Aedes mosquitoes are found to be the main carrier of ZIKV, passing the virus to humans. Originally isolated from patients in Africa in 1954 (African lineage), virus disseminated to Southeast Asia (Asian lineage), establishing new endemic foci, including one in India. Numerous cases of ZIKV infection have been reported in several locations in India and neighboring countries like Pakistan and Bangladesh since mid of the last century, suggesting that the virus reached this part of Asia soon after it was first discovered in Uganda in 1947. Although, the exact means by which ZIKV was introduced to India remains unknown, it appears that the ZIKV strain circulating in India possibly belongs to the “Asian lineage,” which has not yet been associated with microcephaly and other neurological disorders. However, there still exists a threat that the contemporary ZIKV virulent strain from South America, carrying a mutation can return to Asia, posing a potential crisis to newborns and adult patients. Currently there is no specific vaccine or antiviral medication to combat ZIKV infection, thus, vector control and continuous monitoring of potential ZIKV exposure is essential to prevent the devastating consequences similar to the ones experienced in Brazil. However, the major obstacle faced by Indian healthcare agencies is that most cases of ZIKV infection have been reported in rural areas that lack access to rapid diagnosis of infection. In this review, we attempt to present a comprehensive analysis of what is currently known about the ZIKV infection in India and the neighboring countries.Zika virus (ZIKV) is an emerging arbovirus infection endemic in multiple countries spread from Asia, Africa to the Americas and Europe. Previously known to cause rare and fairly benign human infections, ZIKV has become a major international public health emergency after being linked to unexpected neurological complications, that includes fetal brain damage/death and microcephaly in babies born to infected mothers and Guillain-Barre syndrome (GBS) in adults. It appears that a single genetic mutation in the ZIKV genome, likely acquired during explosive ZIKV outbreak in French Polynesia (2013), made virus causing mild disease to target fetus brain. The  Flavivirus genus of the Flaviviridae family, which includes other members, namely dengue virus (DENV), yellow fever virus (YFV), West Nile virus (WNV), Japanese encephalitis virus (JEV), and tick-borne encephalitis virus (TBEV) (Zika virus (ZIKV), is a mosquito-borne virus that belongs to s (TBEV) . ZIKV iss (TBEV) .During the recent large outbreak in Brazil, ZIKV emerged as an infectious agent  affectinZika virus infection in adults is mild and self-limiting and, often, infected individuals do not seek any medical attention . SymptomAedes species mosquitoes, primarily Aedes albopictus, and Aedes aegypti. There is a close relationship between the host and mosquito vector, where mosquitoes become infected while feeding off the host blood and pass the virus to the next host during the blood meal process , while 196 samples were tested for presence of anti-ZIKV neutralizing antibodies. Selection of the sites for the blood collection was made to cover the large geographic area as well as to include locations with high rainfall or forest, which could affect the epidemiology of arthropod-borne virus infection. To ensure that seroprevalence is strictly associated with local infection, individuals traveling any great distances were excluded. Neutralizing antibodies were detected in 33 serum samples out of 196 tested (16.8%). ZIKV seropositive donors were detected in many locations, including those near Bombay , Saurashtra , Madhya Pradesh , Hyderabad and Madras . It appears that there are several locations with high seropositivity rate including Bareja and Nagpur. Interestingly, authors state that the single site showing the highest rate of seropositivity was Bareja, which is located in the Ahmedabad district. The same site, the Ahmedabad city, is where ZIKV positive samples were recently reported by It is believed that ZIKV reached Asia in 1970s, when cases were documented in Pakistan and Indonesia . HoweverA. aegypti mosquitoes collected in Malaysia in 1966 . Two sites, Karachi and Punjab province in Pakistan are in close proximity to the Indian border, while Karachi is near Ahmedabad, India. ZIKV’s association with microcephaly and GBS during the recent outbreak prompted investigations on circulation of the virus in Bangladesh, country neighboring India . One serum sample was found positive for ZIKV RNA when 200 samples collected at various locations in Bangladesh were analyzed , cats (21%), dogs (14%), and birds (7%). These observations point out the importance of Aedes and Culex mosquitoes control.Zika virus surveillance study in Pakistan revealed that many animals were also seropositive for ZIKV, suggesting that different hosts, besides human, could be involved in ZIKV circulation in this region . Also, dThe Asian lineage of ZIKV is characterized by mild form of infection, with symptoms mainly including skin rashes, fever and signs of inflammation. Interestingly, fever of unknown etiology is common diagnosis in the rural hospitals in India and Pakistan. Limited access to clinical diagnostics hampers identification of infectious agent making many ZIKV cases remain undetected. This brings to attention the necessity of simple and low-cost diagnostics for ZIKV infection in the region. These diagnostics should be simple in use and require less complex and expensive equipment. One of the tests which could fit the requirements is the lateral flow immunoassay (LFI), often referred to as “dip stick.” LFI combines unique advantages of bio-recognition probes and chromatography, where results are evaluated visually. Variety of samples could be used for LFI, including, urine, serum, plasma, and saliva . LFI diavia Fc-receptors  . Later, Figure 2). According to the European Center for Disease Prevention and Control (ECDC), invasive ZIKV-vectors, including Aedes aegypti and Aedes albopictus have been reported in several European countries, including Austria, Bosnia, France, Germany, and Italy  are pre-disposed to ZIKV importation and epidemics in the near future, causing a potential public health concern (In this regards, periodic imported cases of ZIKV infection due to South American strains of ZIKV have already been documented in Europe (nd Italy . In addiulations . As thes concern . A closeSK conceived of the original idea and wrote the sections-Introduction and Modes of ZIKV transmission. TU wrote the sections-Historical overview of ZIKV in India and ZIKV genotype in India. EM prepared all the original figures. MB wrote the section-Challenges of ZIKV diagnostics and treatment. AR wrote the section-Will ZIKV microcephaly ever be diagnosed in India. SV did the overall supervision, editing, and managing of the multisite collaborations.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.


Arabidopsis thaliana is one of the best studied plant model organisms. Besides cultivation in greenhouses, cells of this plant can also be propagated in suspension cell culture. At7 is one such cell line that was established about 25 years ago. Here, we report the sequencing and the analysis of the At7 genome. Large scale duplications and deletions compared to the Columbia-0 (Col-0) reference sequence were detected. The number of deletions exceeds the number of insertions, thus indicating that a haploid genome size reduction is ongoing. Patterns of small sequence variants differ from the ones observed between A. thaliana accessions, e.g., the number of single nucleotide variants matches the number of insertions/deletions. RNA-Seq analysis reveals that disrupted alleles are less frequent in the transcriptome than the native ones. Arabidopsis thaliana is a small flowering plant distributed over the northern hemisphere and has become the model system of choice for research in plant biology. In 2000, the genome sequence was released as the first available plant genome sequence [A. thaliana accessions were analyzed by sequencing to investigate, among many other topics, genomic diversity, local adaptation, and the phylogenetic history of this species [sequence . After g species ,4. While species ,6,7, tec species  which re species ,10,11. L species .A. thaliana for basic plant biology research, it is frequently grown in greenhouses under controlled and optimized conditions. Previous studies investigated the mutation rates within a single generation [A. thaliana population in the United States of America, which is assumed to originate from a single ancestor thus showing mutations accumulated over the last decades [−9 substitutions per site per generation [Due to the high value of neration ,13. Mutaneration . Another decades . This stneration . Since nEven further away from natural conditions in the environment is the propagation of cells in suspension cultures. Cells from such cultures can easily be employed for transient transfection experiments . TransieSince most functions of plants are dispensable in suspension culture, it was expected that mutations in these dispensable genes accumulate over time due to genetic drift or even due to positive selection. We sequenced and analyzed the genome of At7 cells which have been propagated in suspension culture for about 25 years and identified massive genomic changes.A. thaliana suspension cell culture At7 [The ture At7  is derivture At7 . All seqTotal RNA was extracted based on a previously described protocol . Based oCells were separated from media through filtration on a Büchner funnel. Next, cell walls were destroyed by treatment in the Precellys Evolution  for 3 × 30 s at 8500 rpm with 1 mm zirconium beads (Roth). The following DNA extraction was based on a previously described cetyltrimethylammoniumbromid (CTAB) protocol . To incrDNA was quantified via a Qubit fluorometer  following suppliers’ recommendation. Sequencing was performed on a total of four flow cells. The Ligation Sequencing Kit SQK-LSK109 , and the Rapid Sequencing Kit SQK-RAD004 (ONT) were used to prepare two DNA libraries each for sequencing based on the suppliers’ protocol. Sequencing on a MinION (ONT) (ERR3445571) and GridION (ONT) (ERR3445572), respectively, was performed using R9.4 flow cells (ONT) based on the supplier’s instructions. Real time base calling for the MinION experiments was performed on a MinIT (ONT) via MinKNOW software (ONT).Sequencing library preparation was performed as described previously  and sequhttps://github.com/bpucker/At7. Completely deleted regions were identified through the identification of zero coverage regions in At7, i.e., regions where no At7 reads are mapped to the Col-0 reference sequence, as described before [Dedicated Python scripts were applied for the genome-wide coverage analysis, the classification of genes based on coverage values, and the investigation of coverage values at variant positions. These scripts are available at Github: d before .ONT reads longer than 3 kb were subjected to genome assembly via Canu v1.8 assembler  using thIllumina sequencing reads of At7 were aligned to the TAIR9 reference sequence of Col-0 via BWA MEM v0.7.13 . Next, GDrosophila cell lines [Mapping of the At7 Illumina short reads and ONT long reads to the Col-0 reference sequence was used to assess genomic changes in At7. The coverage analysis revealed duplications and deletions of large chromosomal segments , File S1ll lines ,37.A. thaliana plant with different centromer-specific H3 histones [An average coverage of 50-fold in a small region at 1 Mbp on Chr1 and in a region between 9.5 Mbp and 11 Mbp on Chr2 indicates hemizygosity, i.e., there is only one copy left and the regions are hemizygous. Small blocks in the northern end of Chr4 show a similar coverage. An average coverage of 150-fold indicates that most other regions are triploid. Although the starting material from Col-0 was diploid 25 years ago, these regions represent a minority in At7. A region on Chr1 (17.5 to 21 Mbp) is present in five copies (pentaploid). There are only 116 regions larger than 100 bp which appeared of being completely deleted in At7. These observations in At7 differ from findings in CHO cells, where large chromosomal segments were found in a hemizygous state . Possiblhistones .A. thaliana accessions revealed equal numbers of insertions and deletions [A. thaliana and the close relative A. lyrata revealed randomly distributed small deletions as one major source for the genome size differences [Besides Illumina sequencing, Oxford Nanopore Technologies’ MinION and GridION sequencer were deployed to generate long reads for the detection of large structural variants. Alignments against the Col-0 reference sequence revealed 160,348 deletions and 5902 insertions . Previoueletions . For comeletions . Artificeletions . The comferences . High nuRead mappings revealed that both, plastome and chondrome, are still detectable in At7 cells. Due to differences in the cultivation conditions and DNA extraction methods, a reliable comparison of plastome coverage to nucleom coverage between At7 and plants is not feasible. Although the loss of chloroplasts may seem likely in plant cells incubated in the dark and with sugar supply through the cultivation media, several central biosynthetic pathways like de novo synthesis of fatty acids ,43 and iA. thaliana accessions [Mapping of all At7 Illumina sequencing reads against the Col-0 reference sequence and following variant calling revealed an almost equal amount of single nucleotide variants (SNVs) and small insertions/deletions (InDels) . This iscessions ,8. The scessions . Variantcessions  as previcessions ,49. The cessions . It is ecessions ,29, whilcessions ,8, the gAbout 94.2 % of all small sequence variants display deviating frequencies. This can be explained by the asexual reproduction of the originally diploid At7 cells. A mutation only occurs in one allele and is consequently passed on to all daughter cells. However, the other allele of the same locus would still show the original sequence. It is very unlikely that the same mutation occurs in all alleles independently. Although exchange between alleles cannot be excluded, it is not a relevant mechanism for the generation of homozygous variants. Many homozygous variants between At7 and Col-0 are clustered in a few regions . In mostA. thaliana genome sequencing project. Errors in the At7 sequencing data and in the Col-0 reference sequence [A. thaliana accessions before and have been considered to be recent events or technical artifacts [A minority of the homozygous variants could also have been contributed by the original material which was used to generate this suspension cell cultures. Based on previous studies of mutation accumulation over generations ,6,8,14, sequence ,51 couldrtifacts .Since extremely large differences in the genome structure of At7 and Col-0 might not be revealed through variant detection tools, genes of the Col-0 genome sequence annotation Araport11 were classified based on their read mapping coverage . About 8The impact of InDels on coding sequences was analyzed by comparing the length distributions of InDels inside protein encoding sequences with the InDel length distribution outside of these coding regions. InDel lengths which are divisible by three (one codon size) are enriched inside of coding sequences. Of all 28,213 analyzed InDels, only 856 are located within the 33.5 Mbp of protein encoding regions annotated in Araport11. This depletion of InDels inside of protein encoding regions indicates that at least residual selection against disruption of these sequences is still ongoing.Assessment of the functional impact of small sequence variants revealed a high impact effect  on a total of 2189 genes . This hip-value = 0). The genomic coverage of these variant positions displays a very similar pattern: Abundance of the reference allele is on average substantially higher than the coverage of the alternative alleles. This indicates that not the nonsense mediated decay pathway [RNA-Seq analysis revealed a substantial difference in the abundance of native alleles and defective alleles. Isoforms with a destructive variant displayed a substantially reduced abundance ( pathway , but dif pathway  and seemA. thaliana genome assemblies [To reveal the sequences of large structural variants, we generated an independent de novo genome assembly of At7. The Canu assembler performed best in our hands. A set of 1.4 million ONT reads longer than 3 kb was assembled into 433 contigs representing 126.2 Mbp of the At7 nucleom . The N50semblies ,10,11. WSolanum tuberosum [After the identification of genomic changes in At7 compared to the Col-0 reference genome sequence, it would be interesting to investigate genomic changes in other cell culture lines. A previous study indicates that the speed of genomic changes could be cell line specific . Based ouberosum . Aneuplouberosum  and in luberosum ,58. CompIn this study, we focused at the genome sequence and excluded other previously reviewed DNA modifications like methylation and chromatin condensation ,60. The In-depth investigation of genomic changes under cell cultures conditions including methylation differences could benefit applications like the development of stable transgenic lines from in vitro cell cultures . Using r


Lates calcarifer, known as seabass in Asia and barramundi in Australia, is a widely farmed species internationally and in Southeast Asia and any disease outbreak will have a great economic impact on the aquaculture industry. Through disease investigation of Asian seabass from a coastal fish farm in 2015 in Singapore, a novel birnavirus named Lates calcarifer Birnavirus (LCBV) was detected and we sought to isolate and characterize the virus through molecular and biochemical methods.In order to propagate the novel birnavirus LCBV, the virus was inoculated into the Bluegill Fry (BF-2) cell line and similar clinical signs of disease were reproduced in an experimental fish challenge study using the virus isolate. Virus morphology was visualized using transmission electron microscopy (TEM). Biochemical analysis using chloroform and 5-Bromo-2′-deoxyuridine (BUDR) sensitivity assays were employed to characterize the virus. Next-Generation Sequencing (NGS) was also used to obtain the virus genome for genetic and phylogenetic analyses.Blosnavirus genus than to the Aquabirnavirus genus within the Birnaviridae family.The LCBV-infected BF-2 cell line showed cytopathic effects such as rounding and granulation of cells, localized cell death and detachment of cells observed at 3 to 5 days’ post-infection. The propagated virus, when injected intra-peritoneally into naïve Asian seabass under experimental conditions, induced lesions similar to fish naturally infected with LCBV. Morphology of LCBV, visualized under TEM, revealed icosahedral particles around 50 nm in diameter. Chloroform and BUDR sensitivity assays confirmed the virus to be a non-enveloped RNA virus. Further genome analysis using NGS identified the virus to be a birnavirus with two genome segments. Phylogenetic analyses revealed that LCBV is more closely related to the These findings revealed the presence of a novel birnavirus that could be linked to the disease observed in the Asian seabass from the coastal fish farms in Singapore. This calls for more studies on disease transmission and enhanced surveillance programs to be carried out to understand pathogenicity and epidemiology of this novel virus. The gene sequences data obtained from the study can also pave way to the development of PCR-based diagnostic test methods that will enable quick and specific identification of the virus in future disease investigations. Lates calcarifer, known as seabass in Asia and barramundi in Australia, is a member of the family Centropomidae that is widely farmed in Southeast Asia, including Singapore [Lates calcarifer Herpes Virus (LCHV) which have been detected and isolated from diseased Asian seabass in recent years [ingapore . Asian snt years , 3.In early 2015, periodic episodes of mortality occurred in a coastal Asian seabass farm in Singapore. Diseased seabass from this farm were submitted to the Animal Health Laboratory, Agri-Food & Veterinary Authority of Singapore (AVA) as part of disease investigation. Gross examination of the diseased fish revealed multifocal “white patch” lesions on the body. Hence, a viral agent was suspected to be involved in the disease outbreak.Lates calcarifer Birnavirus (LCBV) which can be classified within the Birnaviridae virus family. Since its first detection in early 2015, the birnavirus has been detected and isolated from diseased Asian seabass till now.Routine diagnostics for known common viral pathogens  did not yield any positive detection. Further attempts to identify emerging viral pathogens of seabass such as SDDV, were unsuccessful. Further investigation including cell culture, transmission electron microscopy (TEM) observation, chloroform and 5-Bromo-2′-deoxyuridine (BUDR) treatments and Next-Generation Sequencing (NGS) identified a novel birnavirus. The novel birnavirus is named as These findings provide critical information about the presence of a novel birnavirus that could be linked to disease observed in the Asian seabass from the coastal fish farm. This re-iterates the importance for disease transmission studies and enhanced surveillance programs to minimize economic losses in aquaculture. New diagnostic methods for LCBV can also be developed based on the gene sequences to facilitate detection and quick disease diagnosis.Three diseased Asian seabass from a coastal farm were submitted to the Animal Health Laboratory, AVA for examination on 10 February 2015. The submission was identified as case A17/2/15. The fishes had an average bodyweight of 272 g and average body length of 23.5 cm. Samples of skin, muscle, eye, brain, gills, heart, liver, pancreas, kidney and spleen were taken for histopathology, bacteriology and virology examination. On a separate occasion, diseased Asian seabass were submitted for examination under case A11/6/17 on 06 June 2017. The five Asian seabass submitted had an average bodyweight of 23.1 g and an average body length of 10.4 cm. Similar laboratory examinations were carried out.2 tissue culture flasks in 5 ml of growth medium  with L-glutamine, supplemented with 10% fetal bovine serum, 200 IU/mL penicillin G, 200 μg/mL streptomycin sulphate, 0.035% bicarbonate). Growth medium was removed from the flasks before inoculation of the organ homogenates. After an incubation of 1 h, culture medium -1-piperazineethanesulfonic acid (HEPES buffer) was added to the flasks. The flasks were then incubated at 25+/− 2 °C, with daily observation for cytopathic effect (CPE). Virus infectivity was assayed using 96-well microplates containing monolayers of BF-2 cells. The titre was determined by 50% tissue culture infective dose (TCID50) assay with endpoints calculated by the method of Reed and Mu¨ench [Virus isolation in Bluegill fry (BF-2) cell line was carried out using the organ pools of kidney, liver and spleen collected from the diseased fishes under cases A17/2/15 and A11/6/17. The organ homogenates were first filtered through a membrane filter with a pore size of 0.45 μm (Millipore) before inoculating onto the pre-cultured BF-2 cell monolayer. The cells were cultured at 25+/− 2 °C for 1–2 days in 25cmThe infected BF-2 cell culture flasks were frozen and thawed 3 times before centrifugation of the cell culture fluid at 3500 g for 10mins at 4 °C. The supernatant was collected and filtered through a 0.22 μm syringe filter. The filtered supernatant was loaded on top of a 30% sucrose cushion in a centrifuge tube before centrifugation at 36,000 rpm  using Beckman Type 45 rotor for 2 h at 4 °C to pellet the virus. The virus pellet was dissolved in 0.5–1 mL TNE buffer  and aliquots of purified virus were stored at − 70 °C.BF-2 cells were infected with LCBV. Two days after infection, infected cells were pelleted in 1X phosphate buffered solution (PBS)  and fixed with 4% paraformaldehyde. The infected cell pellets were next subjected to dehydration through a series of increasing concentrations of ethanol at room temperature. The cell pellets were embedded with the resin mixture  , and 16% v/v Araldite 502 . The polymerized samples were sectioned with an ultra-microtome , the sections were double-stained with lead citrate  and uranyl acetate , and viewed under the transmission electron microscope (TEM) . The electron micrographs were captured with the Gatan Charge-Coupled Device (CCD) Camera attached to the TEM.The 6th passage of the cell culture fluid from case A17/2/15 was mixed thoroughly with 10% (v/v) chloroform and incubated for 10mins at room temperature to let the chloroform react with the virus [Similarly, monolayer cultures of BF-2 cells, infected with the 6th passage of the cell culture fluid, were exposed to BUDR or to control medium , 7 and iThe total RNA was extracted from the purified virus of case A17/2/15 using Direct-zol™ RNA MiniPrep Plus kit and cell culture fluid of the 5th passage of case A11/6/17 using QIAamp Viral RNA Extraction Kit. The extracted RNA was used as an input for NGS on an Illumina MiSeq platform. RNA library preparation steps were performed at AIT Biotech Pte Ltd. using the TruSeq Total RNA Sample Preparation kit without ribosomal RNA depletion, according to manufacturer’s instructions. Reads that were generated from the MiSeq Nano flowcell were assembled into contigs using the Trinity program. Assembled contigs were used in BLAT to search against the NCBI RefSeq and GenBank viral sequences to align the contig sequences with the available viral sequences present in the library.A total of 1134 amino acid (aa) positions was used for the phylogenetic analysis. The amino acid sequence of contig DN13010 1090 aa) generated from NGS data analysis of case A11/6/17 was translated from the nucleotide sequence using ExPASy Proteomic server. The deduced amino acid sequence was aligned and compared with major capsid protein (VP2) from various viruses using BioEdit v7.1.3 . These s0 aa geneAn experimental fish challenge study was carried out to test the Koch’s postulates: to determine whether the fish injected with LCBV would display similar clinical signs and disease, to re-isolate, grow and characterize the causative agent , 13.5 and 3 × 106 TCID50/mL). Twelve fishes were injected with 0.1 mL of uninfected BF-2 cell culture supernatant as control group. Supernatant of the cell culture was prepared by centrifugation at 2000 g for 15mins and filtered with 0.45 μm syringe filter before inoculation. The fish and water quality were monitored daily. Dead and moribund fish were collected when they have been found. All the remaining fish were euthanised at end of week 5 after injection. The liver, spleen and kidney were kept for Polymerase Chain Reaction (PCR), histopathology, cell culture isolation and routine bacteriology examination.Asian seabass fingerlings  were obtained from Marine Aquaculture Centre, AVA. The fish were maintained under experimental conditions at 28 °C and fed with commercially prepared dry pellets. Twelve fish were sacrificed to test if the fish are free from known fish pathogens. Twenty-four fish were injected using the intra-peritoneal (i.p.) route with 0.1 mL of infected BF-2 cell culture supernatant  containing two different virus concentrations  were exposed to chloroform and the effect of chloroform treatment on infectivity were measured by infectivity assays. A loss of infectivity of less than 1 log10 was observed after BUDR treatment, indicating that the virus had a RNA genome  and the effects of BUDR treatment on DNA replication were measured. A loss of infectivity of less than 1 logChanna lucius virus genomic segment A and Channa lucius virus genomic segment B, respectively. The DN154 contig showed ~ 69% identity over 1638 bp with Channa lucius VP1 gene for RNA-dependent RNA polymerase.NGS was performed for whole viral genome characterization of sample from case A17/2/15. Sequencing reads were de novo assembled into contigs using Trinity. The assembled contigs were then used by BLAT to search against the NCBI RefSeq and GenBank collection of viral sequences to align the contigs with the available viral sequences in the library. Most of the contigs did not match to any genes of aquatic origin with the exception of 2 contigs which were of interest in our study: DN177 (2502 bp) and DN154 (2744 bp) that mapped onto the partial sequences of Channa lucius virus, also known as Blotched snakehead virus (BSNV), belongs to the Blosnavirus genus within the Birnaviridae family which comprises of four genera: Aquabirnavirus, Avibirnavirus, Blosnavirus and Entomobirnavirus [rnavirus , 15. BirNGS was also performed on sample from case A11/6/17. A total of 250,175 reads was used to construct contig DN13010 (3527 bp), and 358,495 reads was used to construct contig DN14359 (2731 bp). There were about only 18–21 nucleotide differences between the contigs of DN177 with DN13010, and DN154 with DN14359, suggesting the presence of the same birnavirus in both cases of A17/2/15 and A11/6/17. The nucleotide sequences of contigs DN13010 and DN14359 were uploaded to GenBank with accession numbers MK103419 and MK103420 respectively.Aquabirnavirus genus  and the other viruses in the nus Fig. . BSNV isn assays . The cluAn experimental fish challenge study was done to check for fulfilment with Koch’s postulates. The supernatant from case A17/2/15 infected BF-2 cell culture (6th passage) was injected via the intra-peritoneal route into Asian seabass fingerlings to observe if infected fish would display similar clinical signs of disease. All of the naïve fish that were injected with the control supernatant remained aclinical while several fish injected with infected BF-2 culture showed clinical signs of localized scale loss at 3 days’ post-injection. A few of the inoculated fish exhibited “white patch” lesions on the body during the first week of infection with lesions disappearing in the later phase of infection, thereby indicating that the fish may successfully mount a protective immune response against LCBV Fig. a and b. Furthermore, the organ homogenates of the LCBV-infected fish were tested positive by reverse-transcription polymerase chain reaction (RT-PCR) using primer set Seg B 1F and 1R (data not shown), while the control fish were tested negative. The organ homogenates of LCBV-infected fish were also inoculated into BF-2 cells and these cultures developed the characteristic CPE, indicative of successful virus isolation. The cell culture passages were continued for 5 passages and all the experimentally-infected samples were tested positive by RT-PCR. CPE was not evident in BF-2 cell culture when the control fish organ homogenates were used for virus isolation. This experimental fish challenge study confirmed that the novel LCBV could infect Asian seabass and be re-isolated from the infected fishes, although the natural route of virus infection remains to be studied and determined.In this study, we report the first detection of LCBV isolated from diseased Asian seabass on coastal farms in Singapore in 2015. LCBV is previously undescribed and attempts to characterize the virus were performed. First, the inoculation of organ homogenates from diseased Asian seabass into BF-2 cells resulted in the appearance of CPE. Subsequent inoculation of the supernatant harvested from these infected cultures into new cell cultures maintained the similar CPE. Second, using TEM, virion-like structures were visualized in infected-cell supernatants as electron-dense particles. TEM analyses, combined with sensitivity studies to chloroform and BUDR, provided evidence that LCBV is a non-enveloped RNA virus. Third, the NGS results and phylogenetic analyses of the virus genome revealed that a novel birnavirus was isolated from the infected cell supernatants of cases A17/2/15 and A11/6/17. Furthermore, when naïve Asian seabass were injected with the supernatant from LCBV-infected BF-2 cell culture under experimental conditions, similar clinical signs such as the “white patch” lesions were reproduced. In addition, the same viral gene sequences could be amplified from LCBV-infected fish and the same virus can be re-isolated in BF-2 cell line, substantiating the fulfilment of Koch’s postulates for this virus.Birnavirus had been known to infect fish as early as the 1940s and the virus can be transmitted both vertically via the eggs and horizontally via water , 18. In Birnaviridae family, the Aquabirnavirus and Blosnavirus genera contain birnaviruses that infect aquatic species. The most well-known fish birnavirus species and the most well-characterised member in the Aquabirnavirus genus is infectious pancreatic necrosis virus (IPNV). This is because IPNV has a very broad host range and can infect many different species of fish as well as molluscs and crustaceans, causing necrotic lesions in the pancreas and kidney [Out of the four genera of the d kidney . IPNV isd kidney . After Id kidney –24.IPNV-infected fish typically demonstrates clinical signs including darkening of the skin, abdominal swelling and abnormal behavioural signs ranging from lethargy to short bursts of erratic swimming . SignifiAquabirnavirus genus, making PCR detection of LCBV using published primers difficult. This may help to explain why PCR detection of LCBV using known primers was unsuccessful [Blosnavirus genus and separated from IPNV and other viruses in the Aquabirnavirus genus. This result is in agreement with the sequence analysis whereby LCBV shared the highest level of sequence similarity with BSNV as compared to viruses in the Aquabirnavirus genus. As BSNV has been classified under the genus Blosnavirus by the International Committee on Taxonomy of Viruses, LCBV could be classified under the genus Blosnavirus together with BSNV [Birnaviridae family, separated from Blosnavirus. This hypothesis requires further confirmation through characterization of LCBV and additional experiments such as 5′ and 3′ rapid amplification of cDNA ends (RACE) can be carried out to obtain the terminal sequences in the 2 genome segments for more in-depth phylogenetic analyses.There also exists a low sequence similarity between LCBV and the known birnaviruses within the ccessful , 26. Theith BSNV . LCBV coThis work re-emphasizes the importance of surveillance programs to keep up with emerging pathogens. With the increased surveillance on marine fish samples, it may be possible to derive the prevalence of the viral disease in our local fish population and may reflect the virus epidemiology in the neighbouring regions. This work also calls for further studies on serotyping and the potential transmission routes of the virus. Taken together, these observations may have important implications for disease containment strategies and be exploited for potential fish vaccine development.A novel birnavirus LCBV having a non-enveloped RNA genome and an icosahedral shape of ~ 50 nm in diameter was detected and characterized using molecular and biochemical methods through disease investigations of Asian seabass from coastal fish farms in Singapore. It was found that LCBV could infect and induce CPE in BF-2 cells. Similar clinical signs can be observed in an experimental fish challenge study and the fish can recover from the mild lesions caused by LCBV infection. NGS was also used to characterize the virus genome and LCBV was shown to be more closely related to BSNV rather than IPNV in phylogeny studies. NGS, when complemented with good bioinformatics analyses, served as a useful tool for genome annotation of unknown viruses. The sequences obtained from NGS could be used for the development of detection strategy using PCR, which is important and can enable timely disease investigation to be carried out and disease containment plans can be executed quickly.


Pomegranate juice (PJ), which is rich in antioxidants and bioactive polyphenols, has been posited as a beneficial dietary supplement to promote cardiovascular health. We hypothesized that maternal supplementation with PJ will improve uterine and umbilical artery function and thereby enhance fetal growth in the eNOS−/− mouse model of FGR. Wild type  and eNOS−/− mice were supplemented from E12.5-18.5 with either PJ in their drinking water or water alone. At E18.5 uterine (UtA) and umbilical (UmbA) arteries were isolated for study of vascular function, fetuses and placentas were weighed and fetal biometric measurements taken. PJ supplementation significantly increased UtA basal tone (both genotypes) and enhanced phenylephrine-induced contraction in eNOS−/− but not WT mice. Conversely PJ significantly reduced UtA relaxation in response to both acetylcholine (Ach) and sodium nitroprusside (SNP), endothelium dependent and independent vasodilators respectively from WT but not eNOS−/− mice. UmbA sensitivity to U46619-mediated contraction was increased by PJ supplementation in WT mice; PJ enhanced contraction and relaxation of UmbA to Ach and SNP respectively in both genotypes. Contrary to our hypothesis, the changes in artery function induced by PJ were not associated with an increase in fetal weight. However, PJ supplementation reduced litter size and fetal abdominal and head circumference in both genotypes. Collectively the data do not support maternal PJ supplementation as a safe or effective treatment for FGR.The eNOS Fetal growth restriction (FGR), where the fetus fails to reach its genetic growth potential, is a prevalent clinical problem affecting 3–5% of pregnancies . FGR is The underlying etiology of FGR is complex but placental dysfunction underlies most idiopathic cases . This ma−/−) mouse provides a well-characterized model of FGR, with phenotypic similarities to human FGR where; fetuses are ∼10% smaller compared with their wild type (WT) counterparts near term  and in vivo  in the placentas of pre-eclamptic patients (Pomegranate juice (PJ) consumption has been demonstrated to have a number of beneficial cardiovascular effects  and is opatients , 2013.Given the promising effects of pomegranate polyphenols on vascular function in non-pregnant animal models and humans, the ability of PJ to promote utero- and fetoplacental vascular function during fetal development and potentially improve fetal growth in FGR is worthy of investigation. Furthermore, despite the beneficial effects of pomegranate consumptions on adult health, little is known about the effect(s) of high polyphenol consumption on fetal development  and it i−/− model of FGR, without any gross perturbations to fetal development.Here, we tested the hypothesis that maternal PJ supplementation would improve uterine and umbilical artery function and increase fetal growth in the eNOS−/− mice were obtained from Jackson Laboratories (strainB6.129P2-Nos3tm1Unc/J). C57/BL6J mice, the background strain, were used as the WT control. Mice were mated and the presence of a copulation plug was denoted as embryonic day (E) 0.5 of pregnancy. All female mice were 10–16 weeks old at time of mating; male mice were 12–26 weeks old. At E12.5 (term E19.5) mice were randomly assigned to receive either PJ administered via the drinking water or drinking water alone for the remainder of pregnancy. E12.5 was chosen as the time-point of intervention to coincide with a gestational age analogous to human second trimester when FGR could reasonably be diagnosed and therapeutic intervention initiated. Animals had free access to food  and water  or PJ diluted in water (see specific dose information below), and were maintained on a 12:12-h light-dark cycle at 21–23°C in individually ventilated cages. All animals were sacrificed by cervical dislocation at E18.5 and the placentas, fetuses, uterine and umbilical arteries harvested.Animal care and experimental procedures were performed in accordance with the UK Animals (Scientific Procedures) Act 1986 under Home Office licenses PPL 40/3385 and PPL 70/8504. The Local Ethical Review Process of the University of Manchester approved all protocols. eNOS−/− = 8.57 g ± 1.05, WT+PJ = 8.9 g ± 3.37, eNOS−/− +PJ = 8.5 g ± 1.46). Median baseline weights at the commencement of treatment was 27 grams, (range 23.5–29.5 g).Pomegranate juice treated dams received a PJ solution of commercially available POM wonderful juice  dilutedFetal and placental wet weights were measured at E18.5 and growth curves constructed as previously described . Biometr−/− and WT dams and fetuses at E18.5 and mounted onto a wire myograph  and bathed in physiological salt solution (PSS) as described previously ; calculated vessel diameters were recorded. Vessels were allowed to equilibrate for 20 min post normalization before the commencement of vasoactive studies. Maximal smooth muscle contraction was measured by exposing vessels to KPSS solution (120 mM KCl substituted for NaCl). Drug-induced contraction was measured using incremental doses of phenylephrine  in uterine arteries and thromboxane mimetic U46619 (10−9 – 2 × 10−6M) in umbilical arteries at 2 min intervals. Endothelium-dependent relaxation to acetylcholine  and endothelium-independent relaxation to sodium nitro-prusside  was assessed in UtAs and UmbAs pre-contracted with 10−5M PE and an EC80 of U46619 , respectively.Main loop uterine arteries (UtAs) and umbilical arteries (UmbAs) were dissected from eNOSeviously . Normalieviously , wherebyN = 14–20 dams (UtA) and N = 11–13 dams (UmbA), and were compared between genotype and treatment groups. Artery contraction is expressed as active effective pressure (kPa) calculated as follows:For myography analysis, average responses from 2 to 4 vessels (UmbA) or 2–4 vessel segments (UtA) per dam were calculated from n = number of vessels, N = number of animals. Statistical analysis is via two-way ANOVA, with Sidak’s post hoc test where appropriate. EC50 concentrations for dose-response curves were calculated for each vessel individually by applying a sigmoidal dose response curve fit to the data. Differences in vessel diameter, basal tone, KPSS response and EC50s were assessed by two-way ANOVA or Kruskal–Wallis test and data are presented as individual data points with median. Sample size calculations were based on a 5% significance level, with 80% statistical power. For an approximate 15% improvement in vascular function , with a standard deviation of 18%, effect size estimate is 0.850. A minimum sample size of 11 animals per group is therefore required.Artery relaxation is presented as active effective pressure in response to the vasodilator expressed as a % of the maximum pre-constriction achieved with PE or U46619 (later denoted as 100%). Data are presented as mean ± SEM with N = 14–20 l. Fetal weight distribution curves are also presented. Statistical analysis was via two-way ANOVA or Kruskal–Wallis test as appropriate.Litter means for placental weight, fetal weight were calculated and the data are presented as scatterplots with median of −/− mice were ∼15–20% smaller in diameter than arteries of WT mice irrespective of treatment . Basal tone of normalized vessels , expressed as mmHg, was not affected by genotype but was increased in vessels from both WT and eNOS−/− mice that had been consuming PJ . The initial contraction to KPSS was lower in eNOS−/− mice (vs. WT), but this effect was not evident in vessels from PJ supplemented eNOS−/− mice. PJ supplementation had no effect on KPSS-induced contraction in arteries from WT mice . UtA contraction in response to PE was similar in eNOS−/− and WT mice, but in eNOS−/− mice, PJ supplementation significantly increased contraction to PE vs. water controls .UtAs of eNOS−/− phenotype reported by us . PJ supplementation reduced agonist induced UtA relaxation to both Ach and SNP in WT but not eNOS−/− mice. This reduced relaxation response was paralleled by a reduced sensitivity to agonists with a significant increase in EC50  in WT mice. There was no effect of PJ supplementation on EC50 for Ach and SNP-induced relaxation in eNOS−/− mice (data not shown).Consistent with the eNOSed by us  and otheed by us , 2013, r−/− mice were smaller than WT  and there was no effect of PJ supplementation on vessel diameter. There was no effect of genotype or treatment on basal tone or KPSS contraction (data not shown).As with UtAs, UmbAs from eNOS−/− mice ; however the sensitivity to U46619 was increased following PJ supplementation, with a significant reduction in EC50 in vessels from WT animals .UmbA maximal contraction to U46619 was not affected by PJ supplementation in either WT or eNOS−/− mice . In addition, relaxation to SNP was potentiated by PJ treatment in UmbAs of both WT and eNOS−/− mice .As previously reported UmbAs contracted in response to increasing concentrations of Ach , an effe−/− mice was significantly reduced by PJ treatment . However there was no corresponding effect on number of fetal resorptions (data not shown). eNOS−/− fetuses were smaller than their WT counterparts ; neither mean fetal weight not fetal weight distribution was altered by PJ treatment in either genotype irrespective of fetal sex (data not shown). Placental weight was comparable between all groups  resulting in a significantly decreased fetal:placental weight ratio in eNOS−/− litters . Consistent with their growth restricted phenotype, eNOS−/− fetuses were smaller than their WT counterparts, with the largest differences seen in abdominal circumference suggesting a degree of head sparing (Table 1). Despite the lack of an effect of PJ on fetal weight, biometric measurements were affected by PJ supplementation (Table 1). These effects tended to be detrimental with significant reductions in both abdominal circumference and head circumference in WT and eNOS−/− mice treated with PJ.Litter size of both WT and eNOS−/− mice. However it did have pronounced effects on vascular function of both UtAs and UmbAs with interesting differential effects in the different vascular beds and in different genotypes. In WT animals, PJ supplementation decreased sensitivity to agonist-induced relaxation (Ach and SNP) in UtAs whereas in UmbAs relaxation responses to SNP were increased, with PJ also increasing sensitivity of UmbAs to contraction in response to both U46619 and Ach. In eNOS−/− animals, PJ supplementation had the same effects on UmbAs (enhancing contractile responses and increasing relaxation to SNP) but in UtAs contraction to PE was enhanced with no observable effect on relaxation responses. These findings, along with the significant reduction in litter size, and the reduction in fetal head and abdominal circumference irrespective of genotype, do not support maternal PJ supplementation as an effective therapy to improve fetal growth in FGR.Contrary to our hypothesis, PJ supplementation from E12.5-E18.5 of pregnancy did not improve fetal growth in eNOS−/− mice, UtA and UmbA diameters were smaller but basal tone was comparable. Vessel reactivity of UtAs in untreated eNOS−/− mice was significantly decreased, with reduced artery contraction to depolarizing concentrations of potassium and blunted endothelial dependent relaxation to Ach, as demonstrated previously  production by endothelial cells in culture  of FGR. PJ had no effect on fetal or placental weight in control animals but was able to improve fetal weight under conditions of hypoxia compared to water drinking controls. However, the PJ concentration used was three times that in the present study and the enhancement in fetal weight was in part due to a restoration of calories to these animals; glucose supplementation used to control for energy intake in this study also improved fetal weight.A recently published paper, , examinein vitro, consistent with a positive effect of PJ on bone cells. However, a bone growth-promoting effect of PJ (administered from E12.5) was not evident in the current study, as PJ did not affect crown rump length. It is possible that crown-rump length might have increased with extended PJ supplementation; however, the effect of PJ to reduce head circumference and abdominal circumference observed in the current study does not support maternal treatment with PJ over a longer term in our model.−/− mice, a striking observation in the present study was a reduction in litter size in both genotypes following PJ supplementation. Interestingly, one of the mechanisms whereby PJ can reduce blood pressure in hypertensive patients is via decreasing angiotensin converting enzyme (ACE) activity (In addition to the lack of improvement in fetal weight in eNOSactivity . Whist tactivity . Specifiactivity . This maThe period of PJ supplementation in the present study can be considered to be roughly equivalent to the second and third trimesters of human pregnancy. The timing of treatment was chosen as it is from the equivalent point in human pregnancy that babies are identified as growth restricted and an intervention could realistically be introduced. However, in the mouse this time also coincides with significant placental growth to E16.5 followed by rapid fetal growth and development . Althoug−/− mouse model of FGR, which results from of a single gene deletion. Whilst the eNOS−/− model demonstrates many phenotypic similarities to human FGR it is important to note that the etiology of human FGR is complex and our results should be interpreted with caution and investigation in alternative mouse models is warranted before extrapolating the results to pregnant women.Our results indicate that pomegranate supplementation may be detrimental to fetal outcomes in this eNOSWhilst in other models pomegranate supplementation can have highly beneficial effects on multiple measures of cardiovascular health and vascular function these improvements were not observed in the current study. Indeed, maternal PJ supplementation was associated with a range of detrimental effects on utero-and fetoplacental vascular function, litter size and fetal growth. Although elevated oxidative stress has been implicated in pregnancy complications such as FGR and preeclampsia, artificially altering the anti-oxidant milieu during pregnancy may not always be beneficial . If the SG, CS, and MW formulated the research question. SF-S, ECC, and SG designed the study. SF-S, ECC, MD, EJC, and MW performed the research. SF-S, ECC, and SG analyzed the data. SF-S wrote the manuscript and all authors approved the final version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Pneumonia severity index (PSI) is an important scoring system that can assess the severity of community acquired pneumonia and determine admission status. However, there is a lack of research on whether this scoring system can be applied to viral community acquired pneumonia. The purpose of this study was to evaluate the usefulness of PSI in viral community acquired pneumonia. This retrospective cohort study included 1,434 adult patients (aged ≥18 years) who were admitted to the emergency department of a university hospital during 2013–2015 because of community-acquired pneumonia. Viral infections were diagnosed by multiplex PCR. Patients diagnosed with non-viral community-acquired pneumonia were included in the control group . The main outcome was 30-day all-cause mortality. multivariate Cox regression analyses were performed to calculate the risk of death. Respiratory viruses were detected in 261 (18.2%) patients with community-acquired pneumonia. Two types of respiratory viruses were detected in 7 cases. Of the 254 cases detected with only one virus, 62 were influenza A, 18 were influenza B, 65 were rhinovirus, 35 were respiratory syncytial virus, 25 were metapneumovirus, 20 were parainfluenza, 17 were coronavirus, 7 were bocavirus, and 5 were adenovirus. Mortality was not significantly different between patients with respiratory virus and those without respiratory virus; the 30-day all-cause mortality rates were 20.3% and 22.4%, respectively (P = 0.45). Mortality rate increased with an increasing PSI score with or without respiratory viral infection. Pulmonary severity index was significantly associated with mortality adjusted for respiratory virus detection . Pneumonia severity index score is an important factor for assessing the prognosis of patients with community-acquired pneumonia, regardless of respiratory virus detection. Viruses are a frequent cause of community associated pneumonia (CAP) . Among CIt is well known that the prognosis of community acquired pulmonary disease is influenced by the pathogen itself. However, co-morbidities of the patient and severity of infection at the time of diagnosis have been regarded as important factors in prognosis , 4. In aSince the introduction of highly sensitive viral detection methods such as polymerase chain reaction (PCR), the incidence of respiratory viruses has been increasing in community-acquired pneumonia . The devWe aimed to investigate the distribution pneumonia severity index (PSI) class and whether PSI play a role in prognostic factors in viral CAP. We studied the 30-day all-cause mortality rate for hospital admitted CAP patients with respiratory viruses in relation to pneumonia severity index (PSI).A retrospective cohort study of hospitalized adults CAP patients with respiratory virus infection was conducted. All patients aged ≥18 years admitted between January 2013 and December 2015, with suspected respiratory viral infections , were studied. If physicians suspected respiratory viral illness, then virus multiplex PCR was performed. We excluded 2,309 subjects who had no identifiable radiologic pneumonia findings . VirusesThe current study was approved by the institutional review board (IRB) at Dongsan Hospital, Keimyung University School of Medicine. The IRB waived the requirement for informed consent. This study was conducted in compliance with the Declaration of Helsinki.Pneumonia was defined as the presence of a new or progressive infiltrate found using either chest radiography or chest CT scan, in addition to two or more of the following: fever, sputum production, rhinorrhea, sore throat, dyspnea, or a diagnosis of pneumonia by the attending physician. The outcome was designated as all-cause mortality up to 30 days after hospital admission.TM Viral DNA/RNA Extraction Kit . cDNA was synthesized from each of the extracted RNA samples with cDNA Synthesis Premix  and a GeneAmp PCR System 9700 thermal cycler . Blood culture was performed at every pneumonia patient. Sputum culture was performed if patients expectorated sputum.During the study period, nasopharyngeal specimens were obtained using flocked swabs and stored and transported using the universal transport medium . Nasopharyngeal specimens were submitted for respiratory virus detection. Nucleic acids were extracted from 300μL specimens using a Viral Gene-spinRespiratory virus (RV) 13 testing was performed to detect the following viruses: adenovirus(A~F), influenza viruses A and B, RSV A, RSV B, parainfluenza viruses 1 to 3, Rhinovirus, metapneumovirus, coronavirus 229E, coronavirus OC43, and bocavirus. During the RV13 test, an internal control was added to each specimen to check the entire process from nucleic acid extraction to PCR, according to the manufacturer's instructions. An Anyplex II RV13 Detection Kit  was used, according to the manufacturer's instructions.This study was performed at Keimyung University Dongsan Hospital, a 867-bed, tertiary care teaching hospital in Daegu, Republic of Korea. If a patient had an episode of acute respiratory infection at an emergency department or outpatient clinic or within 2 days during admission, he or she underwent multiplex RT-PCR testing. Adult patients (≥ 18 years of age) who underwent multiplex RT-PCR testing between January 2013 and December 2015 were identified by electronic medical records. We collected clinical data using the electronic medical record on general characteristics, co-morbidities, presenting symptoms, lower respiratory complications, requirement of supplemental oxygen therapy and/or ventilatory support, hospitalization duration, and all-cause mortality. Chest radiography was performed on all patients admitted to the hospital, and radiographic interpretation was performed for all cases by radiologists. Chest CT scan was also taken 1327 (92.9%) cases among all CAP. Additional laboratory investigations were performed based on the results of a routine blood test. Sputum samples were collected for bacterial culture preparation at admission and during hospitalization. Blood cultures were also performed when indicated. Pneumonia severity index (PSI) consisted four parts, demographics including age and sex, co-morbidity, physical examination, and laboratory findings [Baseline characteristics  at date of admission for cases and control patients were summarized using descriptive statistics, such as proportion and means . A Chi-squared test was applied for comparison between categorical variables, and two-tailed t-tests, for comparison between continuous variables.P values <0.05 were considered statistically significant. All statistical analyses were performed using SPSS V.21.0 .The univariate and multivariate Cox regression models were used to evaluate the risk of death from CAP. We analyzed survival curves for virus detected and virus not detected subjects by the Kaplan–Meier method and compared them using the log-rank test. Multivariate Cox regression was performed with each detected virus as a variable to analyze the difference in 30-day mortality according to the type of viruses. Among 1,434 adult patients diagnosed and hospitalized for CAP, 261 (18%) were found to have a viral etiology and the remaining 1,173 (82%) had no virus detected. The 30-day mortality rate analyzed according to the PSI score . MortaliMultivariate analyses were performed to determine whether the detection of virus in CAP patients affects the mortality rate at 30 days. PSI classes and virus detection were used as independent variables for multivariate analyses. The presence of viruses did not affect prognosis and PSI classes had a significant effect on mortality at 30 days. PSI classes was observed as a significant variable associated with mortality at 30 days. In the PSI class V, the mortality rate was 27 times that of the PSI class I . PSI wasRespiratory viruses were detected in 261 (18.2%) patients with community-acquired pneumonia. Two types of respiratory viruses were detected in 7 cases. Of the 254 cases detected with only one virus, 62 were influenza A, 18 were influenza B, 65 were rhinovirus, 35 were respiratory syncytial virus, 25 were metapneumovirus, 20 were parainfluenza, 17 were coronavirus, 7 were bocavirus, and 5 were adenovirus. Among 7 co-infected patients; two had bocavirus and parainfluenza, one had bocavirus and influenza A, one had bocavirus and influenza B, one had RSV and rhinovirus, one and RSV and influenza A, one had influenza A and B. The mortality rate at 30 days according to the type of virus was analyzed. Seven patients with two or more viruses were excluded from the analysis. Virus type did not have a significant effect on 30-day all-cause mortality in CAP .A sputum specimen from 936 (65.2%) and 1333 (92.9%) of the blood-culture specimens were obtained. A pathogen in sputum was detected in 65 (24.9%) patients among 261 virus detected CAP: gram-positive pathogens were detected in 31 patients (11.9%), and gram-negative pathogens in 34 (13.0%). A pathogen in sputum was detected in 276 (24.9%) patients among 1,173 virus not detected CAP: gram- positive pathogens were detected in 112 patients (9.5%), and gram-negative pathogens in 164 (13.9%). A pathogen in blood was detected in 26 (9.9%) patients among 261 virus detected CAP: gram-positive pathogens were detected in 12 patients (4.5%), and gram-negative pathogens in 14 (5.3%). A pathogen in blood was detected in 118 (10.0%) patients among 1,173 virus not detected CAP: gram-positive pathogens were detected in 62 patients (5.2%), and gram-negative pathogens in 56 (4.7%). Regardless of respiratory virus infection, S. aureus was most common isolated pathogen either sputum and blood .The PSI class indicates mortality, and the higher the score, the higher the mortality rate for both viral and non-viral CAP . The PSIThe virus-detected CAP was significantly lower in the PSI class III group than in the virus non-detected CAP. These results suggest that viral infection has negligible effect on prognosis when co-morbidities are small or clinical features are not severe. Although there is no significant difference, our hypothesis is supported by the absence of mortality in the virus-detected CAP group, which meets the PSI class I to III . PatientSome viral infections have been reported to be associated with prognosis in critically ill patients –20, but As in the Spanish flu, pneumonia due to secondary bacterial infections has been identified as a major cause of death . After iAmong CAP patients, approximately 18% were found to have a virus, and the detection rate was similar or slightly lower than that in other studies , 32. TheIt is known that the frequency of viral CAP increases as age lowers such as children. , 34 In tThis study has several limitations. First, the absence of a virus in the nasopharynx does not exclude the presence of virus in the lower respiratory tract. Thus, the results may underestimate the viral etiology of lower respiratory tract infections. Second, data were retrospectively collected. Missing data and inadequate documentation such as medication start time may have resulted in study analysis bias. Third, there is a need to validate the results with a larger number of hospitals because of limited number of patients resulting in a small sample size.The mortality rate of CAP was significantly increased according to the PSI class for both virus-detected and virus non-detected groups. PSI class is a crucial factor for assessing the prognosis of patients with CAP, regardless of respiratory virus detection.S1 File(XLSX)Click here for additional data file.


Early detection of emerging foreign animal diseases is critical to pathogen surveillance and control programs. Rift valley fever virus (RVFV), Japanese encephalitis virus (JEV), and African swine fever virus (ASFV) represent three taxonomically and ecologically diverse vector-borne viruses with the potential to be introduced to the United States. To promote preparedness for such an event, we reviewed the current surveillance strategies and diagnostic tools in practice around the world for these emerging viruses, and summarized key points pertaining to the availability of existing guidelines and strategic approaches for early detection, surveillance, and disease management activities. We compare and contrast the surveillance and management approaches of these three diverse agents of disease as case studies to emphasize the importance of the ecological context and biology of vectors and vertebrate hosts. The information presented in this review will inform stakeholders of the current state of surveillance approaches against these transboundary foreign animal disease which threaten the United States. Rift Valley fever virus (RVFV), African swine fever virus (ASFV), and Japanese encephalitis virus (JEV), are three vector-borne veterinary pathogens with the potential to invade the United States (US). All of these viruses are notifiable animal diseases , and RifThe risk of introduction and establishment for RVFV, JEV, and ASFV to the US have all been investigated. The emergence of RVFV on the Arabian Peninsula  and receCulex spp. mosquitoes  and is additionally cross-listed as a select agent with the USDA and US Department of Health and Human Services (HHS). Due to the RVF disease severity, sizeable outbreaks, and the significant health and economic impacts, national and international guidelines for RVF surveillance and response activities have been developed.Rift Valley Fever Monitor”  published a framework to support consistency and coordination of disease control activities, referred to as the “Standard Methods and Procedures (SMPs) for control of RVF in the Greater Horn of Africa” . The oveOf particular relevance to a potential introduction of RVFV to the US, the AU-IBAR SMP document lists recommendations for surveillance approaches in areas which have not yet reported RVFV activity, in order to promote early detection and rapid response to a virus introduction. Among these recommendations are a combination of passive (continuous) and active surveillance   Table 2Table 2. The rapid deployment of available vaccines may aid considerably in curtailing virus circulation. RVF vaccines currently being deployed under emergency circumstances in Africa include the formalin-inactivated attenuated Smithburn strain. The Smithburn strain vaccine is “recommended for use in pregnant animals and in RVF-free countries experiencing outbreaks” ; even thThe USDA APHIS has published “The Foreign Animal Disease Preparedness and Response Plan (FAD PReP)—Disease Response Strategy: Rift Valley Fever” (2013)  Table 2Table 2. Cochliomyia hominivorax (Coquerel), which had been eradicated from the US since 1959 ” include virus isolation, reverse transcriptase polymerase chain reaction (RT-PCR)-based assays, an agar gel immunodiffusion (AGID) assay, an enzyme-linked immunosorbant assay (ELISA), and immunohistochemistry , 44. ManVanVuren and Paweska developed an ELISA based on the capture of the nucleocapsid protein (NP) . AlternaThe MAGPIX® and the Luminex xMAP platforms have also been used to simultaneously detect IgG antibodies against a multitude of key emerging viral species and families including RVFV , 62. TheBird et al. developed a robust pan-RVF virus quantitative RT-PCR assay . PrimersRecently, isothermal nucleic acid amplification methods have gained popularity for their ability to more rapidly detect pathogen nucleic acids directly in a clinical or field sample, and not necessarily require RNA extraction. Eular et al. developed an “isothermal ‘recombinase polymerase amplification (RPA)' assay” on an “ESEquant tubescanner device.” While lacking in sensitivity compared to qRT-PCR, the portability of this system could be a potential tool for field, pen side or bedside diagnostics .One innovative new approach to sample preparation, which also addresses sensitivity issues that might arise during surveillance, is the use of nanotrap particles to improve the detection of RVFV in low-titer samples by ~100-fold . InactivCulex mosquitoes were successfully detected by the platform  assay for quick virus inactivation and use in BSL-2 laboratories” . FurtherWhen considering a diagnosis of RVF in animals in the US, the symptoms observed tend to be non-specific. Therefore, the below diseases are recommended to comprise the testing panel for diagnosis: anthrax, bacterial septicemias, bluetongue, brucellosis, heartwater, ephemeral fever, enterotoxemia of sheep, Nairobi sheep disease, ovine enzootic abortion, peste des petits ruminants, toxic plants, trichomonosis, vibriosis, and Wesselsbron disease . Due to Culex tritaeniorhynchus), swine and wading birds, which serve as amplification hosts  is one of the most prevalent encephalitic arboviruses, mainly found in eastern and southern regions of Asia . Japaneson hosts . Adult pon hosts . Infection hosts . People on hosts , with emon hosts , and pigon hosts , howeveron hosts .Culex mosquitoes and sentinel surveillance programs in place for WNV could simultaneously also support vector surveillance for JEV through expansion of the testing panel, and funding should allow for supplementary surveillance activities.Japanese encephalitis virus (JEV), West Nile virus (WNV), and St. Louis encephalitis virus (SLE) share the same serocomplex . There iThe WHO publication, the “Manual for the Laboratory Diagnosis of Japanese Encephalitis Virus Infection” , covers Surveillance for JEV in the US would comprise passive and active surveillance of mosquito vectors, wildlife hosts, and domestic animals as well as people. Considering the genotype diversity of JEV, phylogenetic models have been developed to study JEV host changes and patterns of dispersal for different JEV genotypes using data collected through active and passive surveillance programs. Through this study, the authors concluded that active surveillance of mosquitoes was important in characterizing the circulating strain diversity of JEV, epidemiology, and transmission patterns, which informed virus spatial and evolutionary patterns . YoshikaThe practice of using of sentinel animals to detect active transmission through seroconversion is widespread in arbovirus surveillance programs. Cohorts of sentinel pigs in addition to mosquito collections have been used to determine the rate of JEV transmission to pigs in Cambodia . While vA variety of serological and molecular diagnostics, using both single- and multiplex platforms, have been developed for detection of JEV .Antibody detection (IgM and IgG) by capture ELISA is the recognized standard for JE diagnosis , 84. HowCurrently, the recommended method for JE diagnosis of human infections is the JEV-specific IgM antibody-capture enzyme-linked immunosorbent assay (MAC-ELISA) in CSF and serum . SeveralRecognizing the problem of serological cross-reactivity among flaviviruses and its impact on surveillance and diagnosis, Cleton et al. used sera from naturally and experimentally-infected horses to develop a protein microarray to differentiate flaviviral infections in horses by NS1-antigen . DiffereJEV viral RNA can be detected from viremic vertebrate samples and mosquitoes by many different available assays spanning traditional single-plex RT-PCR , 90, to Asfaviridae: Asfavirus), is transmitted among wild vertebrate reservoir species by soft ticks in the genus Ornithodoros. Infection of wild and domestic suids with this virus is associated with high mortality, and hemorrhagic fever, however infections in African suid species are asymptomatic (African swine fever virus (ASFV)  for concurrent detection of ASFV, classical swine fever virus, and foot and mouth disease virus, and evaluated use of this assay on swine oral secretions . This mRWhile all three of these pathogens are arthropod-borne viruses, the unique biology of these three systems demonstrates that there is no single approach for the surveillance and diagnostics. Vector, animal, and public health infrastructure needs to sustain broad capacity to rapidly detect and respond to these three viruses and more. The NVSL system, including the over 60 NAHNL labs and the FADDL will play a critical role in the diagnostic testing and confirmation of these disease agents. Currently, many NAHLN laboratories are already approved for ASFV testing . While vGH and RK: conceptualization. GH: funding acquisition. RK: original draft. RK, GH, and EA: review and editing.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.


Hypertension represents a high burden of disease in different healthcare systems. Recent guideline published in 2017 by the American Heart Association and the American College of Cardiology has generated a debate between clinicians and policymakers due to the lowering of diagnosis threshold and the subsequent increase of the prevalence and healthcare costs. No empirical research exists addressing the question about the pressure on healthcare costs generated by new standards. This study aims to quantify the impact on the hypertension diagnosis and treatment costs for healthcare system using the new hypertension guideline.We conducted a budget impact analysis from a Colombianhealthcare payer’s perspective with a 3-year time horizon (2018–2020), in which we estimated the difference in total medical care costs between previous hypertension cut-off points (140/90 mmHg) and new guideline cut-off points (130/80 mmHg).Our results show that the impact of the adoption of the new hypertension guideline would represent a decrease close to 22% in total annual high blood pressure costs in Colombia. This reduction is mainly driven by a lower number of cardiovascular complications. It is worth noting that these results should be taken with caution due to local available data.A high-middle income country such as Colombia should carry out an exhaustive revision of the recommendations of the new hypertension guideline, due to its high probability of saving medical treatment costs for the healthcare system.The online version of this article (10.1186/s12962-018-0152-5) contains supplementary material, which is available to authorized users. Hypertension represents a high burden of disease in different healthcare systems. The World Health Organization (WHO) attributes to hypertension at least 45% of deaths by cardiomyopathies, 51% of deaths by cerebrovascular diseases and costs near 1.26 billion dollars in high-middle income countries  for each scenario was obtained from SPRINT study . Notice We assumed that population that survives each year is equal to those individuals who were diagnosed as hypertensive with the new cut-off points and followed physician prescriptions accordingly with medical guide recommendations  , 19. FigFor each scenario, we calculated total average treatment costs that included medications, diagnostic aids and the acute and chronic management of complications  in patients of the target population. Costs for rehabilitation programs and outpatient treatment and follow-up were not included.Blood sugar, complete blood count, creatinine, urinalysis, conventional electrocardiogram, electrolytes, lipid profile, serum calcium and, thyroid stimulating hormone were included as diagnostic aids.Regarding medication costs, the most frequently prescribed antihypertensive drugs in Colombia were identified. Machado et al.  found thThe Drug Price Information System of Colombia (SISMED) along with the average of the frequency of prescription were used to estimate the average daily cost of drugs, for the period of July–September of 2017. Regarding prescription, the values provided by the SPRINT study were taken into account: 2.8 daily medications were estimated for the intensive control scenario and 1.8 daily medications for the standard control .Furthermore, the cost per episode and the cardiovascular complication costs associated with outpatient management  were taken from local studies  by using Colombian Consumer Price Index  . Costs aTwo clinicians from the investigation group conducted the validity face; they evaluated the structure, outcomes and possible treatment routes for patients in each scenario. The model’s internal validity was evaluated by performing univariate sensitivity analysis, black box test and running the model using extreme values , 26. TheThe outputs of the model correspond to the total number of major cardiovascular events and total medical costs for both scenarios. For external validation, the model’s ability to quantify the number of events was evaluated, and contrasted with the number of benefits reported by the SISPRO for years 2018–2020. Events were defined according to ICD-10 codes as I219- Acute Myocardial Infarction, unspecified; I509- heart failure, unspecified and, I64- stroke, not specified as hemorrhage or infarction.The estimated number of events with their corresponding 95% confidence interval was predicted by using a simple exponential smoothing method  . If the Confidence intervals for estimated cardiovascular events are available in Additional file A probabilistic sensitivity analysis was performed by second-order Monte Carlo simulation with 5000 iterations. Table The probability distribution Beta was used to modeling probabilities. In this distribution, alpha values represent the number of patients who presented the event of interest. Beta values represent the number of patients who did not show the event of interest. For modeling quantity of resources and number of episodes, a Poisson probability distribution was used. In this distribution, the lambda parameter represents the average number of events of interest. For modelling costs, we used a Gamma distribution, Beta parameter is the variance divided by expected value and alpha parameter is expected value divided by beta parameter.The model shows that lowering the cut-off points from 140/90 to 130/80 mmHg and the treatment goal (less than 120/80 mmHg) lead to a decrease in the total annual direct cost of approximately USD 108 million, which represents a reduction of more than 20% of total annual HBP costs in 2018 . Cost saving ranged from USD 9.5 million to USD 900 million . Lowering cut-off points and target values would imply an increase of these costs by 31%, corresponding to USD 6.6 million  . HoweverTo our knowledge, there are no budget impact evaluations to compare these findings, although this “paradoxical” pattern of cost savings followed by an increase in prevalence has been described in other studies , 30. SevBesides the apparent reduction in costs attributed to hypertension-related complications, it is interesting the indirect role that adherence to pharmacological treatment would play. It is unlikely to attribute costs reduction to the choice of cost-effective therapies by the physician. Evidence has shown that they do not consider aspects related to costs or reimbursement measures to establishment or choice of antihypertensive treatment since their incentives are mainly in line to achieve HBP target values and reduce complications .Classifying a patient as hypertensive and establishing a lower BP target value would create incentives to initiate intensive management, leading to a decrease in complications risk. Sensitivity analysis supports this finding which suggested that the level of physician and patient adherence were the primary factor to reduce costs significantly. This relationship was also described by Koçkaya et al. , who fouIn contrast, side effects due to stricter HBP treatment goals (120/70 mmHg) might result in overmedication and hence, increase costs. SPRINT study shows that there are no significant differences in some adverse effects between the group of patients with intensive and standard treatment: 38% of patients treated targeting less than 120 mmHg exhibit serious adverse events and 37% of patients treated with a conventional target presented adverse events . HoweverNevertheless, some caveats of this study must be considered. The budget impact model did not consider therapeutic changes in lifestyle. It is well known that these changes contribute to a decrease in blood pressure and a lower requirement for medications . Thus, iIn addition, given the limited available local data, some parameters as was mentioned above, such as increasing hypertension prevalence and the proportion of cardiovascular events were taken from AHA/ACC guideline and SPRINT-study. The population used in these studies might differ from our population, which might result in an overestimation of the budget impact of the adoption of stricter HBP treatment goals. Furthermore, subgroup analysis by sex and age were not considered in the SPRINT study, which is why it was not possible to consider the differences in cardiovascular event proportions by sex and age in the current study.Despite of these data and methodological limitations, this study sheds the new lights of the economic impact of the recently published hypertension guideline in little-research field, estimating the potential budget impact of the new treatment standards in a middle-income country. Hypertension and complications costs represented about 1.8% of total healthcare expenditure in Colombia in 2014. Our results suggest the importance of taking care of future cardiovascular complications by adopting the recommendations of the new hypertension guideline of the AHA/ACC. It would save costs in a high-middle income country which faces a demographic trend towards population aging and significant financial sustainability difficulties.Despite the concern about the economic impact of increasing hypertension prevalence given lower high blood pressure standards, the potential budget impact related to the adoption of the new hypertension guideline is positive. Our results show that adequate and strict control of blood pressure earlier in life might decrease costs associated with complication management. This is relevant for high-middle income countries such as Colombia that exhibits high chronic prevalence and faces significant sustainability healthcare challenges at the same time. Strategies for guideline adherence by medical personnel should be reinforced to enhance the appropriate management and adequate control of blood pressure figures.Additional file 1. Confidence intervals for estimated cardiovascular events, obtained from probabilistic sensitivity analysis.


We isolated Japanese encephalitis virus (JEV) from brain samples of 2 seals with lethal encephalitis at Weihai Aquarium, Weihai, China, in 2017. We confirmed our findings by immunohistochemical staining and electron microscopy. Phylogenetic analysis showed this virus was genotype I. Our findings suggest that JEV might disseminate though infected zoo animals. Flaviviridae, genus Flavivirus). The virus genome contains a 5′ untranslated region (UTR), followed by a 10,296-nt coding region and a 3′ UTR. The polyprotein consists of 3 structural proteins designated capsid, membrane, and envelope (E) and 7 nonstructural proteins  , a 1-year-old male seal and 1.5-year-old female seal, kept in Weihai Aquarium  were found displaying neurologic signs (backward head). The rest of the seals  in this aquarium did not display any clinical signs of illness. All seals were raised in the same pools and fed a diet of frozen smashed fish. The 2 sick seals were found dead the next day; immediately afterward, we performed necropsies. We observed hemorrhages in the brain . PCR results were negative for all tested pathogens except JEV, indicating this virus might have infected these seals.We collected tissue samples to test for seal parvovirus, phocine distemper virus, influenza A virus, coronavirus, canine distemper virus, canine adenovirus, pseudorabies virus, rabies virus, and JEV using commercial real-time PCR kits  to detect viral antigen in brain samples and performed immunohistochemical staining as previously described , panel D2. We observed cytopathic effects  with the fourth passage of brain homogenate supernatant  calf serum, 100 U/mL penicillin, and 100 mg/mL streptomycin at 37°C in 5% COg for 30 min, pelleted virus onto 3-mm grids, and then stained grids with 2% sodium phosphotungstate for 1.5 min. We used the Tecnai G2 BioTWIN Transmission Electron Microscope  operating at 85 kV to visualize virus particles. We observed round-shaped virions morphologically similar to those of the Flavivirus genus (data not shown). Compared with uninfected cells, many mature virions were found in the endoplasmic reticulum of infected cells by transmission EM (We subjected this fourth passage of virus to negative-staining electron microscopy (EM) analysis. We ultracentrifuged cell supernatant at 82,000 × http://www.takara-bio.com), reverse transcribed RNA to cDNA, and amplified the cDNA by PCR using 15 different primer pairs (https://www.thermofisher.com). Sequence analysis indicated that the complete genome sequence of JEV Seal-Anheal-2017 (GenBank accession no. MH165313) was 10,965 nt in length, encoding a 10,299-nt single open reading frame flanked by a 96-nt 5′ UTR and 570-nt 3′ UTR.We extracted total RNA from the supernatant of this fourth passage of virus, which we designated Seal-Anheal-2017, using the NucleoSpin RNA Midi kit  with XJ69, a mosquito-origin JEV isolated in Xianju County of Zhengjiang Province, China  (9). XJ6The JEV E protein is a major structural protein contributing to viral virulence, host tropism, and antigenicity (Our data indicate that the causative agent of these 2 seal deaths was JEV. Why and how disease developed is unclear. In humans, young children are susceptible to JEV infection and often have fatal outcomes. Because the 2 seals that died were in their adolescence and the others with no clinical signs were older, age might have contributed to the fatal outcomes seen with these seals.Culex tritaeniorhynchus mosquitoes were found in the aquarium, and seal keepers reported that they had often been bitten by mosquitoes. No human cases of Japanese encephalitis were reported in the vicinity of the aquarium; however, most of the population received compulsory JEV vaccination. C. tritaeniorhynchus mosquitoes also feed on livestock and birds, which act as bridging vectors leading to infections in other animals (JEV might cause neurologic disease in more organisms than previously thought. Zoo staff should determine the causative agents in animals displaying neurologic disease, so they can better protect their animals from pathogens. Seals might need to be screened for JEV before they are transported to non–JEV-endemic areas.


The role of Easter Island in the dissemination of Zika virus from the Pacific islands into the Americas remains unclear. We analyzed new Zika virus sequences from Eastern Island and found that Zika virus was independently disseminated from French Polynesia into the Americas and Easter Island at around the same time. SP-AM) that arose because of sequential single viral disseminations from Southeast Asia into French Polynesia and from the South Pacific into Latin America  at 1999  and support 2 independent disseminations from Southeast Asia into the Pacific region—the first in Micronesia around 2007, with no further spread, and the second originating the ZIKVSP-AM lineage that fueled epidemics during 2013–2016 in the South Pacific and the Americas.The overall timescale of the phylogenetic tree estimated for Zika virus Asian genotype strains was fully consistent with previous reports – Table 2.SP-AM lineage was traced to French Polynesia (PSP >0.98) at 2013.3 (BCI 2012.9–2013.6). This lineage exhibits strong geographic subdivision; several highly supported island-specific monophyletic subclades nested among basal strains from French Polynesia  contains strains from French Polynesia, the Americas, New Caledonia, and Vanuatu, whereas clade II (PP >0.94) encloses strains from Easter Island and the Cook Islands. Zika virus strains from Fiji, American Samoa, Samoa, Tonga, and the Solomon Islands branched together in a large but not well-supported (PP <0.57) monophyletic group.The origin of the ZIKV>0.98), as well as of the clade II strain introduced into Easter Island (PSP >0.77), refuting the hypothesis of introduction of Zika virus into the Americas through Easter Island. The source location of the clade II strain of the Cook Islands was traced to Easter Island (PSP >0.34) or Easter Island (PSP >0.37). Our analyses indicate that Zika virus was introduced into Easter Island, New Caledonia, the Cook Islands, and the Americas at around the same time (BCI 2013.6–2014.8), supporting a much longer period of undetected Zika virus transmission in the Americas (≈15 months) (French Polynesia was the most probable source location of the Zika virus clade I strains introduced into the Americas (PSP 1), Vanuatu (PSP 1), and New Caledonia  and the Americas from late 2013 to mid-2014, coinciding with a peak in the number of suspected Zika cases in French Polynesia. The long period of cryptic circulation of Zika virus in the Americas, the early detection of Zika virus in the Caribbean in December 2014 (Our results indicate that French Polynesia was the main hub of dissemination of the ZIKVGeographic dissemination of Zika virus Asian genotype throughout South Pacific and the Americas, estimated with asymmetric and symmetric models.


Scientific Data 5:170199 doi: 10.1038/sdata.2017.199 (2018); Published 9 January 2018; Updated 31 July 2018The Data Descriptor incorrectly states that hybridization for the miRNA expression microarray analysis was performed with an Affymetrix 450 Fluidics Station. This step was performed using an Affymetrix GeneChip Hybridization Oven 645.


The trade of glass beads has long been assumed to have been under Islamic dominance during the early centuries following the Arab conquest of the Middle East, judged by the prevalence of Islamic beads in the archaeological contexts from Viking Scandinavia to medieval Morocco. This paper explores the impact of the Byzantine-Slavic transition on the use and by extension trade of glass beads in the Balkans from the seventh to the ninth century CE. A series of 48 glass beads and 4 vessel fragments from two excavated sites in modern day Albania have been analysed morphologically, technologically and chemically by LA-ICP-MS. The seventh-century beads from Lezha have typological parallels among central European assemblages and are made from recycled natron-type glass. The presence of a high lead-iron-natron variant is of particular interest as it potentially reflects a regional production. The ninth-century beads from Komani are made from soda-rich plant ash glass from the eastern Mediterranean and Mesopotamia and correspond to an Islamic typology. The chronological and geographical differences are reflected in the distinctive cobalt sources used for the two groups. While the beads from Lezha are coloured with a cobalt not correlated with any particular element, the cobalt source of the Komani samples is associated with zinc, typical of Islamic glass making. It thus appears that the supply of beads during the seventh century when the Balkans were under Slavic occupation relied on regional production and recycled material, and that a long-distance trade with the eastern Mediterranean was revived following the Byzantine re-conquest of the south-eastern Adriatic in the ninth century. Intriguingly, the Albanian finds confirm the Islamic control of the production and trade of glass beads during this period and highlight the mediatory role of the Byzantine Empire.The online version of this article (10.1007/s12520-017-0583-5) contains supplementary material, which is available to authorized users. The multidisciplinary study of glass beads in early medieval contexts has proved particularly fruitful to elucidate commercial and cultural networks Pion . It has The study of Merovingian and Lombard beads showed that most of the beads produced in central Europe between the sixth and eighth century were wound beads made from natron type glass derived from the primary sources in Egypt and the Levant or from recycled cullet , by contrast, was a much larger town with six churches and an extensive necropolis. There is evidence of two distinct periods of urban development, the first during the sixth and seventh century, and a second phase in the ninth and tenth century. The site was finally abandoned in the thirteen century , monochrome beads with applied filaments, pinched flower-shaped or three-lobed and moulded beads Table . All theThe ninth- and tenth-century beads from Komani and the two medieval beads from Lezha vary likewise greatly in shape  and colour , but they are mostly drawn and hot-cut, with or without a hot finish Table . Similarunguentarium, usually dated to between the tenth and fourteenth century  have a natron-type base glass with elevated levels of magnesium, titanium and iron, associated with slightly increased vanadium, chromium and zirconium. These characteristics correspond to the so-called Foy-2 compositional group  have a peculiar composition with high lead (PbO 28–34%) and iron (FeO 7.7–15.2%) contents, similar to some sixth- to seventh-century Merovingian beads , has very high magnesium contents (MgO > 3.5%), relatively low phosphorus levels and a much lower potassium to magnesium ratio compared to the four beads from Komani that clearly form a separate group  associated with the plant ash component. Two sub-groups can be distinguished on account of their varying lanthanum, chromium, titanium and zirconium contents that reflect different silica sources Fig. a, b. The% associaup Table . These cup Table , an attrup Table . The fouup Table a, b. Intup Table . A possiup Table . It has up Table .Fig. 7ChSome of the colourants represented among the plant-ash glass beads similarly confirm the chronological and geographical attribution of the groups. The cobalt in the blue beads is strongly correlated with zinc Fig. . This tyThe metal sandwich beads are in fact silver leaf glasses  is produced from soda ash glass with low alumina and titanium and higher calcium and zirconium contents. It is coloured with a cobalt colourant associated with indium, zinc and lead, commonly dated to the thirteen century   represents an exception in that it was made from a combination of sixth- to seventh-century Foy-2 type glass, decorated with a white glass of Egypt II characteristics, usually dated to the eighth or ninth century. Morphologically and compositionally similar beads were included in necklaces retrieved from Slovenian graves, where they typically assume a prominent position . The evidence from Lezha and Komani confirm the wider currency of Islamic beads and the strategic position of the Balkans might in fact have been an important vector for this trade.The presence of Islamic beads in south-western Illyricum during the ninth century testifies to a renewed commercial and cultural connectivity of the Balkans after the region had again come under Byzantine dominion Cheynet . SimilarThe chemical characteristics of the four vessel fragments reflect yet another source of supply. Compositionally, they resemble some Byzantine artefacts from a Coptic monastery on the Sinai Peninsula