bar scale indicates genetic diversity.(TIFF)Click here for additional data file.


Virus-host interactions have received much attention in virology. Virus-virus interactions can occur when >1 virus infects a host and can be deemed social when one virus affects the fitness of another virus, as in the well-known case of superinfection exclusion. Virus-host interactions have received much attention in virology. Virus-virus interactions can occur when >1 virus infects a host and can be deemed social when one virus affects the fitness of another virus, as in the well-known case of superinfection exclusion. Coinfection and subsequent social interactions can change viral pathogenicity, host range, and genetic composition, with implications for human health and viral evolution. I propose that this field can be advanced by bringing new perspectives into virology  and uniting disciplinary divides within virology . The development of novel high-throughput tools that meld molecular and evolutionary approaches can harness viral diversity as an experimental asset to understand complex viral social interactions. A greater knowledge of virus-virus interactions will lead to the reformulation of basic concepts of virology and advances in applied virology, with new treatments that harness interactions between viruses to fight viral and bacterial infections. Virology, etymologically, means the study of viruses. However, much of the discipline has studied viruses as disease agents, often focusing on a single infectious strain and host pairing . Perhaps3–In contrast to the potential importance of virus-virus interactions, we still are in the beginning stages of knowing the frequency, mechanisms, and consequences of these interactions. Four questions are the key to determining how virus-virus interactions affect the viral life cycle and to developing fundamental principles of viral social interactions: (i) What is the extent of coinfection and are there specific ecological correlates or molecular mechanisms that promote coinfection?; (ii) What is the frequency of evolved cooperation and cheating and how are they manifested mechanistically?; (iii) To what extent can viral proteins be shared among viruses, representing potential public goods?; and (iv) Are virus-virus interactions context dependent and what are their consequences across scales , especially in natural settings? Below, I suggest two approaches to make progress towards answering these questions and highlight studies from our group and many colleagues that are advancing the field of virus-virus interactions.Studies on virus-virus interactions have been conducted for a long time, with examples ranging from early phage research  to studiThese efforts are designed to blur the sharp disciplinary boundary between “classical virology” (focused on viruses as disease agents) and virus studies that test evolutionary or ecological concepts . A poten13–The opportunity of unifying these approaches is accompanied by a technical challenge. How do we adapt the tools of classical virology to dissect the molecular mechanisms contributing to virus-virus interactions in more than just a few laboratory strains? Conversely, how do we develop additional experimental approaches applicable to the diversity of strains that evolutionary and ecological virology has uncovered in nature? Based on experiences in our lab, I suggest that high-throughput tools provide an opportunity to use viral diversity as an experimental asset to unify approaches and uncover viral social interactions.Virus-virus interactions represent an inherently complex area of virology in that they involve, at a minimum, two or more viruses. Furthermore, these interactions can change due to the ecological setting, molecular mechanisms, and population processes within and between hosts. For instance, cooperation between variants of influenza virus A were detected in cell culture, but not in human clinical samples . A seconA deliberate approach is needed to connect data from biological findings made at different scales . The samOver the next 5 years, I anticipate five major changes in the emergent field of sociovirology. First, high-throughput single-cell and subcellular techniques will yield unprecedented resolution on viruses’ interactions within their hosts. Second, viral surveys will establish the landscape for coinfection in the environment and within hosts. Third, experimental studies will be driven by theory and mathematical modeling, increasingly developing a body of work that establishes general principles of virus-virus interactions and stimulating new developments in social evolution theory. Fourth, social-evolution-based treatments  that work orthogonally to current approaches will move from the proof-of-concept stage to testing and development. Finally, these advances will collectively conspire to bring virologists of all stripes together to establish principles of virus-virus interactions, integrate them seamlessly into virology, and communicate their importance to the broader microbiological, biological, and medical communities. This future will bring a reformulation of basic concepts of virology and lead to advances in applied virology, with new treatments that harness interactions between viruses to treatments of viral and bacterial infections.


Rapid and precise manipulation of magnetic beads on the nano and micro scales is essential in many biosensing applications, such as separating target molecules from background molecules and detecting specific proteins and DNA sequences in plasma. Accurately moving magnetic beads back and forth requires at least two adjustable magnetic field gradients. Unlike permanent magnets, electromagnets are easy to design and can produce strong and adjustable magnetic field gradients without mechanical motion, making them desirable for use in robust and safe medical devices. However, using multiple magnetic field sources to manipulate magnetic beads presents several challenges, including overlapping magnetic fields, added bulk, increased cost, and reduced durability. Here, we provide a thorough analysis, including analytical calculations, numerical simulations, and experimental measurements, of using two electromagnets to manipulate magnetic beads inside a miniature glass cell. We analyze and experimentally demonstrate different aspects of the electromagnets’ design, such as their mutual influence, the advantages and disadvantages of different pole tip geometries, and the correlation between the electromagnets’ positions and the beads’ aggregation during movement. Finally, we have devised a protocol to maximize the magnetic forces acting on magnetic beads in a two-electromagnet setup while minimizing the electromagnets’ size. We used two such electromagnets in a small footprint magnetic modulation biosensing system and detected as little as 13 ng/L of recombinant Zika virus antibodies, which enables detection of Zika IgM antibodies as early as 5 days and as late as 180 days post symptoms onset, significantly extending the number of days that the antibodies are detectable. Small spherical magnetic beads—usually consisting of a paramagnetic core embedded in a non-magnetic matrix, such as a polymer or quartz ,2—are wiTo remotely manipulate magnetic beads, an external magnetic field is applied. A constant magnetic field can rotate the magnetic beads, and a magnetic field gradient exerts a force on the beads that can displace them. The design of external magnetic field sources affects the forces acting on the magnetic beads. In applications that involve separation of a target molecule from background molecules, a single magnetic field gradient source, e.g., a permanent magnet, is used. The gradient pulls the magnetic beads, which are attached to the target molecules, onto the sample holder’s wall, and the rest of the sample is then washed out. However, a single magnet can only pull the magnetic beads, but not push them away . TherefoThe manipulation of a single magnetic bead by either a single or by several electromagnets has been extensively analyzed ,5,15,22.To evaluate different shapes of magnetic pole tips, we used a single electromagnet setup. In 2.We evaluated cylindrical magnetic poles   whose maIn general, magnetic beads’ movement in a fluid is mainly affected by the magnetic force and the drag force, given by ,2,9(1)Fh forces . HoweverTo derive an analytical expression of the force acting on a magnetic bead, two assumptions were made. First, the bead is a single, zero-dimensional object. Second, the magnetic field surrounding the pole tip is sufficiently high that the bead’s magnetic moment is saturated. Without these assumptions, the magnetic moment of the bead becomes spatially-dependent rather than constant, and the calculation of the force is needlessly complicated. However, under these assumptions, the force acting on the bead depends simply on the bead’s saturation moment and the magnetic field gradient at the bead’s specific location. Overall, the force, given by : (2)Fm=(ole tips . Moreove−1) and the following dimensions: To manipulate magnetic beads back and forth along a straight line, two electromagnets that are facing each other are required. To analyze the characteristics of this manipulation, we used two identical electromagnets, labeled A and B in To demonstrate and analyze the movement of magnetic beads, we used ~30,000 magnetic beads  mixed in phosphate buffered saline with Tween-20 buffer (PBST) and placed in a rectangular bottom-sealed borosilicate sample cell whose inner dimensions were 8 mm × 0.4 mm × 70 mm . The sample cell was held between the electromagnets so that the beads could be manipulated along its narrow dimension . When the two electromagnets were alternately turned on and off, the magnetic beads moved periodically between them. The movement of the magnetic beads was imaged to the back plane of an objective lens  and visualized using a CMOS camera . When two or more electromagnets are used to manipulate magnetic beads, mutual magnetizing of the pole tips and the remnant magnetic fields of the ferromagnetic cores affect the forces acting on the magnetic beads. For example, in a two-electromagnet setup, when one of the electromagnets is active and the other is inactive, the beads are expected to move towards the active magnet. However, due to remanence and the influence of the active electromagnet, the ferromagnetic core of the inactive electromagnet is magnetized, and it attracts nearby beads. In −1. To correlate the working distance and the pole tips’ parabolic curvature, we assumed that the distance between the pole tips was 3 mm. For the correlation between the working distance and the electric signal profile, we assumed that the distance between the pole tips was 3 mm and the parabolic curvature of the tips was −1. In addition, we assumed that the electromagnets were activated by two square waves, with a 50% duty cycle and a 180° phase shift between them. The peaks of the square waves were 36 V, and the valleys ranged between 0 V to −36 V. Therefore, the ratio between the voltages of the electromagnets, First, we simulated the correlation between the working distance and different parameters, such as the distance between pole tips, the pole tip’s parabolic curvature, and the electric signal profile. To correlate the working distance and the distance between the pole tips, we assumed that the parabolic curvature of the tips was −1) 3 mm and 4 mm apart and imaged the beads’ movement inside rectangular glass cells that had various inner widths, ranging from 0.4 mm to 0.8 mm . The electromagnets were activated by two square waves, with an amplitude of 36V, a 50% duty cycle, and a 180° phase shift between them. Second, we experimentally validated the correlation between the working distance and the distance between the pole tips. We placed the magnetic poles (−1). Using XYZ translational stages, we changed the distance between the pole tips from 1 mm to 7 mm. For each distance, the beads’ movement was recorded, and the width of the neck was measured.Third, we explored how the magnetic beads move between two electromagnets with parabolic pole tips. When the two electromagnets, positioned opposite each other, were placed in proximity, the magnetic beads moved along a narrow line on the z-axis between the pole tips. This movement resembled an hourglass with a narrow neck. To evaluate the correlation between the width of the neck and the distance between the pole tips, we placed a bottom-sealed borosilicate sample cell with an inner width of 0.6 mm  between two electromagnets with parabolic pole tips  in an MMB system . The MMB−1) and the following parameters: Two electromagnets, positioned 3 mm apart (one on each side of the glass sample cell), apply an alternating magnetic field gradient to the sample, causing the magnetic beads to aggregate and move together in a periodic lateral motion in and out of the laser beam. The modulation frequency of the electromagnets was set to 1 Hz. While the beads are moving, the laser beam remains fixed, causing the beads to form an “on” signal when they are in the laser beam and an “off” signal when they have moved out of the laser beam. The new electromagnets have parabolic pole tips , there is a tradeoff between the winding area width, The dose response curves of the IgM antibodies in the small footprint MMB system (“New MMB”) and the standard MMB system (“MMB”) are depicted in Efficient manipulation of magnetic beads heavily depends on the strength and structure of the magnetic field sources. Electromagnets provide strong and adjustable magnetic fields, and their lack of mechanical movement makes them less susceptible to mechanical malfunction than permanent magnets. Three key factors affect the performance of electromagnets: (1) the magnetic properties of the electromagnet’s pole material , which affect the maximum magnetic field and the response time of the electromagnet, (2) the electromagnet’s physical parameters , and (3) the pole’s tip shape, which affects the strength and direction of the magnetic field near the pole tip. For example, the sharper the pole tip, the higher the magnetic field gradient near the tip. In applications that require high magnetic field gradients at very short distances from the pole tip  ,19, a coWhen two or more electromagnets are used to manipulate magnetic beads in space, the mutual influence of the magnetic fields dictates the maximum working distance over which magnetic beads can be manipulated without being trapped by one of the electromagnets. To enable precise manipulation of magnetic beads over a large area, increasing the working distance is desirable. Here, we showed that, for parabolic pole tips, the working distance can be broadened by increasing the parabolic curvature. However, above a certain parabolic curvature  is on par with the standard MMB system. According to recent studies , this liHere, we provide a thorough analysis, including analytical calculations, numerical simulations, and experimental measurements, of using one or two electromagnets to manipulate magnetic beads. In general, the magnetic field gradient of an electromagnet, and subsequently the force acting on magnetic beads, can be significantly maximized for a specific application by carefully selecting the pole material and the tip’s shape. In addition, the shape of the beads’ aggregate and the distance over which they can be manipulated highly depends on both the position of the electromagnets relative to each other and their relative voltage.In a two-electromagnet setup, to maximize the magnetic force acting on magnetic particles while maintaining the electromagnets’ minimal size and power consumption requirements, the following protocol can be used. First, based on power consumption constraints, select the maximum wire current,


Background: Dementia is one of the public health priorities in China. This study aimed to examine the hospitalization costs of patients with dementia and analyzed the factors associated with their inpatient costs. Methods: This was a prevalence-based, observational study using claims data derived from two urban insurance schemes during the period from 2008 through 2013 in Guangzhou. The extended estimating equations model was performed to identify the main drivers of total inpatient costs. Results: We identified 5747 dementia patients with an average age of 77.4. The average length of stay (LOS) was 24.2 days. The average hospitalization costs per inpatient was Chinese Yuan (CNY) 9169.0 (CNY 9169.0 = US$1479.8 in 2013). The mean inpatient costs for dementia patients with the Urban Employee-based Basic Medical Insurance (UEBMI) scheme (CNY 9425.0 = US$1521.1) were higher than those for patients with the Urban Resident-based Basic Medical Insurance scheme (CNY 7420.5 = US$1197.6) (p < 0.001). Having UEBMI coverage, dementia subtypes, having hypertension, being admitted in larger hospitals, and longer LOS were significantly associated with hospitalization costs of dementia. Conclusions: The costs of hospitalization for dementia were high and differed by types of insurance schemes. Dementia was associated with substantial hospitalization costs, mainly driven by insurance type and long LOS. These findings provided economic evidence for evaluating the burden of dementia in China. Due to the rapidly growing aging population, dementia is becoming a major public health issue worldwide ,2. A cliWith the growing number of patients, dementia imposes a significant financial burden on patients and their families, as well as on the health care systems. For example, in 2015, the estimated costs of dementia in the US was US$818 billion, equal to approximately 1% of the world gross domestic product (GDP) . This fiChina expands health insurance coverage to the entire urban population with two social medical insurance programs—the Urban Employee-based Basic Medical Insurance scheme (UEBMI) and the Urban Resident-based Basic Medical Insurance scheme (URBMI) . The UEBMany researchers have evaluated the direct medical costs of dementia in other countries ,15,16,17Adding to the current literature, the present study aimed to examine the hospitalization costs of patients with dementia using the urban health insurance claims data and analyzed the factors associated with their inpatient costs in Guangzhou City, Southern China.Guangzhou city, the capital of Guangdong Province, is the largest and most developed city in Southern China. Thus, the study sample mainly represents the urban population in Southern China. We chose Guangzhou city in this study for two reasons. First, from a practical standpoint, we are only able to access insurance claims data from Guangzhou city while not from other cities or provinces due to administrative restrictions. Second, insurance policies in China vary between cities, and analyzing the per-person costs from insurance claims of an entire city minimizes potential selection issues caused by population or policy differences between cities. The data for this study were derived from the UEBMI and URBMI claims databases of Guangzhou city during the period from 2008 through 2013. This was the latest and de-identified claims dataset that was available for our research, due to administrative restrictions on access to the insurance claims data. The databases contained information on sociodemographic factors, direct medical costs of inpatient care based on actual payments to providers from a total of 309 medical institutions and explicit classification of medical conditions for a large sample of hospitalized patients receiving dementia treatment in Guangzhou city. The detailed benefits and reimbursement policies of the UEBMI and URBMI schemes were summarized in A prevalence-based and retrospective approach was applied to identify the total medical costs of inpatients with dementia. The study included all inpatient records of people admitted to hospitals in Guangzhou city with the primary diagnosis of dementia. We reviewed all the reimbursement claims submitted for inpatient care during 2008 and 2013 and subjects were selected using the International Classification of Diseases Tenth Edition (ICD-10), which included AD (F03 and G30), VaD (F01) and other types of dementia (G20) . The lasThe UEBMI and URBMI claims data provided information about direct medical costs of inpatients with different kinds of dementia from the healthcare system perspective, including the reimbursement amount paid by the health insurance scheme (UEBMI or URBMI) and out-of-pocket (OOP) spending paid by the patients. According to the classification of costs used in the UEBMI and URBMI schemes, the total inpatient medical costs were categorized as laboratory and diagnostic costs, non-medication treatment costs, medication costs, bed fees and the costs of other fees, including special caring fees and air-conditioning. Laboratory and diagnostic costs were the expenses of physical examinations and biochemical tests. Medication costs were separated into Traditional Chinese Medicine and western medicine spending. Non-medication treatment costs were the costs of any other treatments except for medication, which contained blood transfusion expenses, surgery fees, anesthesia charges, and costs for medical consumables. In this study all costs from 2008 to 2012 were adjusted to 2013 Chinese Yuan (CNY) value considering the urban resident’s Consumer Price Index (CPI) of Guangzhou city . All cosThe dependent variable in this study was total hospitalization expenditures per inpatient. The primary independent variable was types of health insurance and was dichotomized as UEBMI and URBMI. Additional confounders included in the expenditure model were gender, age, disease subtype, comorbidities, hospital levels , length of stay (LOS), and years. Hospitals in China are categorized into three levels: primary ; secondary ; tertiary  .Predictors of total hospitalization costs for patients with dementia were identified based on the Andersen’s behavioural model . IndividGender was dichotomized as male vs. female. Age was categorized into five groups: 18–50 years old, 50–59, 60–69, 70–79, 80–84, 85 and older. Disease type was grouped into three subtypes: AD, VaD, and other types of dementia. Comorbidities were measured as binary variables for the following conditions—whether having a diagnosis of hypertension, diabetes, coronary heart disease. Hospital level was classified into three levels: primary (level I), secondary (level II) and tertiary (level III). The LOS was grouped into five categories: less than 10 days, 11–15 days, 16–30 days, 31–60 days, longer than 60 days. Years were measured as binary variables for controlling the impact of policy changes across the years.Descriptive statistics  and standard deviation (SD)) were used for demographic information and costs. The Kruskal-Wallis test and Friedman’s two-way non-parametric ANOVA test were used to identify the differences in inpatient costs by types of insurance, because the value of medical spending often has a skewed distribution. In order to determine the main drivers of total inpatient costs and account for the skewness of cost data, in this study we applied the extension of generalized linear model (GLM)—the Extended Estimating Equations (EEE) model . AdvantaThe present study was conducted according to ethical standards set by the institutional research committee. This research was approved by the Institutional Review Board of the School of Public Health, Sun Yat-sen University .During the study period, a total of 5747 inpatients with dementia from the UEBMI and URBMI claims data in Guangzhou city were identified see . Female Overall, the mean total inpatient costs of patients with dementia was CNY 9169.0  see ., Non-mep < 0.001). However, the percentage OOP expenses of the total costs for the URBMI scheme patients (30.0%) was nearly 2 times that for the UEBMI scheme patients (18.1%), indicating the underlying differences in benefit packages of the two insurance schemes. Regarding cost composition, the biggest cost component in the UEBMI group was non-medication treatment costs (38.7%), while the biggest cost component in the URBMI group was medication costs (37.9%).When comparing the hospitalization costs by insurance status, the mean total inpatient costs for patients with dementia under the UEBMI scheme (CNY 9425.0 = US$1521.1) was higher than the patients under the URBMI scheme (CNY 7420.5 = US$1197.6) (p < 0.01)  see . Among ap < 0.01) for the overall sample (see p < 0.01).This study found that insurance type, dementia subtypes, comorbidities, hospital levels and LOS were significantly associated with inpatient costs of dementia (mple see . Comparep for interaction <0.01)  and LOS  categories (.01) see . The modp < 0.05). Among the UEBMI subgroup, the hospitalization costs for VaD inpatients and patients with other types of dementia were CNY 1317.3 (US$212.6) and CNY 1567.3 (US$253.0) significantly higher respectively compared to the AD inpatients (p < 0.01). Patients with hypertension among the UEBMI subgroup incurred significantly higher inpatient costs (CNY 642.7 = US$103.7) (p < 0.01), while patients with diabetes among the URBMI subgroup had significantly higher hospitalization expenses (CNY 3724.5 = US$601.1) (p < 0.05). Dementia patients stayed at tertiary hospitals had CNY 6153.0 (US$993.1) higher medical expenditures among the UEBMI subgroup and CNY 5097.8 (US$822.8) higher among the URBMI subgroup, compared with patients staying at primary hospitals (p < 0.01). Among the UEBMI and URBMI subgroups, patients with longer LOS incurred significantly higher inpatient costs after adjusting for other factors. Compared with LOS less than 10 days, hospitalization costs for the longest LOS group (>60 days) was CNY 16,933.1 (US$2732.9) higher among the UEBMI patients and CNY 7500.0 (US$1210.5) higher among the URBMI patients (p < 0.01).Different findings were observed between the UEBMI subgroup and URBMI subgroup see . Gender It was strategically important to understand the hospitalization costs of patients with dementia and the key drivers of the medical expenses in China due to the high burden of the disease. The present study was an observational study conducted with a large dementia sample in Guangzhou city. We found that the mean total direct inpatient costs of dementia were CNY 9169 (US$1479.8). The non-medication treatment costs and medication costs were the biggest contributors to the total expenses. The type of insurance schemes (UEBMI), different dementia subtypes, comorbidities (having hypertension), being admitted in the secondary and tertiary hospitals, and longer LOS were significantly associated with total inpatient costs of dementia patients. When further exploring the interaction effect between insurance type and LOS, we found that the UEBMI enrolees had significantly higher hospitalization costs as compared to the URBMI enrolees when the LOS exceeded 30 days. This was the first study using data from two urban health insurance claims data of an entire city to investigate the healthcare costs for hospitalization of dementia and compare the costs under two different urban insurance schemes as well as to identify the key drivers of inpatient costs for dementia in China.Comparing our findings to studies in other developed countries, a significant wide gap was observed in the direct medical costs incurred in dementia patients. To compare the costs of different countries in different study period, we derived 2013 US dollar value by using consumer price indices of study countries in the years of costs and purchasing power parity (PPP) exchange rate in 2013 from the Organization for Economic Co-operation and Development (OECD) . The aveRegarding the cost composition, the non-medication treatment costs 38.5%) and medication costs (38.4%) took up the biggest proportion of the inpatient costs for dementia patients in this study. The non-medication treatment costs for dementia patients included cognitive behavior therapy , bright-8.5% and When comparing the average costs of dementia reported in this study with previous China-based studies, our finding was higher. Wang et al.  reportedThis study also examined the differences in hospitalization costs of dementia between two urban health insurance schemes for the first time while the previous China-based studies did not mention. The patients enrolled in the UEBMI scheme incurred higher inpatient costs than the URBMI patients, but the UEBMI patients had a lower percentage of OOP expenses. This finding was consistent with previous research showing how different types of insurance affected health care utilization and costs among patients with other diseases in China ,33,34. TIn the present study, we found that compared with AD patients, the inpatient costs for VaD patients were significantly higher. Societies where hypertension was the major problem seemed to have a proportionally high prevalence of VaD . More vaConsistent with the previous literature , hyperteWe found that longer LOS was related to higher medical expenditures, which was consistent with previous studies . After eThere were some limitations in this study. First, this study only examined hospitalization costs. The costs of outpatient care, indirect costs due to loss of productivity and family members’ informal care were not analysed since our dataset did not include this information. Thus, we may underestimate the total medical expenditures of dementia in China. Second, the study population was limited to urban insurance beneficiaries in one city of China and did not include rural patients, from which the sample cannot represent the whole Chinese population. Third, disease severity, which was often measured by the Mini-Mental State Examination and an important predictor of costs, was not included in the analysis because such data were not available in the claims data.The costs of hospitalization for dementia were high and differed by types of insurance schemes in China. Dementia was associated with substantial hospitalization costs, mainly driven by the insurance type and lengthy hospital stays. The findings of this study could provide valuable information for understanding the burden of dementia and evaluating the health insurance policy in China. Future studies could consider including outpatient costs, direct non-medical costs, and indirect costs to measure the societal costs of dementia for a better understanding of the economic burden of dementia in China. In addition, studies could also consider analysing dementia patients covered by both urban and rural insurance schemes, as well as including patients from other cities in China, and collecting information on disease severity in future analysis.The findings of this study have important policy implications for reducing the costs of hospitalization for dementia patients and improving the health insurance system in China. First, given the differences in reimbursement rates and benefit packages between the UEBMI and URBMI schemes, we suggest that these two urban health insurance schemes should eventually be consolidated to be an integrated insurance program in China. Second, efforts to reduce hospital LOS such as establishing more community-based care facilities, day care centres and promoting home-based care could be viable measures to contain hospitalization costs of dementia in China. Third, besides the basic health insurance schemes, China should establish a long-term care insurance program to support dementia patients who receive long-term institutional or residential care, which could reduce the economic burden of China’s basic health insurance funds through reducing the lengthy hospital LOS.


Chrysochromulina parva virus BQ2 labeled as “Phaeocystis globosa virus (group II)” should have been labeled “Phaeocystis globosa virus (group I).” Similarly, the two branches labeled as “Phaeocystis globosa virus (group I)” and “Phaeocystis globosa virus” adjacent to Chrysochromulina parva virus BQ1 should have both been labeled as “Phaeocystis globosa virus (group II).”In the original article, there was a mistake in The authors apologize for this error and state that this does not change the scientific conclusions of the article in any way. The original article has been updated.